tions of g r o w t h t h a n are most anaerobes since t h e y require a n Eh of approximately 0.2 millivolts for growth. Cysteine or N a2S accelerates growth, p r e s u m a b l y b y lowering t h e E/z sufficiently; reducing agents of m o r e oxidizing E0' values such as ascorbate or ferrous ions are less ef-fective. Desulfovibrio desulfuricans will utilize S 04" , S03"~, S203*"~, S406 -- , S204~", S205~", a n d even colloidal sulfur. Postgate (232, 233) showed t h a t t h e kinetics of substrate reduction in h y d r o g e n b y whole cells w e r e consistent w i t h sulfite being a n intermediate. T h i s was con-firmed b y Millet (187) w i t h labeled sulfur-32. A u b e r t suggested t h a t sulfur entered organic combination at t h e sulfite level of oxidation ( 1 3 ) . Sulfate does not appear to p e n e t r a t e t h e bacterial cell easily, a n d
Reviews b y Starkey ( 2 7 9 ) , Updegraff a n d W r e n ( 3 0 0 ) , Butlin a n d Postgate ( 4 5 ) , Postgate ( 2 3 3 ) , ZoBell ( 3 3 3 ) ; a n d Kluyvel a n d v a n Niel (136) h a v e considered aspects of t h e n u t r i t i o n a n d physiology of the bacteria reducing sulfate. T h e culture media r e c o m m e n d e d for growing these bacteria, u s u a l l y Desulfovibrio species, contain a con-siderable a m o u n t of iron, p r e s u m a b l y to act as a n indicator since black iron sulfide is formed d u r i n g growth. T h e mesophilic sulfate-reducing
bacteria are m o r e exacting in their r e q u i r e m e n t s for anaerobic
condi-it h a s been suggested t h a t t h e anion m i g h t require a p r e l i m i n a r y ender-gonic activation a n d t h a t t h e activated sulfate would t h e n enter t h e cell. Proof for this theory is lacking a n d a r y l sulfatases w h i c h activate sulfate h a v e n o t been found i n D. desulfuricans. Thiobacillus species oxidize sulfide to sulfuric acid a n d sulfate-reducing bacteria reduce sulfate to sulfur ( 2 3 3 ) .
F u n g i t h a t utilize sulfate a r e u s u a l l y g r o w n i n media t h a t contain between 0.0001 a n d 0.0006 M sulfur. Most fungi reduce sulfate a n d incorporate it into t h e sulfur-containing a m i n o acids a n d glutathione.
Aquatic Phycornycetes, Saprolegniales, a n d Blastocladiales (47) do not u s e sulfate, b u t other Phycomycetes, including Chytridiales, L e p
-tomitales, Peronosporales, a n d Zygomycetes utilize sulfate ( 5 8 ) . M u t a n t s r e q u i r i n g reduced sulfur compounds h a v e been obtained from Neurospora crassa ( 1 1 4 ) , Aspergillus nidulans (231a, 2 6 5 ) , Pénicillium chrysogenum ( 1 1 0 ) , Ophiostoma multiannulatum ( 5 8 ) , a n d Ustilago zeae ( 2 2 8 ) . A s u m m a r y of sulfur sources for fungi is given i n T a b l e X I . Most fungi utilize sulfide, thiosulfate, a n d t h e m o r e oxidized forms of inorganic sulfur (except dithionite), b u t because of t h e instability of these compounds i n culture solutions, t h e data on t h e reduced forms are questionable. Steinberg found t h a t Aspergillus niger grew better on aged t h a n on fresh samples of sodium sulfide or disulfide, p r e s u m a b l y since it utilizes t h e m o r e oxidized forms of these compounds ( 2 8 5 ) . T h e p a t h w a y suggested for sulfate reduction is: sulfate ( S 04~ ) - » sulfite (S03—) - » sulfide ( S " ) or thiosulfate ( S203— ) - » cysteine -> methio-n i methio-n e .
Spencer a n d H a r a d a (276) h a v e recently p u t forward a tentative scheme for sulfate utilization i n fungi (Scheme I ) .
SCHEME I
ATP + s o ; ATP sulfurylase adenosine - 5 - sulf atophosphate +
phosphatase pyrophosphate
adenosine-3' -phosphate*5' - sulfatophosphate SOa cysteine + choline + adenosine 3',5-diphosphate
choline sulfokinase choline sulfate
T h e y suggest t h a t all fungi w h i c h utilize inorganic sulfate as sole source of sulfur for growth c a n produce adenosine-3'-phosphate
5'-3. I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 411
sulfatophosphate. W o r k w i t h m u t a n t strains of Aspergillus nidulans h a s shown t h a t t h e reduction of sulfate to sulfite is a n obligatory step in t h e formation of cysteine from inorganic sulfate ( 2 6 5 ) . T h e reaction
(Eq. 1 0 ) .
S 04— + 2H -> SO,— + H20 (10)
is endergonic a n d has a standard free e n e r g y change of + 1 4 kcal.
Choline sulfate, a n ester widely distributed in fungi, is also utilized b y t h e m as sole source of sulfur (64a, 128). Since m u t a n t strains of Aspergillus nidulans a n d Pénicillium notatum do not utilize inorganic sulfate or choline sulfate as sulfur sources, a n d t h e p a r e n t strains grow readily on either compound, it is assumed t h a t t h e two h a v e common metabolic p a t h w a y s ( 2 6 5 ) . E g a m i a n d Itahashi (72) showed t h a t Aspergillus oryzae utilize choline sulfate m o r e readily t h a n inorganic sulfate; competitive metabolism experiments w i t h two sulfur com-pounds w e r e interpreted to m e a n t h a t choline sulfate is a n intermediate i n t h e utilization of sulfur of inorganic sulfate.
Blastocladiella emersonii grows w i t h either cysteine or m e t h i o n i n e a n d is p r e s u m a b l y blocked prior to cysteine synthesis ( 1 7 ) . Allomyces arbuscula, however, can utilize m e t h i o n i n e only ( 9 9 ) . Reduction of sulfate to sulfide is n o t effected b y fungi as it is i n some bacteria.
Candida species reduce elemental sulfur to h y d r o g e n sulfide (184, 259) a n d Neurospora crassa reduces selenite to selenium ( 3 3 1 ) . Sulfur a n d its compounds a r e oxidized b y fungi, e.g., Pénicillium luteum a n d Aspergillus niger (192) a n d sulfur-containing a m i n o acids b y Micro-sporum gypseum ( 2 7 7 ) . Cysteine sulfinic acid h a s been suggested as a n intermediate i n cysteine oxidation ( 2 7 7 ) .
E. I R O N
It is well k n o w n t h a t iron is a constituent of respiratory e n z y m e s a n d is found chiefly i n association w i t h p o r p h y r i n s in t h e form of h e m e compounds. A n u m b e r of e n z y m e s also r e q u i r e inorganic iron for their activity.
W a r i n g a n d W e r k m a n (318) studied t h e effect of iron deficiency on a n u m b e r of heterotrophic bacteria. T h e growth of Aerobacter indolo-genes, Escherichia coli, Pseudomonas aeruginosa, a n d Klebsiella pneu-moniae w a s e n h a n c e d w h e n iron was added to a m e d i u m previously treated w i t h 8-hydroxyquinoline. Pseudomonas aeruginosa required three to four times m o r e iron t h a n t h e others, probably because of its complete four-band cytochrome a n d cytochrome oxidase system a n d its catalase a n d peroxidase activity. I n Aerobacter indologenes ( = A.
cloacae v a r . ) , hydrogenase, formic dehydrogenase, formic
hydrog-enylase, a n d cytochrome w e r e dependent on the a m o u n t of iron in the medium- T h e oxidation of a m m o n i a to nitrite in e n r i c h m e n t cultures of Nitrosomonas species was accelerated b y 6 m g iron per liter, after use of quinoline purification methods ( 1 8 0 ) . It is n o w k n o w n t h a t this extraordinarily high r e q u i r e m e n t is due to residual quinoline i n t h e culture solution since a large p a r t of the added iron complexes w i t h the chelate. T h e m e t a l is also essential for the growth of Nitrobacter
( 3 , 1 7 5 ) .
Bacteria not containing h e m e compounds also r e q u i r e iron for their metabolism, e.g., in Clostridium pasteurianum, the m e t a l is essential for nitrogen fixation a n d also for hydrogenase action ( 2 1 8 ) .
A n obligate r e q u i r e m e n t for iron has been shown w h e n bacteria a n d fungi dissimilate nitrate, as discussed u n d e r nitrogen. I n some cases h e m e compounds are required for n i t r a t e reduction, as in Haemophilus influenzae (97) a n d in a strain of Staphylococcus aureus ( 1 4 1 ) . I n H. influenzae, only p o r p h y r i n compounds w i t h a v i n y l side chain func-tioned whereas S. aureus could utilize h e m a t i n but not protoporphyrin.
F u n g i usually r e q u i r e between 0.1 a n d 0.3 p p m iron for growth a n d are able to utilize either ferrous or ferric iron. Iron-containing con-stituents of fungi include t h e h e m e compounds, as in bacteria. T h e p i g m e n t p u l c h e r r i m i n in Torulopsis pulcherrima contains iron (58, 136, 248) as does aspergillin, t h e black p i g m e n t in spores of Aspergillus niger, w h i c h contains 0 . 2 5 % of the m e t a l ( 2 3 9 ) . Some fungi, especially species of Aspergillus, release large a m o u n t s of organic acids, including citric a n d gluconic acids, w h i c h bind ferric iron; the ecological value of this is obvious. Metabolic events r e q u i r i n g iron in fungi include organic acid formation ( 8 7 ) , penicillin production (137, 2 9 9 ) , sporula-tion, (202, 2 0 3 , 2 8 2 ) , b u t it is m o r e t h a n likely t h a t these a r e non-specific a n d indirect effects. It is claimed t h a t t h e production of penicillin a n d streptomycin r e q u i r e m o r e iron t h a n is needed for m a x i m u m growth ( 5 8 ) .
Most of t h e critical work on iron r e q u i r e m e n t s in algae has been done w i t h Chlorella (112a,b), Scenedesmus, a n d Anabaena, b u t it is likely t h a t all algae require iron for their metabolism since t h e types so far examined contain functional h e m e compounds (86, 197, 2 9 3 ) .
F . C O P P E R
Copper is a well-known constituent of a n u m b e r of enzymes, poly-phenol oxidase, monopoly-phenol oxidase, laccase, ascorbic acid oxidase, nitrite reductase, discussed fully in Chapter 3 of V o l u m e IA.
Copper is a well-known inhibitor of growth in microorganisms and has been used extensively as a fungicide. It is required for growth in
3 . I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 4 1 3
FIG. 1 7 . Trace metal contents of Pseudomonas aeruginosa grown under various oxygen tensions. Various gas mixtures ( N2; 1 % 02 in N2; 5 % 02 in N2; air; and Oa) were dispersed through Pyrex glass sinters under a pressure of 3 pounds per square inch and the flasks were shaken at 1 0 0 oscillations per minute through a horizontal displacement of about 2 inches. -quirements of Rhodospirillum rubrum a n d Rhodopseudomonas a n d showed t h a t copper w a s o n e of t h e essential trace metals ( 1 1 6 ) . I n Pseudomonas aeruginosa, copper is required for denitrification,
espe-deficient media. I t is t h e author's experience t h a t t h e a m o u n t s of copper used i n several of t h e r e c o m m e n d e d m e d i a for microorganisms are often unnecessarily high, a n d in some at toxic concentrations.
T h e r e is evidence t h a t copper is required for nitrogen fixation. T h u s Gribanov (101) showed t h a t o p t i m u m rates of nitrogen fixation b y Azotobacter chroococcum w e r e achieved at t h e extraordinarily high level of 5 m g . copper p e r liter. H i g h e r concentrations reduced t h e fixa-tion process. Loginova (155) observed t h a t t h e addifixa-tion of 10 kg of copper sulfate p e r hectare h a d n o effect on the n u m b e r of nodules in vetch b u t m a r k e d l y increased their weight since copper increased t h e nitrogen-fixing capacity of y o u n g nodules. W i t h aged nodules t h e effect of copper decreased. Greenwood (100) showed t h a t w h e n clover w a s supplied w i t h sufficient combined nitrogen to suppress t h e symbiotic fixation of t h e gas, withholding copper produced copper-deficiency symptoms b u t w h e n the plants w e r e dependent on atmospheric nitro-gen, a deficiency of t h e m e t a l resulted i n s y m p t o m s of nitrogen de-ficiency. It is claimed t h a t copper affects nitrogen fixation b y depressing hemoglobin synthesis.
Lees (145) showed t h a t removal of the m e t a l b y sodium diethyl-dithiocarbamate inhibited microbial nitrification i n a soil percolated w i t h a m m o n i u m sulfate a n d this could be completely restored b y add-ing copper sulfate or m a n g a n e s e sulfate. T h i s effect is, therefore, non-specific. O n t h e basis of a thiourea inhibition of a m m o n i a oxidation b y Nitrosomonas, Lees (150) suggested a possible copper r e q u i r e m e n t for the reaction, b u t this is not unequivocal evidence. Lees a n d Meiklejohn (146) showed t h a t the addition of 14 or 28 copper to cultures of Nitrosomonas increased yield b y about 1 7 % . T h i s effect, however, is not v e r y significant.
Copper is required b y fungi for n o r m a l g r o w t h at about 0.01-0.1 p p m . H i g h e r concentrations a r e toxic although some fungi grow in saturated copper sulfate solution (58, 87, 2 7 8 ) . T h e u p t a k e of t h e m e t a l is somewhat greater at moderate p H t h a n at low values. I n Aspergillus niger, copper affects the pigmentation of t h e spores: at low levels t h e y a r e p a l e yellow a n d w i t h increasing a m o u n t s of t h e m i c r o n u t r i e n t t h e y appear b r o w n a n d eventually black. T h e effective r a n g e is 0.05-2 /*g copper p e r 50 m l basal culture solution (193, 2 0 2 ) . A similar effect on spore color b y copper is found i n Trichoderma viride ( 3 7 ) . A deficiency of copper in Aspergillus niger does n o t greatly depress t h e yield; in this respect it differs m a r k e d l y from a deficiency of either iron, zinc, or m o l y b d e n u m . Copper r e q u i r e m e n t has also been shown for derma-tophytic fungi, e.g., Trichophyton species (58, 87, 2 4 6 ) .
Algae so far examined r e q u i r e copper for growth, b u t t h e
require-3 . I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 4 1 5
m e n t is small a n d varies in a m o u n t in t h e different algal groups. I n some blue-green algae t h e concentration of copper w h i c h inhibited Phormidium tenue stimulated Spirulina species ( 2 3 0 ) . As i n bacteria, the limiting r a n g e between deficiency a n d toxicity for copper is v e r y n a r r o w . T h u s 1 0 ~7 M copper inhibited photosynthesis in Chlorella as did a series of copper inhibitors; yet at a lower concentration, copper is a n essential element for Chlorella ( 9 8 , 1 9 7 , 2 3 0 , 2 9 3 ) .
G . Z I N C
A deficiency of zinc in microorganisms results in a n upset metab-olism. T h u s in Neurospora crassa, alcohol dehydrogenase a n d t r y p t o p h a n synthetase a r e reduced b u t other e n z y m e s h a v e increased activity, e.g., diphosphopyridine nucleotidase ( D P N a s e ) ( 1 9 9 ) . Hexokinase is also reduced in felts deficient in zinc ( 1 7 8 ) , a n d this is i n accord w i t h the reported accumulation of inorganic phosphate in tomato leaves w h i c h suggested a possible role for t h e element i n t h e hexokinase e n z y m e ( 2 3 0 ). P y r u v i c carboxylase w a s limiting i n Rhizopus nigricans déficient i n zinc. Vallee h a s shown t h a t alcohol, lactic a n d glutamic
dehydrogenases contain zinc ( 3 0 1 ) .
V e r y little is k n o w n about t h e zinc r e q u i r e m e n t s of bacteria since there h a v e been few studies on t h e subject. T h e r e q u i r e m e n t is u s u a l l y quite low a n d can b e demonstrated only b y using rigorous purification methods.
R a u l i n showed t h a t zinc is required b y Aspergillus ( 2 4 2 ) , a n d this was confirmed b y B e r t r a n d a n d Javillier ( 2 9 ) . Steinberg ( 2 8 2 , 2 8 4 ) showed t h a t 0 . 1 8 m g zinc p e r liter w a s required for m a x i m u m growth, a n d Nicholas a n d Fielding ( 2 0 2 ) gave 0 . 2 m g zinc p e r liter as t h e optimal value. T h e addition of 1 Χ 1 0 ~3 μ% zinc to 1 liter of a purified culture solution increased the d r y weight twofold. I n yeast, zinc stim-ulates growth at concentrations from 0 . 2 to 2 0 0 p p m , a n d M c H a r g u e a n d Calfee ( 1 6 0 , 1 6 1 ) claim 1 0 p p m stimulated production of carbon dioxide. T e x e r a reported t h a t t h e zinc content of cultures determines the n a t u r e of antibiotics produced b y Fusarium hyperoxysporum ( 2 9 9 ) . I n t h e absence of t h e m e t a l t h e culture filtrates inhibited both grampositive a n d gramnegative bacteria, b u t w i t h zinc present only g r a m -positive ones w e r e inhibited.
T h e respiratory coefficient ( g r a m s C 02 evolved) : ( g r a m s d r y weight of felt) is u s u a l l y increased b y traces of zinc in species of Aspergillus, Pénicillium, Trichothecium, Rhizopus nigricans a n d in others. It is claimed t h a t zinc is required for a m o r e complete oxidation of carbohy-drates a n d t h a t w h e n t h e m e t a l is deficient ( 5 4 , 5 5 ) organic acids ac-cumulate. It is unlikely, however, t h a t this is a specific effect since
de-ficiencies of other trace metals h a v e similar effects. T h u s oxalic, glu-conic, citric, lactic, a n d fumaric acids accumulate in Aspergillus a n d Pénicillium species owing to a variety of causes other t h a n a zinc de-ficiency ( 5 8 ) . I n Ustilago sphaerogena, zinc is required for t h e syn-thesis of cytochromes ( 2 0 1 ) .
H . M A N G A N E S E
M a n g a n e s e can substitute for m a g n e s i u m in a n u m b e r of reactions in-volving adenosine triphosphate since it combines w i t h the pyrophosphate
2 01 1
1 —ι
1H H
M n+ +, ppm
FIG. 18. The effect of Ca*f and M g++ requirements of Lactobacillus plantarum (L.
arabinosus). Density of a suspension measured. Redrawn by permission of the Jour-nal of Bacteriology.
component of the nucleotide as does m a g n e s i u m . It can thus operate in the glycolysis of sugars a n d in t h e decarboxylation reactions in t h e citric acid cycle. H y d r o x y l a m i n e reductase requires m a n g a n e s e for its activity, a n d it is also essential for photosynthesis in algae (see Chapter 4 ) . T h e m a n g a n e s e r e q u i r e m e n t s for o p t i m u m growth in Lactobacillus plantarum, L. casei, a n d Streptococcus faecalis are 0.1, 0.03, a n d < 0 . 0 3 p p m , respectively ( 1 6 4 ) . I n the former the m a n g a n e s e r e q u i r e m e n t is greatly decreased in the presence of m a g -n e s i u m while calcium or stro-ntium h a v e a defi-nite b u t spari-ng effect as shown i n Fig. 18. T h e effect of m a n g a n e s e on acid production is shown in Fig. 19.
3 . I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 4 1 7
B e r t r a n d a n d Javillier, i n 1 9 1 1 , showed t h a t m a n g a n e s e is required for sporulation i n Aspergillus niger ( 2 9 ) , a n d since t h e n their w o r k has been a m p l y confirmed. T h e r e q u i r e m e n t i n fungi is between 0 . 0 0 5 a n d 0 . 0 1 p p m , a n d it is clear t h a t the n u t r i e n t is essential for all genera thus far studied ( 5 8 ) . A deficiency of t h e m e t a l can be demonstrated in Neurospora without removing it from the m e d i u m , b u t i n Aspergillus, since t h e r e q u i r e m e n t is less, m a n g a n e s e m u s t be removed from con-stituents of t h e m e d i a before it can be shown to be essential for growth ( 2 0 3 ) . A deficiency of t h e m e t a l reduced t h e yields b y o n l y one-half since t h e mycelia usually coalesce into r o u g h colonies in the absence
FIG. 19. The response of Lactobacillus plantarum ( L . arabinosus) to low ( A ) and high (B) concentrations of M n++ in manganese-deficient medium. From MacLeod and Snell, (162); reproduced by permission of the Journal of Biological Chemistry.
of m a n g a n e s e . I n yeast t h e n u t r i e n t concentrated in cells even from m e d i a containing v e r y m i n u t e a m o u n t s of it. About 1 0 p p m m a n g a n e s e stimulates growth i n yeast a n d toxic effects a r e noted only at concentra-tions as high as 5 0 0 p p m ( 5 8 ) .
Chlorella pyrenoidosa requires 1 0 ~7 M m a n g a n e s e for heterotrophic g r o w t h a n d 1 0 ~4 M for autotrophic growth. Autotrophic growth, Hill reaction, a n d photosynthesis responded equally w h e n increments of t h e m e t a l w e r e added to manganese-deficient cultures ( 1 3 0 , 1 3 1 ) . Similar results w e r e obtained w i t h Scenedesmus quadricauda, Nostoc muscorum, a n d Porphyridium cruentum. Chlorophyll content of 1-day-old auto-trophic cultures of Chlorella a n d Scenedesmus was not affected b y m a n g a n e s e deficiency, b u t after being illuminated for 3 days, deficient cultures of Chlorella h a d m u c h less chlorophyll t h a n similarly treated nondeficient cultures. Chlorella i n a m e d i u m containing m a n g a n e s e
just sufficient for m a x i m u m photosynthesis a n d Hill reaction h a d a m a n g a n e s e : chlorophyll m o l a r ratio of 1:600 ( 1 3 1 ) .
I. M O L Y B D E N U M
It is well established t h a t m o l y b d e n u m is required for n i t r a t e re-duction i n bacteria, fungi, a n d algae since it is a constituent of the assimilatory a n d dissimilatory n i t r a t e reductases ( 7 9 - 8 4 , 2 0 4 - 2 0 8 , 209, 2 1 1 , 2 1 2 ) . I n fact, w h e n e v e r n i t r a t e is utilized t h e r e is a n obligatory r e q u i r e m e n t for the m i c r o n u t r i e n t ( 8 2 ) . T h e trace m e t a l r e q u i r e m e n t although m u c h reduced w h e n a m m o n i a is t h e sole source of nitrogen is n o t completely eliminated i n Neurospora ( 2 0 4 ) , as it is i n t h e alga Scenedesmus obliquus ( 1 1 ) . I n Neurospora crassa a deficiency of m o l y b d e n u m resulted in decreased a m o u n t s of catalase a n d peroxidase.
T h e e n z y m e s w e r e restored to n o r m a l a m o u n t s b y r e t u r n i n g the element in vivo to t h e deficient felts. T h i s effect on the iron e n z y m e s is probably indirect, resulting from a decrease in t h e activity of molyb-d e n u m - molyb-d e p e n molyb-d e n t flavoproteins w h i c h promolyb-duce h y molyb-d r o g e n peroximolyb-de, the c o m m o n subtrate for catalase a n d peroxidase ( 2 1 0 ) .
Since t h e classic w o r k of Bortels ( 3 4 ) , it has been k n o w n t h a t m o l y b d e n u m is r e q u i r e d for nitrogen fixation in Azotobacter species a n d t h a t v a n a d i u m can partially replace it in this process. H o r n e r et al.
( 1 1 3 ) , J e n s e n ( 1 2 5 ) , Nicholas et al. ( 2 1 8 ) , Bové et al. ( 3 5 ) , all con-firmed t h a t m o l y b d e n u m is essential w h e n Azotobacter is utilizing atmospheric nitrogen although its partial replacement b y v a n a d i u m varies w i t h t h e species. T h e m o l y b d e n u m r e q u i r e m e n t for nitrogen fixation i n A. chroococcum is illustrated i n Fig. 20a, a n d its replacement to the extent of about 7% b y v a n a d i u m is illustrated in Fig. 20b. I n other strains of Azotobacter t h e sparing action of v a n a d i u m w a s n o t ob-served. T a k a h a s h i a n d N a s o n (294) a n d Keeler a n d V a r n e r (129) showed t h a t tungstate is a competitive inhibiter of m o l y b d a t e i n nitro-gen fixation, a n d t h e latter demonstrated t h e incorporation of tungstate into t h e same protein fractions as m o l y b d e n u m . Bershova showed t h a t active cells of Azotobacter absorb m o r e M o " from t h e m e d i u m t h a n do resting cells a n d t h a t some of the absorbed m o l y b d e n u m is secreted later into t h e m e d i u m , b u t this could result from autolysis ( 2 5 ) . A similar re-q u i r e m e n t for m o l y b d e n u m was found in Clostridium species (218) w h e n fixing nitrogen gas.
M o l y b d e n u m is also required for t h e fixation of nitrogen b y bacteria in t h e root nodules of legumes a n d nonlegumes, b u t its mode of action is not k n o w n (109, 3 2 3 ) . It is of interest t h a t m u c h m o r e of t h e micro-n u t r i e micro-n t is required for micro-nitrogemicro-n fixatiomicro-n t h a micro-n for micro-n i t r a t e reductiomicro-n.
Blue-green algae also r e q u i r e 0.2 m g m o l y b d e n u m per liter w h e n
3 . I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 4 1 9
ρ» ' ι » I I _ L _ I I I I I I I I I I L 0001 0 003 0005 001 1 0 20 3 0 40 5 0
pq. Mo/ml. culture mg. Mo /ml. culture
FIG. 20a. Effect of molybdenum content of culture medium on the growth of Azotobacter chroococcum 8003. Ordinate: % growth based on turbidity measure-ments; abscissa; micrograms or milligrams of molybdenum per milliliter of medium.
From Nicholas et al. (218).
FIG. 20b. Effect of sodium vanadate in replacing sodium molybdate for the growth of Azotobacter chroococcum 8003. From Nicholas et al. (218).
t h e y utilize either gaseous nitrogen or nitrate nitrogen: 0.2 m g m o l y b -d e n u m per liter (86, 197, 2 3 0 ) . I n Fig. 21 a r e shown the results of a n experiment w i t h Anabaena cylindrica in w h i c h the basal m e d i u m w a s freed from traces of m o l y b d e n u m b y coprecipitation with F e a n d 8-hy-droxyquinoline in the presence of acetic acid as described b y Nicholas a n d Fielding ( 2 0 2 ) . M o l y b d e n u m is required for t h e utilization of nitrogen gas or nitrate b y the alga, but not w h e n g r o w n on a m m o n i u m only. Both figures show t h a t the rate of assimilation a r e greater at
FIG. 21. The effect of molybdenum concentration on the growth of Anabaena cylindrica. a. With gaseous nitrogen, potassium nitrate or ammonium chloride as nitrogen source; cultures grown for 18 days (N2, N03~) or 14 days ( N H4+) . b. With gaseous nitrogen or potassium nitrate as nitrogen source in the presence of ethylene-diaminetetraacetic acid (0.05 gram per liter; cultures grown for 14 days). From
Fogg and Wolfe (86).
lower concentrations of m o l y b d e n u m w h e n t h e nitrogen source was n i t r a t e t h a n w h e n molecular nitrogen was utilized. It is the growth of the organism only w h i c h is reduced in t h e absence of t h e trace m e t a l ; t h e r a t e of n i t r a t e u p t a k e from t h e m e d i u m is unaffected b y molyb-d e n u m concentration. Similar results w e r e obtainemolyb-d w i t h t h e yeast Hansenula anomala ( 2 6 9 a ) . T h e addition of t h e element to
molyb-denum-deficient cultures resulted i m m e d i a t e l y i n nitrate reduction.
This suggests a role for m o l y b d e n u m in n i t r a t e reduction.
Root nodules of Leguminosae a n d nonlegumes contain several times the m o l y b d e n u m content of their roots. Nodule bacteria contains a particularly active nitrate reductase (52, 5 3 ) .
3. I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 421
T h e essentiality of t h e m i c r o n u t r i e n t for Aspergillus niger w a s established b y t h e careful work of Steinberg (282, 284) ) . T h e require-m e n t s are srequire-mall, as illustrated i n Fig. 1. T h e A. niger assay require-method detects as little as 1 X 10~4 μg m o l y b d e n u m a n d is m o r e sensitive t h a n the M o " radioassay method. All fungi a p p e a r to r e q u i r e the element w h e n t h e y are utilizing n i t r a t e ( 8 2 ) .
A report b y Z a v a r z i n t h a t m o l y b d e n u m is required for oxidation of nitrites in nitrifying bacteria has not been confirmed i n other laboratories ( 3 3 2 ) .
J . V A N A D I U M A N D G A L L I U M
V a n a d i u m can p a r t l y replace m o l y b d e n u m in nitrogen fixation in Azotobacter chroococcum as shown in Fig. 20b. T h i s is not a general p h e n o m e n o n in Azotobacter since in A. indicus a n d A. vinelandii, v a n a d i u m has no sparing action on m o l y b d e n u m . A r n o n a n d Wessel
(10) showed t h a t v a n a d i u m was a n essential m i c r o n u t r i e n t for growth of Scendesmus obliquus. T h e a m o u n t required, u p to 100 μg p e r liter, is m u c h higher t h a n its need for m o l y b d e n u m at 0.1 μg per liter. A deficiency of v a n a d i u m does not reduce t h e chlorophyll content as m a r k e d l y as does a shortage of m o l y b d e n u m . I n strong light, photosynthetic oxygen production in cells deficient i n v a n a d i u m was inhibited, b u t it could b e reactivated slowly b y r e t u r n i n g v a n a d i u m . A deficiency of v a n a d i u m appears to affect the dark reaction in photo-synthesis. According to B e r t r a n d t h e element is widely distributed in microorganisms, especially those in m a r i n e habitats ( 2 8 ) . Bertrand claims t h a t v a n a d i u m stimulated growth in Aspergillus niger, b u t the effect is small a n d needs confirmation ( 2 7 ) .
Steinberg suggested t h a t gallium was required for o p t i m u m growth b y Aspergillus niger at 0.01 μg per liter, a n d n o n e of 27 other elements tested replaced it ( 2 8 1 ) . H e w a s u n a b l e to substantiate this finding be-cause his supply of purified sugar was used u p . T h i s w o r k has not t h u s far been confirmed in other laboratories. Steinberg also claimed t h a t s c a n d i u m w a s beneficial w h e n glycerol w a s t h e carbon source, b u t again n o further reports are available to substantiate this observa-tion ( 2 8 2 ) .
A t this stage it should be pointed out t h a t although trace metals m a y not b e essential for n o r m a l growth processes t h e y m a y exert a n in-fluence on metabolic processes b y stimulating or inhibiting them. T h u s in Pénicillium japonicum, colombium, m o l y b d e n u m , tungsten, a n d c h r o m i u m increase fat production. T h i s m a y be due to a nonspecific activation of enzymes b y a variety of metals of equivalent valency, or
t h e y m a y block certain reactions resulting in a " s h u n t " metabolism to fat synthesis ( 5 8 ) .
K . C O B A L T
V i t a m i n Bi2 (cyanocobalamin) a n d hydroxocobalamin (vitamins B i2 a
a n d Bi2b) a r e present in a n u m b e r of microorganisms including bacteria a n d algae. V i t a m i n B1 2 is believed to b e r e q u i r e d i n m e t h y l a t i o n processes a n d for synthesis of nucleic acids.
Strains of lactic acid bacteria a r e k n o w n to r e q u i r e v i t a m i n Bi 2, as do algal flagellates, a n d these h a v e been used to bioassay the v i t a m i n a n d its analogs. It w a s estimated t h a t 0.01 mμg Bi 2 is required to form 880,-000 Euglena cells or 1.13 X 10~17 g m Bi 2 p e r cell, w h i c h is equivalent to approximately 5000 molecules of v i t a m i n B12 per Euglena cell. F r o m this H u t n e r suggested a r e q u i r e m e n t of 6 X 10"1 3 g m cobalt p e r milli-liter of culture m e d i u m (117, 118, 2 3 5 , 2 3 6 ) . T h e task of removing t h e trace m e t a l to these low a m o u n t s is beyond t h e capacity of puri-fication techniques i n c u r r e n t use. T h e element h a s been shown to be required for t h e growth of n u m e r o u s blue-green algae ( 9 3 ) .
Recently a cobalt r e q u i r e m e n t h a s been found for Bacillus circulons w h e n g r o w n in n i t r a t e m e d i u m ( 2 2 0 a ) .
T h e synthesis of cobalamins occurs i n certain bacteria a n d especially i n species of Streptomyces a n d Nocardia of t h e Actinomycetales. T h e addition of cobalt to t h e culture m e d i u m increases t h e yield of c y a n o b a l a m i n from Streptomyces griseus. I n addition to cyanoco-b a l a m i n , species of Streptomyces form hydroxococyanoco-balamin ( v i t a m i n B i2 a, B1 2 b) i n w h i c h t h e cyanide of cyanocobalamin is replaced b y h y d r o x y l a n d v i t a m i n B i2 c containing a nitrite radical in place of c y a n i d e ; all t h r e e forms h a v e been isolated from Streptomyces griseus ( 5 8 ) .
Synthesis of v i t a m i n Bi 2 in filamentous fungi has n o t been shown conclusively, b u t substances active for Lactobacillus leichmannii a r e formed b y Ashbya gossypii (58) a n d Aspergillus niger ( 2 0 3 ) . I t ap-pears t h a t l,2-dimethyl-4,5-diaminobenzene is a precursor of cobalamins as well as flavins ( 5 8 ) . T h u s Streptomyces olivaceus incorporates the labeled 5,6-dimethylbenzimidazole into t h e cobalamin molecule.
Cobalt accumulated in Neurospora crassa; the degree of concentration was 2 3 times t h a t i n t h e external m e d i u m . M o r e t h a n 4 0 % of t h e cobalt w a s b o u n d to cell protein. T h e iron content of t h e m e d i u m in-fluenced the u p t a k e of cobalt b y the fungus ( 1 6 ) .
Nicholas (203) showed t h a t v i t a m i n B1 2 i n Aspergillus niger was reduced b y a deficiency of cobalt although t h e d r y w e i g h t yield of t h e felts w a s not depressed as shown i n T a b l e X I I . T h i s result suggests either t h a t v i t a m i n B1 2 is not required b y t h e fungus for its metabolism
3 . I N O R G A N I C N U T R I E N T N U T R I T I O N O F M I C R O O R G A N I S M S 4 2 3
Cobalt is required for t h e growth of Rhizobium japonicum outside t h e host plant. I n this respect it resembles Bacillus circulons, w h i c h also requires t h e m i c r o n u t r i e n t w h e n g r o w n o n nitrate. Nicholas (220a) has shown recently t h a t w h e n Azotobacter is fixing atmospheric nitro-gen or utilizing n i t r a t e its v i t a m i n B1 2 content is m o r e t h a n w h e n it is -duce cobalt to a deficiency level. T h e latter interpretation is m o r e likely to b e correct.
A h m e d a n d E v a n s ( 1 , 2 ) showed t h a t t h e addition of cobalt to cultures of soybean plants g r o w n u n d e r symbiotic conditions resulted in m a r k e d increase i n t h e d r y weight of shoots a n d prevented t h e de-velopment of nitrogen deficiency symptoms t h a t w e r e present i n plants not given t h e micronutrient. T h e experiments of Reisenauer ( 2 4 4 ) showed t h a t g r o w t h of alfalfa w i t h o u t combined nitrogen w a s stimu-lated b y cobalt. H a l l s w o r t h et al. ( 1 0 3 ) showed a r e q u i r e m e n t for both
Nicholas, et al. (220b) found t h a t t h e n i t r a t e reductase e n z y m e w a s m a r k e d l y reduced i n cells of Rhizobium japonicum deficient i n cobalt.
T h e r e q u i r e m e n t for growth on n i t r a t e is less t h a n 2 /xg/liter. Since the m i c r o n u t r i e n t (labeled w i t h C o5 8) did n o t concentrate i n purified
T A B L E X I I I
EFFECTS OF COBALT ON THE GROWTH OF Rhizobium japonicum*
ON NITRATE NITROGEN Turbidity
Nitrogen 5 Days 7 Days content6
Treatment (O.D.) (O.D.) (mg/flask) Experiment I
0 cobalt6 0 .02 0 .35 0.53 0.5 ppb cobalt 0. 19 1. .27 4.75 5.0 ppb cobalt 0. .21 1. .40 5.00 Experiment I I
0 cobalt0 0 .39 0.62
0 . 5 ppb cobalt 1 .33 5.50
0 . 5 ppb cobalt 1 .33 5.50