A. P H O S P H O R U S
Phosphorus is indispensable for all microorganisms since it is r e -quired for t h e utilization of glucose (see C h a p t e r 3, V o l u m e I A, b y Goddard a n d B o n n e r ) a n d is a n i m p o r t a n t constituent of essential cell metabolites including nucleotides of a d e n i n e , p y r i d i n e , flavin, a n d u r i d i n e , a n d other coenzymes, including pyridoxal phosphate a n d t h i a m i n e pyrophosphate. I t is a basic constituent of all nucleic acids.
I n culture solutions monobasic or dibasic phosphates a r e usually supplied a n d t h e o p t i m u m concentrations v a r y w i d e l y depending on t h e composition of t h e m e d i u m . T h u s w h e n o n l y a m m o n i u m salts a r e used t h e n t h e dibasic phosphate is preferred to offset t h e drop i n p H associated w i t h t h e u p t a k e of a m m o n i u m ions. T h e m o l a r i t y of culture solutions varies between 0.001 a n d 0.005 M phosphate.
Orthophosphate can be replaced for some fungi b y other forms of inorganic phosphate (70) a n d either p h y t i c acid ( 2 6 6 ) , adenosine phosphates ( 2 7 2 ) , or casein (30) can also be used. Phosphites a n d h y p o -phosphites a r e not readily metabolized b y t h e microorganisms.
P h o s p h a t e is absorbed r e a d i l y b y fungi d u r i n g t h e e a r l y stages of growth since it is required for glycolysis a n d e n e r g y systems of t h e cell. Its absorption in aerobes is dependent on oxygen, a n d inhibitors
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 399
of respiration also depress phosphate intake. T h e phosphate uptake in relation to growth in Aspergillus niger is illustrated in Fig. 8. Combined forms of phosphate are usually hydrolyzed by phosphatases in the fungal mats. Both alkaline and acid phosphatases have been reported in fungi, but their specificity is in doubt ( 1 6 9 ) .
Bacteria and fungi, often associated with the rhizosphere (cf. Chapter 6) of plants, are able to dissolve calcium hydrogen phosphate. Phos-phorus was more readily released from tricalcium phosphate and pyro-phosphate by bacteria than b y fungi ( 1 7 0 ) . It has been suggested that
.1
OL^ 1 1 1 l . i ι
\Dry weight _ 7minute Ρ \
-\
/ in mycelium \/
' \ P in medium / ^/ 1 1 1 X / l 1 12
FIG. 8. Growth and phosphorus changes in Aspergillus niger. Seven-minute phosphorus is organic phosphorus mineralized by acid hydrolysis of 7 minutes' duration. From data of Mann (169).
the liberation of phosphorus from organic compounds is due to the action of enzymes of the esterase type and that liberation from insoluble mineral phosphates is due to effect of carbon dioxide or organic acids originating in microbial metabolism ( 1 9 6 ) . Streptomyces species, Asper-gillus, and Pénicillium have all been shown to solubilize soil phosphates
( 2 9 2 ) . Harley ( 1 0 3 a ) , using labeled phosphate, has studied the uptake of phosphate b y excised mycorhizal roots of beech, and Swaby and Sherber (292) showed that species of the genera Arthrobacter, Pseudo-monas, XanthoPseudo-monas, Achromobacter, Flavobacterium, Streptomyces, Aspergillus, and Pénicillium released phosphate from hydroxyapatite by producing organic acids. Numerous bacterial types including Bacil-lus subtilis, B. cereus var. mycoides (B. mycoides), B. megaterium, and
"B. mesentericus" release soluble phosphorus from calcium and iron
phosphates, calcium glycerophosphate, a n d lecithin ( 1 0 3 a ) . I n Pinus radiata, phosphorus u p t a k e from plants inoculated w i t h m y c o r h i z a w a s greater t h a n in those not containing the microorganisms ( 1 0 3 a ) .
B . P O T A S S I U M A N D S O D I U M
Potassium is universally required b y microorganisms, b u t t h e r e is evidence t h a t it can be replaced b y r u b i d i u m in Streptococcus faecalis
(Fig. 9 ) . I n other bacteria potassium is only partially replaced b y
12 14 4 6 8 10
Micromoles Κ + or R b+p e r 10 ml
FIG. 9. Comparative effects of K+ and Rb+ on growth of Streptococcus faecalis as measured by density of a suspension. Redrawn by permission of the Journal of Biological Chemistry (162).
r u b i d i u m , a n d this is often referred to as t h e sparing effect of one ion for another. A n example of this is in Leuconostoc mesenteroides; as shown in Fig. 10, p r e s u m a b l y r u b i d i u m substitutes for potassium in some b u t not in all its metabolic functions.
T h e counteraction of t h e stimulatory or inhibitory effect of one ion b y another is usually k n o w n as ion interaction. T h i s p h e n o m e n o n has been widely studied since antagonisms v a r y greatly between organisms ( 1 9 5 ) . M a c L e o d a n d Snell (163) showed t h a t m a n y instances of ion interaction could be explained b y assuming t h a t ions w h i c h suppress growth do so because t h e y interfere w i t h other ions involved in m e t a b -olism. I n Fig. 11 is shown t h e effect of sodium on the potassium require-m e n t of Lactobacillus casei ( 1 6 2 ) . H e r e sodiurequire-m is antagonistic since it
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 0 1
FIG. 11. The effect of N a+ on the K+ requirement of Lactobacillus casei as measured by density of a suspension. Redrawn by permission of the Journal of Biological Chemistry (162).
increased the requirement for the essential ion potassium. These effects are frequently complex since a single ion m a y replace another for some function but antagonize it in others and the two effects m a y or m a y not be observed independently at different concentrations. Thus at low concentrations rubidium decreased the requirement of Leuconostoc mesenteroides for potassium, but at higher concentrations it inhibited growth by competing with potassium for sites that specifically require the latter ion ( 1 6 4 ) . Similar phenomena also occur between essential cations and related nonessential ions. A rubidium-potassium effect has also been observed b y Lavollay in Aspergillus niger ( 1 4 2 ) . Barton-Wright (18) showed that a regular dose-response curve to potassium could be obtained with Streptococcus faecalis, and he used this organism for determining potassium. In this bacterium rubidium also has a spar-ing action on potassium. Leuconostoc mesenteroides P-60 has also been used to assay potassium, but sufficient rubidium is put into the medium to eliminate the sparing effect of this ion on the potassium requirement.
Levels of potassium used for fungal cultures vary between 0.001 and 0.004 M, which is regarded as adequate. A shortage of the element in some fungi results in oxalic acid formation (189) and also poor utiliza-tion of the carbon source, especially carbohydrates (58, 2 4 5 ) . Sodium only partially replaces potassium in Aspergillus niger, and under certain conditions rubidium and cesium and other alkali metals have slight ef-fects only ( 2 8 8 ) . Burris and Harris (39) showed that Azotobacter chro-ococcum was able to grow in media deficient in potassium and sodium.
Allen and Arnon (8) have shown that sodium is essential for the blue-green alga Anabaena cylindrica and that 5 ppm was sufficient for optimum growth. Neither potassium, lithium, rubidium, nor cesium replaced sodium for growth, and there is no evidence that larger amounts are harmful. It is likely that sodium is generally required b y blue-green algae, and Allen (6) has found that 25 cultures of various Cyanophyceae grow well in a sodium-containing medium in the absence of added potassium, but no attempts were made to remove potassium from the constituents of the culture solutions. Gerloff, et al. (93) report a beneficial effect of sodium on Microcystis aeruginosa, and Kratz and Myers (139) found that logarithmic growth of Anabaena variabilis, Anacystis mdulans, and Nostoc muscorum cannot be maintained with-out sodium.
C . M A G N E S I U M A N D C A L C I U M
Although calcium is required in small amounts by microorganisms, it will be considered here together with magnesium since in the literature interactions between the two nutrients are often reported.
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M a g n e s i u m is u s u a l l y required b y microorganisms in larger a m o u n t s t h a n calcium. A calcium r e q u i r e m e n t is difficult to demonstrate in some bacteria a n d fungi since it a m o u n t s to o n l y a few parts p e r million.
T h u s , calcium is a m i c r o n u t r i e n t for microorganisms. T h i s is u n d e r standable w h e n one considers t h e function of t h e two elements. M a g -n e s i u m is r e q u i r e d i -n phosphorylatio-n systems, especially i -n t h e gly-colytic p a t h w a y s , often acting as a link between substrate, e n z y m e , a n d coenzyme, e.g., adenosine a n d u r i d i n e triphosphates. N o precise func-tion however h a s been assigned to calcium in microorganisms.
0,1 0.2 0.3 0.4 0 . 54 1 10 Mg + +, ppm
FIG. 12. The comparative response of Streptococcus faecalis to M g+ + in the presence and absence of citrate (20 mg citrate ion per milliliter) as measured by density of suspension. Redrawn by permission of the author and Journal of Bac-teriology (164).
W e b b demonstrated t h a t for m a n y bacteria m a g n e s i u m is needed for cell division (320, 3 2 1 ) . H e n r y a n d Stacey (107) h a v e shown it to be required for t h e formation of t h e gram-staining complex. T h e com-parative response of Streptococcus faecalis to m a g n e s i u m i n t h e pres-ence a n d abspres-ence of citrate, shown in Fig. 12, demonstrates t h a t a chelating agent reduces t h e availability of t h e m e t a l to the fungus.
Shooter a n d W y a t t (267) showed t h a t Staphylococcus pyogenes ( = S.
aureus) r e q u i r e d m a g n e s i u m a n d calcium after t h e m e d i u m h a d been passed t h r o u g h a n ion exchange resin. T h e y suggest t h a t calcium is r e q u i r e d for t h e formation of proteases. T h e effect of m a g n e s i u m only a n d t h e interaction between m a g n e s i u m a n d calcium on growth a r e shown i n Figs. 13 a n d 14.
M a x i m a l yield was obtained after 18 h o u r s ' growth w i t h 0.2 m g ions of m a g n e s i u m per liter. W h e n calcium o n l y was added growth was less t h a n t h a t obtained with m a g n e s i u m only. T h i s effect w a s noted w h e n 0.005 m g ions of calcium per liter was added a n d increased slowly u p to 2.5 m g ions calcium per liter. W h i l e both m a g n e s i u m a n d calcium stimulated growth singly, t h e two elements together w e r e m o r e effective a n d optimal growth was obtained w i t h 0.02 m g per liter of m a g n e s i u m a n d 0.05 m g ions per liter of calcium.
60r-0008 002 004 008 0-2 0-4 08 mg. ions/1. magnesium added
FIG. 13. Effect of the addition of M g++ on the growth of Staphylococcus pyogenes between 6 and 24 hours. From Shooter and Wyatt (267).
Norris a n d Jensen (223) showed t h a t calcium w a s essential for nitro-gen fixation in Azotobacter vinelandii, A. chroococcum, A. beijerinckii, a n d A. insignis in media w i t h or w i t h o u t combined nitrogen in t h e form of a m m o n i u m acetate. T h e y showed t h a t strontium could replace cal-cium. A. agilis did not appear to r e q u i r e calcium, b u t this m a y be re-quired at a lower level.
Strains of Rhizobium r e q u i r e trace a m o u n t s of calcium only a n d larger a m o u n t s of m a g n e s i u m for growth (224, 2 2 5 ) . Bush a n d Wilson
(42) showed t h a t Azotobacter vinelandii, g r o w n in a nitrogen-free
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m e d i u m , required calcium for nitrogen fixation. Azotobacter agilis grew well i n a calcium-deficient m e d i u m a n d its growth w a s only slightly stimulated b y adding t h e m i n e r a l n u t r i e n t . Several strains in-cluding A. chroococcum, A. beijerinckii, a n d A. macrocytogenes r e q u i r e calcium for nitrogen fixation b u t not for t h e assimilation of a m m o n i a . Azotobacter indicus grew better in t h e absence of calcium.
Δ Λ Δ Δ.
mg ions/1 calcium added
FIG. 14. Effect at 18 hours on the growth of Staphylococcus pyogenes of the ad-dition of Ca+ +, Ca++ and arginine, and Ca++ and M g++ (broth batch L . 2 ) . O, Ca+ +;
· , Ca++ and 0.0005 M arginine; + , Ca++ and 0.004 mg ions per liter of M g+ +; X, Ca++ and 0.008 mg ions per liter M g+ +; Δ , Ca++ and 0.08 mg ions per liter Mg+ +. From Shooter and Wyatt ( 2 6 7 ) .
T h e first report on the calcium r e q u i r e m e n t s of fungi is b y Young a n d Bennett in 1 9 2 2 ( 3 3 0 ) . Since t h a t t i m e t h e r e has been m u c h con-fusion in regard to claims for essentiality of this n u t r i e n t since t h e
effects of adding calcium h a v e not been disentangled from those of p H . I t is clear, however, t h a t about 1 9 genera of fungi respond to additions of calcium, b u t often at v e r y low levels between 0 . 2 a n d 2 0 p p m . Some fungi, e.g., Aspergillus species, Neurospora species, do not a p p e a r to h a v e a calcium r e q u i r e m e n t ( 2 8 2 ) . T h e g r o w t h response of Coprinus ephemerus to calcium is shown in Fig. 1 5 .
I n several fungi s t r o n t i u m replaces t h e calcium r e q u i r e m e n t , e.g., Allomyces arbuscula ( 1 2 0 ) . Calcium is k n o w n to protect m a n y or-ganisms from t h e injurious effects of h y d r o g e n ion or potassium, a n d t h e r e is some evidence t h a t in bacteria it offsets zinc toxicity ( 5 8 ) . T h u s caution is r e q u i r e d in i n t e r p r e t i n g data indicating a n absolute a n d specific r e q u i r e m e n t for t h e n u t r i e n t . A calcium r e q u i r e m e n t should be evident, irrespective of p H or of constitution of media, a n d this can be achieved i n most cases o n l y b y prior r e m o v a l of calcium from t h e basal media. T h i s has only been done in v e r y few instances. Calcium,
FIG. 15. The growth of Coprinus ephemerus at different levels of calcium (milli-grams per liter). Redrawn from Fries (91) by permission of the Svensk Botanisk Tidskrift.
like m a g n e s i u m , m a y be m a d e less available to t h e organism b y chelating agents in t h e m e d i u m , e.g., citrate or t a r t r a t e .
T h e m a g n e s i u m content of t h e media for fungi is u s u a l l y about 0.001 M , b u t this varies w i t h t h e t y p e of carbon source a n d t h e r a t e at w h i c h it is utilized (282, 2 8 3 ) . Aspergillus niger requires about 10 m g m a g n e s i u m p e r liter of culture solution for o p t i m u m g r o w t h (202, 2 0 3 , 2 8 2 ) . Aspergillus terreus seems to r e q u i r e m o r e m a g n e s i u m w h e n g r o w n i n still c u l t u r e t h a n it does i n shake culture. E n z y m e s activated b y m a g n e s i u m in vitro are often stimulated to a lesser extent b y m a n -ganese, b u t u n d e r u s u a l growth conditions m a g n e s i u m is t h e physiologi-cally active element. T h e absorption of m a g n e s i u m is u s u a l l y slower in n e u t r a l t h a n i n acid solution since it is r e a d i l y precipitated u n d e r
150
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