Utilization of calcium phosphates for microbial growth at alkaline pH

Soil Bid. Eiochem. Vol. 22. No. 7. pp. 887-890. All rights reserved

1990

R-tntcdm Great Bntain.

Copyright

0038-0717,90 $3.00 + 0.00 C 1990 Pergamon Press plc

UTILIZATION OF CALCIUM PHOSPHATES FOR MICROBIAL GROWTH AT ALKALINE pH H. 0. HALVORSON, A. KEYSAN* and H. L. KoRxwRGt Marine Biological Laboratory. Woods Hole, MA 02543, U.S.A. (Accepred 5 June 1990) Summary-A strain of aerobic yeast isolated from marine environment, and a number of spore-forming bacteria isolated from soil and from sewage, were grown on defined media at pH 8 containing various calcium phosphates as sole sources of phosphorus. Direct measurements confirm that, at this pH, the concentrations of inorganic phosphate in solution. released through the dissociation of the calcium phosphates. are more than adequate for the microbial phosphate uptake systems to function efficiently. This conclusion is supported by the observation that growth is even more rapid when the calcium phosphates were enclosed in dialysis bags and thus not in direct contact with the micro-organisms.

solubilization of calcium phosphate would occur even in the absence of micro-organisms (Brown, 1973). It is the main purpose of this paper to show that a variety of micro-organisms, including a yeast isolated from a marine environment and some bacteria isolated from soil and from sewage, grow readily at alkaline pH and can derive their phosphorus needs from calcium phosphate trihydrate or from various natural apatitcs. No contact between the organisms and the IMP is required: growth was even better if the IMP was enclosed in dialysis tubing. These results suggest that, provided a utilizable carbon source is available, many microorganisms could solubilize rock phosphates if they were also capable of taking up inorganic phosphate (P,) in the low concentrations that would be in physico-chemical equilibrium with IMP at alkaline pH values.

INTRODCCTION

Since the early work of Gerretsen (1948). Sperber (1957), Katzenelson and Bose (1959) and Duff ef al. (1967). it has been assumed that inorganic insoluble mineral phosphate (IMP) can be solubilizcd by micro-organisms mainly through the production of organic acids and of chelating keto acids from carbohydrates. The evidence for this assumption is based on findings that samples of soil. and of fresh or sea water. when incubated in media also containing sucrose or glucose, yield many micro-organisms capable of using various types of IMP (calcium phosphate trihydrate or a variety of apatitcs) as sole sources of phosphorus. It has further been postulated that bacterial utilization of rock phosphates from soils c;Ln be of agricultural importance (Alexander, 1967; Ayyakkannu and Chandramohan, 1971; Cosgrave, 1977; Stevenson, 1986); the importance of this process in the rhizosphcre has, however, been questioned (Tinker and Sanders, 1975). Moreover, it must be borne in mind that cu one-third of agricultural soils are alkaline (Chrn and Barak, 1982) and this includes most soils in arid and semi-arid zones. That micro-organisms might also be able to utilize IMP as a phosphorus source at alkaline pH was first indicated by the work of Azcon er al. (1976). who showed that lavender plants could take up phosphorus from IMP if placed in sterilized soil supplemented with mycorrhiza and bacteria but not if the micro-organisms were omitted. Naik ef al. (1982a, b) further showed that various types of yeast. isolated from estuarine environments, were also adept at deriving their phosphorus from IMP; however, the optimum pH for this process was between 6 and 6.5, when considerable *Present address: Department of Biological Chemistry. Institute of Life Sciences. Hebrew University of Jerusalem, Israel. tTo whom all correspondence should be addressed at: Department of Biochemistry. University of Cambridge, Tennis Court Road. Cambridge CBZ IQW, England. saa2X7-A

MATERIAIS

Micro -organisms

AND

METHODS

used

Yeast “D” was isolated from the marine environment as a colony able to produce a clearing in a layer of calcium phosphate trihydrate embedded in 1.5% (w/v) agar supplemented with glucose and ammonium chloride. It grew only under aerobic conditions, produced an orange red pigment on all media, and (on rich medium) grew well at pH values ranging from 2 to 8. On defined media, the yeast grew with acetate, citrate or glucose as carbon source, and ammonium ions as nitrogen source. A variety of aerobic spore-forming bacteria were isolated, from soil and from sewage, as colonies able to solubilize calcium phosphate from agar plates, containing this IMP as sole phosphorus source and supplemented with glucose and ammonium chloride. Those isolated from soil were thick rods, morphologically similar to B. cereus; those from sewage were thinner rods. No attempts were made to classify these organisms.

887

888

H.O. HALVORBN

ef al.

Media

All liquid media for growth of micro-organisms contained 12.5 mM ammonium chloride. 0.5-I rn.w sodium glutamate, “essential salts” (Ashworth and Komberg, 1966). washed calcium phosphate or rock phosphate, in 250 mM Tris. pH 8.1 (except when specified otherwise) at 0.3-1.2 mg dry mass ml-‘, and appropriate carbon sources, each at l&-Z5 mM. Growth of micro-organisms

0.1

Defined media (IO-20 ml), contained in sterilized Erlenmeyer flasks (250 ml). were inoculated with 50-100~1 of suspensions of micro-organisms that had been scraped off the surface of agar plates containing a defined medium with acetate as carbon source, and solidified with 1.5% (w/v) agar. The flasks were shaken at 140 rev min-’ in a waterbath at 30°C. At specified times, samples (0.2-0.5 ml) were taken aseptically and growth was measured, as turbidity at 660 nm. with a Shimadzu u.v.-visible spectrophotometer. When such samples also contained calcium phosphates. 0.2 ml of I N HCI was added to the cuvette contents which were then made up to I ml final vol with water. It was established that this procedure reducer&completely the extinction due to calcium phosphate, and virtually completely that due to the various rock phosphates used, without damaging Yeast “D” cells; however. this procedure could not be used with the bacteria tested, since they were lysed by the acid treatment. When the calcium phosphates were enclosed in dialysis tubing (which was then sterilized by dipping into 95% ethanol and air-drying), it was not necessary to add acid prior to the measurement of extinction at 660 nm; and it was thus possible directly to use such measurements for the determination of bacterial growth. Determination I$ phosphate

Samples (I ml) of various media were centrifuged for 3 min in a Micro-Centrifuge (Allied Fisher Scientific Co., Model 235 C) which was found to be sufficient to sediment insoluble calcium phosphates. The supernatant solutions were removed with a Pasteur pipette and their phosphate content was measured by the procedure of Chen et al. (1956). RESULTS

AND

s 8

DISCUSSION

Yeast “D” grew readily in an unbuffered medium containing glucose as carbon source, and calcium phosphate as source of phosphorus. There was, however, a long lag before growth began. This lag was largely overcome by the addition to the medium of small amounts (0.02-0.05%) of yeast extract, or of amino acids directly derived from the tricarboxylic or glutamate acid cycle, such as aspartate (0. l-l .Omi*r), which suggested that these materials played an anaplerotic role (Kornberg, 1966). Since the yeast extract used was found to contain over 6 mg of P, g-‘. all growth media used in this work were supplemented with I mM glutamate. It is evident from Fig. 1 that, in medium relatively weakly buffered at pH 8 by inclusion of 5 m.w Tris, the rate of growth of yeast “D” on IO ItIM glucose

0

5

1s

10 Time

20

25

30

(hl

Fig. I. Effect of pH on the rate of growth of Yeast “D”. on glucose, with calcium phosphate as the source of phosphorus. The yeast suspensions were shaken at 3OC in a growth medium containing essential salts, 12.5 mht ammonium chloride, IO mM glucose. I mM glutamate. washed calcium phosphate (0.6 mg ml-‘) and Tris buffer, pH 8.0. either at a final concentration of 5 mM (0. A) or 500 mM (0.0). Growth (0, 0) was measured as OD,.,. as described in the Materials and Methods section; pH (A. A) was measured directly with a glass electrode.

accelerated as the pH fell. This acceleration of growth was accompanied by dissolution of the calcium phosphate: all the solid material, added initially in suspension. was dissolved at pH < 4.5. This result accords with the findings of other workers, who correlated the utilization of IMP with the metabolic production and excretion of organic acids derived from the catabolism of various sugars (see Introduction). On the other hand, it is also apparent from Fig. I, that yeast “D” grows, albeit less rapidly (doubling time: IO-16 hr). on IO mM glucose and calcium phosphate when the pH is maintained at pH 8 by inclusion of Tris buffer in adequate concentrations (500mM); under these conditions, solubilization of the solid mineral did not occur to any quantitatively significant extent. This indicates that, even at pH 8, the supposedly insoluble calcium phosphate dissociates sufficiently to provide P, in concentrations sufficient for the phosphate uptake systems of the yeast to effect its entry into the cell. In order to test this possibility, but to obviate the need for buffers to neutralize acids produced, sodium acetate was used instead of glucose in the growth medium. Again the rapid onset of growth of yeast “D” on this carbon source required supplementation with yeast extract or with Cd- or C,-dicarboxylic acids; again, I mM glutamate was routinely added to supply the anaplerotic function. The initial pH was adjusted to 8.1; in unbuffered medium, this rose as growth proceeded to over 9, which then tended to slow further growth of the yeast. Consequently, Tris buffer at pH 8.1, to various final concentrations, was also included in the acetate medium. As shown in Fig. 2. the growth of the yeast, with calcium phosphate as source of phosphorus, was logarithmic over at least 3 doublings (mean doubling time co 15 h); the pH at beginning and end was 8.05 and microscopic examination confirmed that the culture contained only yeast cells. A strikingly similar curve was obtained when well-washed calcium phosphate trihydrate was replaced by powdered “rock

Calcium

phosphate

Time(h)

Fig. 2. Growth of Yeast “D” at pH &I on acetate, with different forms of IMP as phosphorus source. The media for growth were as described in the legend to Fig. 1 except that 25 mst sodium acetate was used in the place of glucose; the Tris buffer pH 8.1 was at 100 IIIM. Calcium phosphate trihydrate (0) and finely-ground “rock phosphate” (0). as sole sources of phosphorus, were at 0.6 mg ml-‘.

phosphate”, which had also been washed well in 0.5 mM Tris pH 8 before use. Again, growth was logarithmic over at least 3 doublings, and the mean doubling time was only slightly longer (ca 18 h). Similar results wcrc obtained when other forms of apatitc wcrc used. For example, one type of apatitc, (Sydcnham, Ont.; 2677 from the Pennsylvania State Collection) when coarsely powdered. supported the growth of Yeast “D” on acetate at pH 8 to over 3 doublings in 42 h. No growth was obscrvcd when the mineral phosphates wcrc omitted. The findings that supposedly “insoluble” mineral phosphates could supply the phosphorus requirement of growing yeast at pH 8 implies that these types of calcium phosphates dissociate to yield significant concentrations of P, in media at this pH. In order to invcstigatc this point, suspensions of calcium phosphated trihydratc and “rock phosphate”, which had been washed three times with 0.5 M Tris buffer, pH 8, and had been suspended in this bulfcr at I2 mg dry mass ml-‘, were shaken at 30°C for I8 h. The P, content of supcrnatant solutions, obtained after centrifuging samples (I ml) for 2, 3 and 5 min in a Micro-Centrifuge were found to be II6 &-I nmol ml-i from calcium phosphate, and phosphate”. 27.4 * 0.4 nmol ml-’ from “rock According to the solubility isotherms at 25’C of calcium phosphates at various pH values (Brown, 1973). it would be expected that calcium phosphate trihydrate would be in equilibrium with P, at ca 130 nmol ml-’ and “rock phosphate” [given as a hydroxyapatite Ca,(PO,),OH] with P, at cu 30 nmol ml-‘. Our findings are in good agreement with these expectations. Moreover, it is well established that the K, for the uptake of P, by yeasts and by many bacteria is below 5pM (Borst-Pauwels and Peters. 1987). These findings therefore also suggest that IMP, if in a physical state to permit adequate mixing with an aqueous environment, can through normal physio-chemical processes and quite independently of any biological agent release P, at concentrations more than sufficient to permit microbial or

and microbial

a89

growth

plant uptake systems to take up this P,. This conclusion is further supported by the observation that samples of calcium phosphate, or various types of apatite, suport the growth of yeast “D” on acetate at pH 8 even better if the minerals are enclosed in sealed bags of dialysis tubing. The P, that is released diffuses into the medium and, as the yeast escapes adsorption on the solid mineral granules, growth is uniform and rapid. Under these conditions, the doubling time on 25 mM acetate at pH 8.1 was reduced to 9 h and that on 25 ITIM glucose was also 9 h; microscopic examination confirmed that the yeast cells did not exhibit the “clumping” (i.e. adsorption onto calcium phosphate granules) seen during growth on powdered mineral phosphates. It should be stressed that the ability to utilize calcium phosphates as phosphorus source under alkaline conditions is not peculiar to yeast “D”: a variety of bacteria isolated from sewage and from soil also readily grew on acetate and calcium phosphate at pH 8. Many of these organisms grew only slowly on glucose at pH 8. although their growth was rapid at lower pH values. This suggests that the gene cloned by Goldstein and Liu (1987). that permits enteric bacteria to utilize IMP efficiently, may specify an enzyme primarily conccrncd with carbohydrate catabolism (and, possibly, acid production from that process), rather than with the solubilization per sc of IMP. Although the conditions in the soil arc much mom complex than those in the laboratory. the results reported in this paper suggest that. under the slightly alkaline conditions found in many soils. organic anions (possibly dcrivcd from carbohydrate catabolism by plants) may scrvc as carbon sources for the growth of micro-organisms and that the conscqucnt, albeit slow, utilization of mineral phosphates may play a role in the global phosphorus cycle. The role of thcsc microbial components of the rhizosphere, both in this cycle and in competing with plants for available phosphates at pH values above 7 (Azcon rf 01.. 1976). remains to bc evaluated. thank Dr H. Jannasch (Woods Hole Oceanographic Institution), Professor H. D. Holland (Harvard University) and Mr M. Spector (Lehigh University) for gifts of various types of rock phosphate and Miss Sara Pratt for assistance in the isolation of the organisms used. This work was supported by a research grant from the Tennessee Valley Authority. Acknowledgemenrs-We

REFERENCES Alexander M. (1967) Microbial transformation of phosphorus. In lntroducrion IO Soil ,Cficrobiology, pp. 353-369. Wiley, New York. Ashworth J. M. and Kornberg H. L. (1966) The anaplerotic fixation of carbon dioxide by Escherichia coti. Proceedings of rhe Royal Socierv B 165. 189-205. Ayyakkannu K. and ehandramohan D. (1971) Occurrence and distribution of phosphate solubilizing bacteria and phosphatase in marine sediments at Porto Novo. Marine Biology

I, 201-205.

Azcon R., Barea J. M. and Hayman D. S. (1976) Utilization of rock phosphate in alkaline soils by plants inoculated with mycorrhizal fungi and phosphate-solubilizing bacteria. Soil Biology and Biochemirrry 8, 135-138.

890

H. 0. HALVOUONer al.

Borst-Pauwels G. W. F. H. and Peters P. H. J. (1987) Phosphate uptake in Saccharom,vces cereuisiae. In Phosphate Metabolism and Cellular Reguialion in Micro-organisms. American Society of Microbiology. Washington. Brown W. E. (1973) Solubility of phosphate and other sparingly soluble compounds. In Encironmenral Phosphorus Handbook (W. J. Griffith, A. Beeton. J. M. Spencer and D. E. Mitchell, Eds), pp. 203-223. Wiley, New York. Chen Y. and Barak P. (1982) Iron nutrition of plants in calcareous soils. In Advances in Agronomy, Vol. 35, pp. 217-240. Academic Press, New York. Chhonkar P. K. and Subba-Rao N. S. (1967) Phosphate solubilisation by fungi associated with legume root nodules. Canadian Journal of Microbiology 13. 749-753. Cosgrove D. J. (1977) Microbial transformation in the phosphorus cycle. In Advances in Microbiology Vol. I, (M. Alexander, Ed.). pp. 95-134. Plenum Press, New York. Duff R. B.. Webley D. M. and Scott R. 0. (1963) Solubilization of minerals and related materials by 2-ketogluconic acid producing bacteria. Soil Science 5. 105-I 14. Gerretsen F. C. (1948) The influence of micro-organisms on phosphate intake by the plant. Plant and Soil I, 51-81.

Goldstein A. H. and Liu S. T. (1987) Molecular cloning and regulation of mineral phosphate solubilizing gene from Erwinio herbicola. Biotechnology 5, 72-74.

Katzenelson H. and Bose B. (1959) Metabolic activity and phosphate-dissolving capability of bacterial isolates from wheat roots, rhizosphere and non-rhizosphere soil. Canadion Journal of Microbiology 5, 79-85.

Komberg H. L. (1966) Anaplerotic sequences and their role in metabolism. In Essays in Biochemistry, Vol. 2. (P. N. Campbell and G. D. Greville, Eds). pp. l-3 I. Academic Press. London. Naik M. V.. d’douza J. and Araujo A. (1982a) Phosphorus solubilizing yeasts in estuarine environments. Indian Journal of Marine Sciences Il. 197-198. Naik M. V., d’bouza J. and Araujo A. (1982b) Nutritional studies on phosphorus solubilizing estuarine yeast Zorulopsis glabrata. Indian Journal of