Sodium-potassium ionic ratio correlates with yeast induction from Mucor circinelloides

A critical feature of fungal dimorphism is the morphogenetic conversion from mold to the yeast form. In the conversion of a fungus to the yeast form, little is known about the morphological changes that occur prior to the latter. A previous study from our laboratory showed that K + plays important role in the generation of protoplasts from sporangiospores; other morphologies exhibited included thallo-arthric-, holothallic-, holoblastic conidia as well as septate hyphae with provenant vesicular chains of conidia and yeast cells but phenotypic variabilithy of stable forms also exhibited in synthetic broth in a study conducted at pH 4.5 and temperature 20°C. This was confirmed in this study. We further show that phenotypic modification resulting in several transient forms occurred during the early growth phase that led to conversion of germ cell to neoplast, then through protoplast to prevegetative cell and nascent yeast. On modulating K + concentrations with Na + , growth pattern exhibited was either biphasic or sigmoid, which was at optimal at yeast induction sigmoid curve and its phases correlated with dynamic changes in ionic flux, with a Na + /K + ratio of 0.78 at lagand 2.90 at exponential phase. It was thought that in the pH profile two-phase anisotropic growth environment, transmembrane proton ion gradient favoured ionic circulation that triggered phenotypic modification in which protoplasts were generated and, subsequently, prevegetative cells evolved from simultaneous with metabolic adjustment. Subsequently, cells became vegetative in the yeast form. Our data suggest that diminishing logarithmic growth in the sigmoid pattern was triggered by rapid Na + extrusion from intracellular medium of the induced yeast.


INTRODUCTION
The phenomenon of fungal dimorphism has been a subject of investigation not only because it serves as a model for morphogenesis in the life sciences but has heightened because many dimorphic species exist that are clinically and agriculturally important, as they become invasive in one expressive growth habit or the other.Structural forms that are subject to conversion include hyphae, conidia, spores, germ tube, pseudohyphae, spherule and yeast cells.
The precise morphological form taken, filamentous or unicellular, has been attributed to environmental factors.Although elevated temperature is trigger for morphogenetic switching of many pathogenic fungi, including Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Penicillium marneffei, Sporothrix schenckii, factors like pH (Dede and Okungbowa, 2009), or transition metal ions (Yamaguchi,1975) contribute to Candida albicans dimorphism.In the case of Ustilago maydis, sexual elements are involved as fusion of compatible unicellular forms switch to invasive dikaryotic hyphal phase sustaining growth as such in the presence of the host plant (Bolker, 2001).
Although, for another agriculturally important pathogen, Ceratocystis ulmi, a nutritional factor proline triggered the dimorphic switching to yeast form (Kulkarni and Nickerson, 1981).The ability to undergo morphogenetic transition was thought to be essential for pathogenesis.Since the concern here is the development of a disease, the chain of events leading to establishment of a stable morphology, filamentous or unicellular, is essential to the development of the disease.
Prying into the switching to unicellular growth habit becomes an intriguing exercise as preliminary electrophysiological study indicated that proton flux across the biomembrane of a growing cell in a biphasic anisotropic growth environment permitted the morphogenetic conversion (Omoifo, 2012).Within such milieu, ionic circulation occurred between the bulk and intracellular media (Omoifo and Awalemhen, 2012).Thus, Na + and K + antiport transport mechanism ensued on each side of the divide and this coincided with the transient cryptic forms that accretion ally yielded at optimum conditions, the stable but non-persistent unicellular morphology, which is terminal budding yeast cell.
In this transformation process, we observed a directional assembly of morphogenetic sequences; such cryptic demarcations were modeled as such: sporangiospore-germ sphere-neoplasm-neoplasts-protoplasts-nascent yeasts-terminal budding yeasts (Omoifo, 2009).There is the need to understand the nature of these discontinuities and how they are associated with Na + and K + flux through the biomembrane of the growing microorganism in this aforsaid biphasic anisotropic environment.
In this study, we confirm that K + is absolutely necessary for the generation of protoplasts in phase 1 and, also Na + accumulation played a role in yeast form evolvement in phase 2 of the pH profile when there was correspondingly Omoifo 47 enhanced level proton-release intensity followed by a diminishing level, attributable to activities of the growing microorganism, but fundamentally, following the establishment of transmembrane proton ion gradient, the Na + /K + ratio was more critical in the induction of yeast cells from sporangiospores.A deviation from the optimal ratio for yeast cell induction possibly led to differential expression of other morphological components including the thallic subtypes initiated at the germ sphere composite.

Fungal strain and maintenance
The organism, M. circinelloides Tieghem, used in this study was first isolated from decayed fruit of soursop, Annona muricata L., obtained from the floor bed of the tree.It has been used in earlier studies (Omoifo, 2006a;b, 2012;Omoifo and Awalemhen, 2012).It was maintained as glucose-yeast extract-peptone (GYP: 10: 03: 5 g/l) solid cultures where it exhibited filamentous growth habit.A fresh culture was prepared after seven days.

Inoculum preparation for growth studies
Inoculum was obtained by pouring deionized sterile distilled water over aerobic growth and a sterile glass rod gently passed over the surface so as to dislodge the spores.The suspension was poured into centrifuge tubes and spores washed by centrifuging at 5000 rpm for 7 min at 25°C in an MSE 18 centrifuge.The wash was decanted, sediment re-suspended and further washed with two changes of sterile distilled water.Spore count was taken with Neubauer haemocytometer (BSS No. 784, Hawksley, London vol.1/4000) and the suspension was adjusted to 1 million spores per ml in deionized sterile distilled water, using a tally counter in the quantification of spores with the aid of a tally counter.
In the first set of experiments, the effect of K+ was tested; eac h of duplicate flasks was incorporated with the various concentrations of KH2PO4: 0.0, 0.50, 0.70, 0.90, 1.00, and 1.10 g/l.In the second set of experiments, three levels of K+ which were found outstanding to yeast induction were chosen and varied with four levels of Na+.Therefore, to test the effects of K+ and Na+, each of duplicate flasks was incorporated with the various concentrations of KH2PO4: 0.90, 1.00, and 1.10 and of NaCl: 0.05, 0.10, 0.15, and 0.20 g/l.The pH was adjusted to 4.5 with 2 N NaOH or 1 N HCl, using a Cole-Parmer pH Tester model 59000, in the 5000 ml beakers before dispensing in an 80 ml of broth in each of duplicate 250 ml Erlenmeyer flasks for each test.The solution in each flask was made up to 100 ml with glass distilled deionized water and sterilized at 121°C for 15 min.Means were separated using l.s.d., p < 0.05, 0.2691.No yeast cells were observed in the control treatment; although 0.5 g/l treatment had the highest biomass, there was a preponderance of thallic subtypes, including determinate thallic conidia, holothallic conidia, holoblastic conidia; yeast cells constituted the greater propotion of morphologies in the 1.0 g/l K + treatment.

Inoculation, growth conditions and sample collection.
A 1 ml of spore suspension was drawn and inoculated into each broth flask using a 0.5 ml rubber suctioned pipette in a laminar flow chamber, model CRC, HB-60-180.The inoculum flask was shaken at each operation so as to keep the spores in suspension.Each culture flask was then shaken for 30 s and thereafter incubated at 20°C in a preset cooled Gallenkamp incubator.At 24 h interval the culture flasks were brought to the inoculating chamber.The flasks were shaken; 10 ml of broth was withdrawn and deposited into factory-sterilized plastic sample tubes, pre-labeled for each experiment.The culture flasks were returned for further incubation and samples kept at -18°C until analysis.

Biomass determination
Culture broth samples were thawed up to room temperature before biomass determination.This was done by measurement of optical density at 520 nm.This wavelength was chosen because ordinarily FeSO4 impacted greenish coloration on culture broth.Absorbance was determined with a Grating spectrophotometer CE 303 (Cecil Instruments, Cambridge).

Cellular concentration of cations
For determination of intracellular ion content, the method followed was that of Camacho et al. (1981) with modifications.The culture suspensions were centrifuged at 5000 rpm for 10 min, at 25°C (MSE 18).The supernatant was each decanted and 5 ml of 20 mM MgCl2 solution added and centrifuged for 10 min, in a very rapid operation.A 5 ml of 0.2 M HCl and 5 ml of 10 mM MgCl2 were added to re-suspend the cells.These were then poured into factorymade sterile plastic bottles thereafter left for ion extraction for 24 h.The extract was centrifuged for 15 min and the supernatant was obtained for cation determination using Digital Flame Analyzer (ref. FGA -350-L; Gallenkamp, England).

Statistics
Results were subjected to analysis of variance (ANOVA) test for a split-plot format for combined factors and considered significant if p < 0.05 and comparison between means was performed using the Genstat 5 package.

Effect of K + on biomass
Growth of M. circinelloides occurred at all K + levels tested.The experimental values for mean biomass, as optical density, for the cultures with sporangiospores as inoculums, are shown in Table 1.Mean biomass did not increase as the concentration of K + .Variability in growth in the K + supplemented batch broths was greater than that in the control study as reflected in the corresponding standard errors (Table 1).
A 2-way analysis of variance showed that K + had significant effect on biomass production at p<0.05.But time and, the interaction of time with K + -level did not (Supplementary Table 1).Sifting of means in order to know the contributors to the difference at this level (L.S.D. at p<0.05, 0.2691) gave two homogeneous subsets (Table 2).The least value, which was obtained in the control study, shared a similarity with 0.70, 0.90 and 1.10 g/l K + .The second subset that contained the highest biomass value had two concentration levels with similar significance; 0.50 and 1.0 g/l K + .
In a biotechnological process, assumption of regular growth pattern by a microorganism lends itself to determination of parameters like growth rate, specific product concentration necessary in order to know the basic route to increasing productivity and or improving physiological relationship.The lag phase, exponential phase and stationary phase are well known underlying orientation for microbial growth.Biomass profiles (not shown) however were a-typical of sigmoidal growth pattern at all the K + -levels tested.

Effect of K + on morphological expression
Microscopic examination showed that variability was highly reflected in the morphologies induced.Thallic growth, septate, and did not form meshwork, occurred in different subtypes, including conidiophore terminating in conidiogenous vesicle with concurrent catenate conidia, determinate thallic conidia, holothallic conidia and holoblastic conidia.Similar morphologies also occurred at 0.50 g/l K + , but a striking difference was the occurrence of granular/neoplastic units and emergent/nascent yeasts unbudded), although scanty at this K + -level.At 0.70 g/l K + supplementation, neoplasts which were more preponderant became larger in sizes in contrast to the preceding concentration.Few terminal budding yeast cells, which assumed various shapes and sizes, were observed.Preponderance of thallic subtypes as observed in the control broth diminished still in the 0.90 g/l K + supplemen-Omoifo 49 ted broth while neoplasts became more numerous and larger in sizes; they assumed a tender membrane and were globose or rod shaped.These were the protoplasts.Also there were a few enlarged yeast cells.At 1.0 g/l K +level, thallic presence became scantier.Protoplasts assumed internal dimensions and were very robust.
Although there were few nascent yeast types, enlarged and matured terminal budding yeast cells were preponderant.At the highest K + supplemented broth, protoplasts were globose to subglobose in shape and were not very numerous; nascent/emergent yeast cells were rods, subglobose, ovoidal, obpyriform to ellipsoids.
Other morphologies observed were germlines, septate thallic growth with vesicular conidial head groups, holoblastic-and holothallic conidia, which became more robust.

Effect of combined ionic incorporation on morphological expression
Like the single factor K + supplemented broth in experiment 1 above, reproductive structures formed in the Na + and K + modulated broths included the various thallic subtypes.But the predominant morphology was terminal budding yeast cells, observation of which showed to be of higher magnitude than what occurred in the single factor K + supplemented broths.This perhaps indicated an additional effect as a result of Na + modulation.

Effect of combined ionic incorporation on yeast induction
A 3-factor analysis of variance indicated the significance of individual or combined effects of the elements on growth.Results (Supplementary Table 2/Table 4) show that the main factors Na + , p<0.05, and time, p<0.001, contributed significantly to growth, but K + alone or the 3, 2-way interactions and 1, 3-way interaction combining K + , Na + and time did not.A separation of means was done using the l.s. d. 0.04944, p<0.05, to locate what level of Na + contributed to the significance.This resulted in two homogeneous subsets, with 0.10 g/l Na + level making a distinct subset.This is shown in Table 3. Histogrammatic represen-tation further confirms this (Figure 1a).It also shows that beyond this level of Na + incorporation, biomass was suppressed.What this suggested is that it was the incorporation of Na + that further enhanced or, added value to the induction and proliferation of yeast cells in the combined factor broths but in a time dependent manner.
When plots of interaction of the significant factors with the other elements were thus obtained (Figure 1), patterns generally exhibited 2-phase growth.At the 0.9 g/l K + treatment, O.D. did not differ from 1.0 g/l K + supplemen- tation when modulation was with 0.05 g/l Na + , but it was lower at 0.10 and 0.15 g/l Na + and thereafter upshot the latter treatment at 0.20 g/l Na + (Figure 1a).At the higher level treatment (1.10 g/l K + ), 0.05 g/l Na + modulation stimulated higher growth in comparison with the same level when K + supplementation was with 0.9, or 1.0 g/l K + ; growth was however at par with 1.0 g/l K + supplementation when modulation was with 0.10 g/l Na + .Growth subsequently diminished to a lower value than that at 0.9 or 1.0 g/l K + treatment.
What this indicate is that at 1.0 g/l K + -level, broth modulation higher than 0.10 g/l Na + suppressed biomass accumulation.The fluctuation in growth on modulation of the 0.9 g/l K + broths with Na + made this treatment unreliable for further conduct of our study on fungal dimorphic switching.
Therefore, 0.1 g/l Na + modulation became the optimum for biomass production with growth value lower at either side of this level.

Effect of significant factor incorporation on growth pattern
A 2-phase growth pattern was very conspicuous at time versus K + : 0.9, 1.0 g/l (Figure 1b) and time versus Na + : 0.15, 0.20 g/l (Figure 1c) interaction.However, subtle sigmoid pattern was exhibited at time versus K + : 1.10 g/l (Figure 1b) interaction.Variation between these two growth habits occurred at the other levels.

Na + modulation and growth profile
We decided to look at growth profiles at specific K +levels with the various Na + modulation.Although only one peak occurred at 0.05 g/l Na + -and 0.15 g/l Na + , growth patterns were not sigmoid (Figure 2a).Growth at the other Na + -levels were bi-phasic where the 2 nd peak was still rising at termination of experiment (Figure 2a).The bi-phasic pattern was very conspicuous at time versus K + : 1.0 with 0.15 g/l Na + modulation, while at 0.05 g/l Na + the 2 nd peak was still rising at termination of experiment.However, striking sigmoid pattern debuted at 0.10 g/l Na + modulation, and less strikingly so at 0.20 g/l Na + incorporation (Figure 2b).
At 1.10 g/l K + treatment, approximation of bi-phasic patterns were also observed at 0.15 Na + and 0.20 Na + incorporation, although the 1 st peak at 0.20 g/l Na + occurred where there was a median trough for the 0.15 g/l Na + profile.At 0.10 g/l Na + , modulation growth was still rising at termination of experiment, while profile at 0.05 g/l Na + was a deviant sigmoid, where at lag phase there was an initial fall in growth before subsequent apparent exponential growth (Figure 2c).Variation between these two growth habits occurred at the other levels.
Two main patterns were pronounced when biomass profiles were obtained for the Na + versus K + interactions (Figure 2a-c).Near-perfect sigmoid curves were obtained at 1.0 g/l K + versus 0.10 g/l Na + and 1.0 g/l K + versus 0.20 g/l Na + (Figure 2b) while near-perfect bi-phasic growth profile exhibited at 1.0 g/l K + versus 0.15 g/l Na + (Figure 2b) and 1.10 g/l K + versus 0.15 g/l Na + (Figure 2c).Exhibited growth patterns at the other levels were variations of these two identified patterns, sigmoid and bi-phasic.

Regression analysis of optical density on external and internal ionic composition
The observation on the two standard patterns led us to regress the principal variate, optical density, on the major external and internal ionic composition which had determinable values including K + ext , Na + ext , K + int , Na + int at p<0.05.This was done in order to partition the contribution of the factors to biomass build-up, and possibly form development.Results (Supplementary Table 3) show that these factors apparently did not make significant contribution at p<0.05 as only 1.9% of the variability within was accounted for (std error, 0.186).
Yet ionic circulation was inherent during the growth of the microorganism.This shows that the analysis of variance was unable to account for the behaviour of the microorganism in our minimal medium.

Transmembrane proton gradient and ionic flux
Thereafter, we looked at the differential of pH of bulk medium from the initial value of 4.5; profiling clearly showed the same regular bi-phasic pattern, no matter the exhibited growth habit, which was either bi-phasic or sig-sigmoid.Figure 3 illustrates this.There was initial decrease of bulk medium pH, possibly indicating H + extrusion from intracellular medium and a subsequent reversal after the point of inflection, also showing a possibility of indicating H + uptake by each cellular unit.This could mean a change in direction of transport process for H + through the biomembrane of the growing microorganism.Thus, in each phase, the medium was anisotropic, possibly permiting transmembrane proton gradient.
If we look at the treatment, 1.0 g/l K + : 0.10 g/l Na + where near-perfect sigmoid growth habit exhibited, ionic communication involving particularly H + , Na + and K + occurred between the external and intracellular media.For Na + and K + , Figure 4 illustrates this.At the lag phase, there was simultaneous influx of K + and efflux of Na + .A reversal of this occurred after 48 h of growth.This was followed by very rapid influx of Na + up till mid log phase where K + efflux was slow, but became dramatically steeply, correspondingly with efflux of Na + from mid log phase till 96 h after inoculation.There from, influx of both K + and Na + occurred at the stationary phase of growth.

Intracellular ionic accumulation, growth habit and morphological expression
Histogram of ionic data generated was juxtaposed with that of biomass and pH differential.This showed that the H + -release intensity was highest at 72 h after inoculation (Figure 5a).This was at mid log phase when there was also the highest intracellular accumulation of Na + (Figure 5b).Such H + -release stabilized until biomass peaked at 96 h after inoculation (Figure 5a), but by this time intracellular Na + content diminished; although there was a rise in this level at stationary phase, it was not half as much as that at mid log phase.On the other hand, the H + level diminished at the stationary phase.Using this as standard, Figure 6 shows mid log phase accumulations of Na + in the various treatments and the growth habit thereupon exhibited.At 0.90 g/l K + level, maximal Na + accumulation occurred at 0.05 g/l Na + modulation.This decreased with increase in Na + incorporation.Growth pattern at 0.05 g/l Na + broth was single phase, peaking at 96 h after inoculation.This treatment also induced the least proportion of yeast cells, but proportion of yeast cells increased with rise in Na + incorporations as growth pattern became bi-phasic.
In comparison with the 0.9 g/l K + level, yeast cells were more preponderant in treatments with 1.0 g/l K + supplementation.There were bi-phasic growth expressions in media incorporated with 0.05 and 0.15 g/l.Na + and intracellular Na + accumulations respectively were 8.40 and 11.0 mg/kg, which were much lower than intracellular accumulations at 0.10 g/l Na + modulation that promoted sigmoid growth habit and induced predominantly yeast contents increased with increase in Na + modulation and yeast form induction decreased oppositely, indicating that increase in intracellular Na + content suppressed yeast induction.Still, the proportion of yeast cells induced at the 1.10 g/l K + treatments was never as high as that which occurred at the 1.0 g/l K + treatment.

+ influx and yeast induction
Generally, addition of Na + to culture broths stimulated the induction of yeast cells.At K + treatments where there was optimal intracellular Na + accumulations (0.9 g/l K + : 0.05 g/l Na + , 1.0 g/l K + : 0.10 g/l Na + , 1.10 g/l K + : 0.15 g/l Na + and 1.10 g/l K + : 0.20 g/l Na + ), the Na + influx rate also showed high values, but influx rate was lower with rise in Na + incorporation, and negative at 0.20 g/l Na + modulation, where intracellular accumulation of Na + was also the least (Supplementary Table 4).
This means that there was Na + extrusion at the log phase of growth in the latter treatments.Since observation showed that yeast cell induction was least with 0.90 g/l K + : 0.05 g/l Na + modulation where Na + influx was highest, it probably means that Na + accumulation was not the sole determinant of yeast induction (from provident protoplast -to -formation of nascent yeast) at lag phase, and yeast proliferation (from matured yeast -to -terminal budding yeast) at log phase.
This was corroborated by the fact that optimal yeast induction occurred with treatment (1.0 g/l K + : 0.10 g/l Na + ) intracellular accumulation of 17.95 mg/kg with an influx rate of 0.473 mg/h and, yet a sigmoid growth pattern exhibited.Still, the 1.0 g/l K + : 0.20 g/l Na + treatment with Na + extrusion at log phase (-0.0646 mg/h) and a 2.5 mg/kg intracellular accumulation of Na + , supported sigmoid growth habit.
Perhaps, the lag phase composite entities, including germ cells, spheroid/-neoplasm, neoplastic units and protoplasts (which progressively increased in sizes), was also contributory to yeast induction.

Na + /K + ratio, yeast induction and sigmoid growth pattern
At the K + (1.0 g/l) treatment that supported near perfect sigmoid growth habit, Na + modulation stimulated yeast proliferation.The Na + /K + ratio was 2.9 at the yeast proliferative stage in the treatment combination; 1.0 g/l K + : 0.1 g/l Na + (Figure 7).Treatment combinations with Na + /K + ratios approximating this sigmoid curve-associated ratio promoted high yeast inductive/proli-ferative capabilities no matter whether they exhibited a single, sigmoid or biphasic growth habit, although the relative proportions varied.Again, high Na + / K + ratio at log phase was not the sole determinant of yeast proliferation, for at 1.0 g/l K + : 0.20 g/l Na + treatment combination, the Na + /K + ratio at log Intracellular ion content (m mol/Kg) phase was comparatively low (0.56) yet growth habit was sigmoid and yeast cells preponderant.However, in the 1.0 g/l K + : 0.20 g/l Na + treatment Na + /K + ratio at lag phase, 0.81, was higher than that at the primary sigmoid habit expressive treatment (0.78) (1.0 g/l K + : 0.1 g/l Na + ).
Although there was efflux of K + at lag phase in the former, in contrast to its intracellular accumulation at the primary sigmoid habit expressive treatment, yeast cells still sequentially induced.As microscopic examination showed in the first set of experiments reported above, and in comparison with the other K + treatments, the 1.0 g/l K + treatment stimulated the induction of a higher proportion of yeast cells.This was further enhanced with the incorporation of Na + in such K + supplemented media (second set of experiments).
Following this, the proportion of yeast cells in broth peaked at 0.10 g/l Na + modulaion.In a similar manner, intracellular accumulation of Na + was higher at the 1.0 g/l K + treatment and it reached a peak at 0.10 g/l Na + broth modulation, in comparison with other Na + -levels.This indicated that the presence of Na + intracellular in particular, rather than K + , stimulated the yeast form, thus confirming the results on biomass analysis shown in Table 3 and deductions made from Figure 1a.Therefore, if we use Na + influx rate as a parameter for measuring yeast induction (meq of Na + accumulated/time at specific growth phase, lag or log), we find that the value of rates was parallel with increase in intracellular Na + accumulate and consequently yeast induction.It was however not sequentially when it was viewed against Na + modulation in the minimal medium.
Since K + accumulation appeared to trigger lag phaserestricted neoplasm-protoplast generation and, Na + accumulation triggered vegetative capability, that is, from the provident protoplast through prevegetative-to vegetative .cell, the yeast form acquisition, which was both lag phase and log phase event, and in each phase both ions were antiported in a sigmoid-oriented growth pattern; it possibly meant that Na + /K + ratio could be a more valid means of assessment of yeast induction, that is, at both the inductive stage and the proliferative stage in this study.
This also means that it was not the absolute levels of K + or Na + at this inductive stage, that is, lag phase, but the cellular Na + /K + ratios that were the main determinants that promoted the induction of the yeast morphology.If we realized that the inductive stage encompassed several transient forms, including germ cells, spheroids/neoplasm, neoplasts, protoplasts, prevegetative cells (Figure 8) then the Na + /K + ratio progressively impacted on these forms in the unidirectional milieu, that is, during the H + extrusion phase.Thus, as the H + -release intensity increased against a concentration gradient in the bulk medium, being buffered at a specific acidic pH-level and hence proton ion gradient, there was anti-parallel decrease in the magnitude of Na + /K + ratio: from high in the bulk medium to low intracellular.The Na + /K + ratio at lag phase at which yeast induction was stimulated varied from 0.41 -2.93 in the treatment combinations 0.90 g/l K + : 0.05 g/l Na + to 1.10 g/l K + : 0.20 g/l Na + (Figure 7).

Deviation from optimal Na + /K + ratio and thallic expression
At lag phase, it appeared that a deviation from the sigmoid curve permissive Na + /K + ratio, (0.78), inchoately enabled the occurrence of other morphological structures.Thus, with high Na + /K + ratio, (2.93) at 1.10 g/l K + : 0.10 g/l Na + treatment, polarization of germ cell could be triggered, prolongation of which gave rise to thallic growth, which initially could be coenocytic, determinate and subsequently thallo-arthric on the one hand, and septate which could give rise to conidiophore with apical vesicle bearing concurrent catenate conidia, on the other.
Variation in thallic expression occurred as germ tube converted to conidiogenous structure, which produced conidia in an acropetal manner, or holoblastic conidia as all the walls of a growth sphere participated in septum formation.The higher Na + /K + ratio at lag phase possibly gave rise to the vigorous thallic growth at this treatment (1.10 /l K + : 0.10 /l Na + ), in comparison with the others, and made the thallic subtypes more robust, as already These persistent conidia remained in the growth milieu till termination of experiment.However, since it was presumably affected by the decreasing time-dependent Na + /K + ratio, protoplasm of growth sphere or, conidia converted to neoplasm.Perhaps, as a result of internal pressure, witness conidia burst or cell wall rupture, thereby releasing neoplastic units or protoplasts.These thereafter assumed individual life form.

Ionic circulation and sigmoid growth phases
In the 1.0 g/l K + : 0.10 g/l Na + treatment combination where there was optimal induction of terminal budding yeast cells, growth profile showed a sigmoid curve.This is shown in Figure 9. Demarcation of the growth phases also correlated with cryptic phases of ionic flux as shown in Figure 4, a feature anchored on transversal through the biomembrane, an indication of involvement of electrical signaling and free energy generation inherent in the concentration gradients of Na + and K + created across the membrane of the growing microorganism.At lag phase (Figure 9a), K + accumulated intracellularly as Na + decreased in a time dependent manner (24 -48 h) (Figure 4), indicating a fall from high action potential to a low level across the membrane.
Starting with sporangiospore (Figure 8a), it coincided with the transition from germ cell through neoplasm, then neoplasts to protoplasts (Figure 8b to e), a clear demonstration of phenotypic modification at the lag phase.
At exponential growth phase (Figure 9b), the extrusion of K + observed and rapid intracellular accumulation of Na + (48 -72 h) (Figure 4) as the emerging prevegetative cell (Figure 8f) subsequently assumed autocatalytic (from protoplast-to-prevegetative-to-vegetative) growth.This is further illustrated in Figure 8g to o which showed a wallless membrane bound pre-vegetative cell become polarized at one locus, then it became directionally elongated on its axis until a wall-less vegetative cell initiate was formed.This indicated that through the period of linear growth, biomebrane of induced yeast cells became more permeable to Na + as the vegetative cell matured and subsequently budded; hence there was rapid influx and intracellular accumulation of the ion and perhaps, its inherent use in driving bioenergetic and biosythetic activities, including cell wall construction, increasing macromolecular syn-thesis and mitosis thus leading to yeast cell proliferation (Figure 8p and q), marked by constant specific growth rate, and hence the exponential growth phase (Figure 9b).
In this study, the period of declining logarithmic growth (Figure 9c) coincided with very rapid extrusion of Na + and K + from the intracellular medium (Figure 4) (72 -96 h).This also coincided with the period of pH stability (Figure 5a).It could also be said to be the period of nutrient exhaustion, overcrowding and toxic product accumulation.If this was assumed, then that there was pH stability probably meant that a steady state condition was created between the bulk and intracellular media for a biochemically redirected programme and, or divergent physiological activity which occurred intracellularly.Here, notice that rapid Na + extrusion from intracellular medium occurred, an indication of membrane repolarization arising after a possible log phase-associated membrane Figure 10.Flask-cultures of induced yeast cells of M. circinelloides after 2 weeks of growth.At termination of experiments conducted at pH 4.5, 20°C, the flasks were transferred to the laboratory side bench at 28±1C, ambient.the reversion to aerial mat that was re-induced in each of the flasks was observed following transfer of cultures of induced thallic subtypes and terminal budding yeasts, which sedimented, to the laboratory side bench.depolarisation in the induced yeast cell, which perhaps should normally incur a rise in membrane potential at this declining specific growth rate phase, but simultaneously there was a rapid or heightened K + efflux from intracellular medium in this anisotropic environment but in a segment where the pH steadied.That there was K + efflux at exponential growth phase, but this was further heightened at declining specific growth rate phase, perhaps indicated that membrane K + conductance played a more significant role in maintainng the upward trend of specific growth rate, but beyond optimum level, or threshold, that is, of conductance perhaps in the steady state, such support broke down and the specific growth rate set on a decline, a possibility that led to the assumed diverted biochemical and, or physiological event.Ionic transport into the intracellular medium was again unidirectionally reversed (Figure 4) (96 to 120 h) at the stationary phase (Figure 9d), where there was no further increase in the population level, but the assumed apoptotic programme set it on a steep fall.

Reversion to filamentous growth habit
At termination of experiments, culture flasks of induced yeast cells and thallic subtypes were shifted to the laboratory bench.Observation after 2 weeks of such transfer showed that filamentous growth formed compact matt on the surface of each culture broth.This is shown in Figure 10.

DISCUSSION
This study shows that M. circinelloides exhibited a multifaceted growth habit in synthetic broth, exhibiting both transient and stable morphologies.That neoplasm, derived from plasm of germ cells reorganized into multiple individual units, including neoplasts and protoplasts, which were not observed in the control tests except when K + was incorporated, confirmed finding that K + was of absolute necessity for protoplast formation (Omoifo, 2012).As it has been shown in several studies, a sequential development occurred with the protoplast being the cross-over point of a process that led to the emergence of the yeast form (Omoifo, 2003(Omoifo, , 2009)).This was confirmed in this study.This study's finding differ from that of earlier workers (Lubberhusen et al., 2003;McIntyre et al., 2002) who obtained multipolar budding yeastlike cells from dimorphic M. circinelloides under 30% CO 2 pressure but thallo-arthric conidia on oxygen stimulation.
Yeast induction has been attributed to three main parametric effects, viz: critical concentration of ions, chemical potential and Na + influx rate; these combined in a cooperative manner to effect a streamlining of the phenotypic expression (Omoifo and Awalemhen, 2012).In the present study, the concentration of ions has been found to be most significant in the early process of induction and subsequent proliferation of the induced yeast form.
This has been expressed as the Na + /K + ratio.While a diminishing magnitude of this parameter, from the bulk medium to the intracellular medium led to reorganization of the stable spore-morphology through cryptic transient forms, it was thereafter reversed after the cross-over form, that is, the protoplast, and with an increasing effect until a stable phenotype was achieved.This was the yeast cell.However, the Na + influx rate assumed greater significance in expounding the yeast form differentiation, from protoplast through prevegetative cell-to-nascent yeast, then through maturation until it became terminal budding.
Thus, in a sigmoid growth expressive habit, as occurred in the 1.0 g/l K + : 0.10 g/l Na + treatment, a laglog sequential parametric relationship, or cooperativity was essential for the yeast form induction.Lysed germ cell envelop, thereby 'freeing' the individual neoplasts, which dispersed within the medium, was the main method of generation of these units.
Beside this, a secondary type of ontogeny was when hyphal compartment ruptured or conidium burst, whence encompassing wall remained as carcass or ghost cell, an effect seen mainly at commencement of the 2 nd phase of the pH profile involving oppositely unidirectional H + influx (Omoifo, 2012; this study), which (conidial burst or wall rupture) could have resulted from osmotic effects and, or an increase in turgor pressure within cellular compartments.The reversal in direction in H + transport also involving membrane bound transport mechanism and, hence Mitchellian proton pump (Haris, 1977;Voet and Voet, 1995) directed into the protoplast interior, would affect other radicals concentration, including phosphate group (Lehninger, 1975).Na + and K + as demonstrated in this study, thus confirming the earlier findings (Omoifo and Awalemhen, 2012) on intermedial ionic circulation, and consequently alter the inherent physiology (Calcot, 1981).But in a carbon-substrate growth milieu, this led to H + -substrate symport (Alderman and Hofer, 1981;Mitchell, 1967;West and Mitchell, 1972, 1973, Slayman and Slayman, 1977) into the protoplast interior, thus providing glucose intracellular as used in our study, for bioenergetics and biosynthetic purposes.
At Na + efflux, there was simultaneous influx of K + , a condition reversed at 2nd phase of the pH profile, when protons were translocated into the cellular interior, and as Omoifo 61 found in Escherichia coli, driven by the membrane potential generated as a result of this reverse movement (Setty et al., 1983) which corresponded with initialization of polarization of prevegetative cell, formation of nascent yeast and subsequent yeast proliferation in the study.Now, since K + is exchanged for H + mediated by membrane-bound proton pumps (Conway and O'Malley, 1946;Rothstein and Enns, 1946;Pena, 1975;Ryan and Ryan, 1972;Albert et al., 1994), then presumption of an anaerobic growth environment in a medium mediated by unidirectional H + influx into protoplast cytosol, and based on Mitchell's hypothesis on H + translocation in the absence of oxygen (Mitchell, 1979), would call for further inductive enzymes for a directional physiology; for instance, a reduction in Fe 3+ is a condition that favored glycolytic breakdown of phosphate esterified glucose at carbon 6 (Michelson, 1978).Phosphoglucomutase, an enzyme that converts phosphate esterified glucose to glucose-1-phosphate, has been found in higher proportions in the yeast cells of dimorphic Paraccoccidioides brasiliensis, in comparison with the mycelial form (Kanetuna and Carbonell, 1966).Glucose-1-phosphate is a key intermediate in the pathway to the formation of beta-1, 3 glucan, the key structural material of the yeast cell wall.Assuming that these activities occurred during the growth of M. circinelloides, from this, it can be seen that the possible induction of the enzyme phosphoglucomutase could lead to conversion of co-H +symported glucose to the yeast cell wall structural material, a platform for formation of nascent yeast (Figure 8i-m) copiously observed in this study.
The cell wall generative capacity of protoplast is not in doubt, as Carbonell et al. (1973) showed that the protoplasts of dimorphic H. capsulatum prepared by enzymatic lysis of cell wall from whole yeast cell, could re-generate the cell wall.It could be inferred from the above discussion that catabolic breakdown of the presumed H +symported glucose within the protoplast was glycolytic.This has been demonstrated in our laboratory (Omoifo et al., 2013) by the use of the well-known glycolytic inhibitor, NaF, which exhibited three main effects on the conversion process of sporangiospores of Rhizopus stolonifer to terminal budding yeast cells in minimal medium, viz: (a) complete inhibition of yeast induction, (b) delayed induction of yeast cells and (c) apoptosis of induced yeast cells.In the complete absence of yeast cells, protoplasts were copiously produced.When yeast cells were induced prior to NaF challenge, the inhibitor caused the death of cells.It was strongly suggested that NaF challenge had profound effect on substrate level phosphorylation, which is primarily executed at the enolase enzyme activity step, where NaF irreversibly combines with the enzyme thereby preventing it from performing the significant role of enolization of phosphorglycerate to pyruvate thereby terminating substrate level phosphorylation, being that fluoride ion forms a complex with Mg 2+ that competitively locates the Mg 2+ -requiring enolase active site (Cinnasoni, 1972), an effect enhanced with high levels of phosphate (Bassetti et al., 2004) also copiously used in our medium, (note that Mg 2+ was part of elemental composition of our multiionic system in the present study), and thus prevents the dehydration of 2phosphoglycerate and, hence the formation of phosphoenolpyruvate (Voet and Voet, 1975;Christophe et al. 2001), as this is a critical step in the generation of ATP through substrate level phosphorylation.This implied that protoplasts through prevegetative cell per-formed energy generation through the physiological mechanism of substrate level phosphorylation, which could be terminated with the glycolytic inhibitor.But when the inhibitor challenge was prior to generation of a functional envelop, the transient forms remained as protoplasts.
It is demonstrated here that the exhibition of sigmoid growth curve by induced yeast cell of M. circinelloides is similar to the life pattern of Saccharomyces cerevisiae.It is demonstrated here also that a classical sporangiospore-producing filamentous microorganism, M. circinelloides, converted through multiple transient forms debuting as prevegetative cell and subsequently proliferating neither as sporangiate thalli nor conidiate thalli, but terminally buds unicellular cells, which describe a sigmoid growth pattern.Such should have properties that could be used for comparative purposes.This study confirmes earlier report (Omoifo and Awalemhen, 2012) that M. circinelloides exhibited sigmoid growth when 1.0 g/l K + treatment was modulated with either 0.10 or 0.20 g/l Na + but with higher specific growth rate in the former.It was also shown that broth where the exponential growth phase was more steeply oriented permitted a near perfect sigmoid curve at 1.0 g/l K + : 0.10 g/l Na + treatment.An earlier study (Omoifo, 2012) showed that the H +released intensity from intracellular medium of the growing microorganism was maximal in the 1st phase of the pH-profile, that is the extracellular medium reached the maximum degree of acidification.This is akin to increase in external medium pH changes when Escherichia coli is suspended in acidic medium whereby the internal pH approaches neutrality (Padan et al., 1976;Booth et al., 1978Booth et al., , 1979;;Zilberstein et al., 1979), a system that is energy dependent (Padan et al., 1976;Mitchell, 1979;Setty et al., 1983).As the present study shows, this was when Na + ions were correspondingly extruded from the intracellular medium.In the 2nd phase of the pH-profile when there was diminishing protonrelease intensity (Omoifo, 2012; this study), the Na + influx rate was optimal, which was 0.47 mg/h for that treatment in comparison with the 1.0 g/l K + : 0.20 g/l Na + treatment, the less permissible sigmoid curve-oriented growthmedium where the Na + influx rate was 0.12 mg/h (Omoifo and Awalemhen, 2012).The lower sodium influx rate also therefore appeared to correspond with lower proton ion electrochemical potential.As this study shows, the difference in sodium accumulation and inherent proton potential was also reflected in the yeast induction capability of the media as the former (1.0 g/l K + : 0.10 g/l Na + treatment) induced comparatively higher population level of terminal budding yeast cells.
That the biphasic pH profile was similar whether growth was sigmoid or 2-optima habit or the variant was of either, suggested that physicochemical activities permissible with transmembrane-proton ion -gradient were also similar during the patterned growths.However, because of imbalance in ionic content at lag phase, that is, deviation from the treatment 1.0 g/l K + : 0.10 g/l Na + ratio (0.78), biochemical activities possibly promoted localized polarization of growth spheres; tubular growth therefore ensued.This was probably the origin of the thallic subtypes observed in this study.Although, inherent biophysical phenomena took place and these included neoplasm formation, cell envelop rupture and release of neoplastic units, protoplasts formation, wall-less prevegetative cell formation and its polarization until a wall-less vegetative cell initiate was formed, which were all lag phase events.
Since lyses of cell wall of thallic subtypes were not complete, carcasses of same were conspicuous and they became additional to biomass quantitation at the lag phase.Hence they contributed to the first optimum of the 2-phase growth habit.As it was obvious, post cell envelop rupture modifications of protoplasts were subjected to the same ionic effects as occurred in the sigmoid-related log phase that induced terminal budding yeast cells.This was possibly why terminal budding yeast cells were also induced, along with, and after the first phase thallic growth cessation, in the two-phase growth pattern and became more preponderant during the second phase.

Conclusion
In the morphological species concept, it does not appear that neoplasm, neoplast, protoplast, prevegetative cell and nascent yeast have been characterized for filamentous microorganisms.Specifically, transformation of spores of M. circinelloides has been significantly influenced by the establishment of transmembrane proton ion gradient and this led to demarcations resulting in transient forms observed in this study in which ionic circulation correlated with morphology.This thus suggested that the morphological, biochemical and physiological sequences herein described were tightly coupled to each other and thus illustrated a process that appeared dependent on self assembly with inherent membrane restructuring at key junctions but driven by a transmembrane proton ion gradient.It was argued here that, active reactions led to energy generation through the electrochemical proton gradient in phase 1 favouring phenotypic modifications and this was sequentially followed by substrate level phosphorylation in the ultimately adopted morphological form, the yeast cell.
It further showed the significance of Na + /K + ratio in determining the early growth phases of M. circinlloides in a sigmoid oriented growth pattern and deduced that a deviation from the sequential parametric relationship led to expression of morphologies other than the yeast form, which physiologically catabolized glucose-carbon through substrate level phosphorylation; but at the stationary phase, perhaps a diminished effect of the Na + /K + ratio led to equal generation and subsequent apoptosis of the induced terminal budding yeast cells.Thus, it could be surmised that the process of yeast induction from aerially borne spores of M. circinelloides occurred in a highly functional proton gradient driven cooperative system in which modulation by phase-determining sodium-potassium ionic ratio became significant.But exposure of such induced yeast to atmospheric oxygen led to a reversion to the filamentous growth habit.
Although this study is only exploratory, it offered opportunity to view the issue of fungal dimorphic switching with a consideration given to electrophysiological relationships.The specific effects of Na + and K + however could not be taken as absolute as discrepancies exist.For instance, in the study of Omoifo and Awalemhen (2012), it was shown that at the declining specific growth rate phase K + mode was promoted, which is incongruous with the present deposition.However, if this study suggested a transmembrane proton ion gradient as another paradigm for dimorphic switching of M. circinelloides, then the dynamics of K + conductance on membrane activation, lysis of germ cell envelop and the process of polarization of prevegetative cell, as well as it's possible effect on Na + conductance and inherent organelle mobilization on one hand, and yeast cell proliferation on the other, call for closer attention, especially as Setty and co-workers (Setty et al., 1983) showed that the relative concentration of K + and membrane permeability are major factors that control the kinetics and degree of H + movement.

Figure 1 .Figure 2 .
Figure1.Effect of K + and Na + supplementation on biomass generation of M. circinelloides cultlivated in multiionic broth.A, extracellular K + -time profile; B, extracellular Na + -time profile; C, K + & Na + simultaneous supplemention of broth.Although there were multiple phenotypic expression, observation showed that protoplasts and yeast cells predominated at 1.0 g/l K + : 0.1 g/l Na + .

Figure 3 .Figure 4 .
Figure 3. Treatments showing representative growth patterns, sigmoid and two-phase habit, and pH profiles during the cultivation of M. circinelloides in synthetic broth.Note: whatever the growth habit, the pH profile showed similar pattern.

Figure 5 .
Figure 5. Histograms showing biomass accumulation and bulk medium pH variation in relation to intracellular ion content.

Figure 6 .
Figure 6.Histogram showing Na + accumulation after 72h of growth and growth patterns exhibited by M. circinelloides cultivated in synthetic broth.

Figure 7 .
Figure 7. Treatment combinations and intracellular Na + /K + ratio (r) in inductive (lag) and proliferative (log) phases during the cultivation of M. circinelloides in synthetic media.

Figure 9 .
Figure 9. Sigmoid curve and possible electrophysiological processes that occurred in induced yeast cells of Mucor circinelloides when cultivated in glucose-substrate 1.0 g/l K + : 0.10 g/l Na + broth at pH 4.5 and at 20C.

Table 1 .
K + supplementation, biomass estimates with their respective standard errors and form of growth during the cultivation of M. circinelloides in synthetic broth for 120 h at pH 4.5 and 20C, ambient temperature.

Table 2 .
Homogeneous subsets of mean biomass estimates obtained during sporangiospore-yeast transformation of M. circinelloides cultivated in K + -incorporated synthetic broth for 120 h at pH 4.5 and ambient temperature of 20°.