Environ. Sci. Technol. 1994, 28, 1502-1505
Acid-Base Properties of a Soil Fungus, Trichoderma harzianum Chrlstlna Krantz-Rulcker,' Bert Allard, and James H. Ephralm
Department of Water and Environmental Studies, Linkoping University, S-581 83 Linkoping, Sweden The acid-base properties of mycelium from Trichoderma harzianum were studied as a function of mass, state of mycelia, i.e., starved or nonstarved, and ionic strength of the aqueous phase. The nonstarved mycelia exhibited an extensive buffering behavior, which was attributed to the production of chemical species participating in the resultant equilibrium. Production of such "acidic" moieties resulted in higher acid capacities for the nonstarved mycelia (500-800 mequiv/kg mycelia)while for the starved mycelium,negative acid capacitieswere obtained ( -120 mequiv/kg mycelia) indicating a basic surface. Plots of charge balance versus pH showed an insensitivity to ionic strength changesand facilitated the estimation of the point of zero charge (pzc) for the starved mycelia. Similar plots for the nonstarved mycelia showed that this system was sensitive to ionic strength changes and yielded no distinguishable pzc values. It was assumed that the nonstarved mycelium possibly allowed transport of ions into its cell wall structure or even intracellularly whereas the starved mycelium did not. N
Introduction Fungi have a high capacity to accumulate metals, and there are numerous studies concerning the use of fungal biomass for removal and possible recovery of metals from aqueous effluents (1-4). However, an almost neglected area of metal accumulation in soil is the effects of fungi on their speciation and transport. Fungal metal accumulation of up to 20-30 % of the dry weight is reported ( 5 ) , which is comparable to the capacity of conventional ion exchangers and far above the capacities of anionic abiotic materials (6). There is little knowledge about the processes behind metal accumulation by fungi. The cell walls are composed of polysaccharides, proteins, and lipids which contain functional groups with potential metal binding capacities such as phosphate, carboxyl, amine, hydroxyl, and sulfhydryl groups (7). The chitin content of fungal cell walls has been shown to be important in, for example, uranium accumulation (81, and this was also found to be the case for a range of other fungal species and metals (9). Other wall components such as phenolic polymers and melanins are also reported to exhibit metal-chelating properties (10). The accumulation of metals by microorganisms is generally a rapid process (11). The observed accumulation can in most cases be accounted for by surface adsorption processes, the much slower process of intracellular accumulation (if any) being of little significance. More has to be known about the surface properties of the fungi in light of existing functional groups to explain the mechanisms behind metal accumulation. Efforts have been made to determine such functionalities via the use of potentiometric titrations. Reported experiments have mostly been performed at a single ionic strength (12).
* E-mail address:
[email protected]. 1502 Envlron. Scl. Technol., Vol. 28. No. 8, 1994
The objective of this study was to describe the acidbase properties of a fungal mycelium under different conditions, e.g., varying state of the mycelium (starved versus nonstarved) as well as ionic strength of the solution phase. These studies are envisagedto provide information to facilitate a comprehensive description of metal accumulation by fungal mycelia. A common soil fungus, Trichoderma harzianum, was selected as a suitable system. It has previouslybeen shown that this fungus can accumulate considerable amounts of cadmium, mercury and zinc (13). The pH dependence of the accumulation differs between a starved mycelium and a nonstarved one. The adsorption of metals at low concentrations, relevant for natural systems, is largely independent of pH in the pH range 3-9 for the starved system, while an increased accumulation of zinc is observed at low pH (3-5) for the nonstarved system. A fungal production and release of a compound in a certain pH range in the nonstarved system has been suggested. Thus, a considerable H+capacity must be expected, as well as the occurrence of different functional groups and substantial differences between starved and nonstarved fungi. Potentiometric titrations would constitute a suitable method for characterizing such a system which would be expected to behave as a polyelectrolyte. Theoretical Background The ionicstrength of the solution phase has a significant effect on the potentiometric titration response of a polyelectrolyte. This has been employed to gain insight into the configurationlconformation of polyelectrolyte molecules in solution (14). Plots of apparent dissociation constants pKappversus the degree of neutralization (a) have essentially zero slope and are insensitive to ionic strength changes for a monomeric acid. However, if the molecule exists as a linear polymeric assembly, there will be a considerable effect of the ionic strength on the slope of such PKapp versus a plots. The value of pKappwilldeviate most at highest a,with the deviation being largest at the lowest ionic strength. As the value of a is decreased, the ordinate value (pKapp)is also lowered, the difference between the experimental points at the different ionic strengths becoming less and less until they coincide on the ordinate axis where a equals zero. This behavior is exaggerated in gels where pKappversus a curves are essentially parallel, the uppermost curve corresponding to the lowest ionic strength (for a negatively charged polymericsurface). The difference between PKapp for two ionic strengths is proportional to the concentration of the ratio of the separate ionic strength at any given a (15). Such observationshave been described and explained using the Gibbs-Donnan theory (15). Application of this concept has shown that the linear analogs of cross-linked weak acid polyelectrolyte gels exhibit a counterionconcentrating region next to their surface that simulates the separate phase properties of the gels (16). In a typical polymeric solution at equilibrium, the electrochemicalpotential for each diffusible species in and 0013-936X/94/092S-1502$04.50/0
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out of the polymer subphase may be equated. By assigning the same standard state to the components in the two phases and assuming that the osmotic pressure term is small enough to neglect, the following important relationship between the activity ratios of the exchangeable counterions, e.g., K+ and H+,in the two phases is obtained (17): PH, - PK, = PH,
- PKg
In the above equation, the subscripts s and g represent the solution and gel phases, respectively. The terms pK, and pKg in eq 1 denote the respective negative logarithm of the potassium ion concentration in the solution and gel phases. A critical observation of the equation shows that if the pH, - pK, term in the solution is fixed so is the pH, - pKg term in the gel. The development of eq 1has been possible by envisaging a situation where components diffuse into the polymer domain. In such instance, a plot of the parameter, A- versus pH - pK for the various ionic strength must yield a unique curve since pH, - pKg is invariant during the course of the titration. Here A- is defined by
where m is the molarity and V is the volume of the base (b) and acid (a), respectively. In instances where components diffuse into a gel, a curve of A- versus pH would yield parallel lines separated by the concentration ratios of the various ionic strengths. A plot of A- should be a unique function of pH at different ionic strengths for systems where transport of the components is resisted.
Materials and Methods Growth and Preparation of Fungal Mycelium. The fungus used, Trichodermaharzianum, was obtained from the Department of Microbiology,University of Agriculture, Uppsala, Sweden (strain 565). The stock culture was stored on malt extract agar slants at 4 "C and transferred to fresh agar slants at 3-month intervals. The fungus was grown in a glucose mineral salts medium with the following composition (per liter): magnesium glycerophosphate (C3H7MgOsP.2H20),0.5 g; (NH&S04, 2.0 g; KC1, 0.5 g; CaCl2.6H20, 0.25 g; 2-(N-morpholino)ethanesulfonic acid (MES buffer), 9.76 g; glucose, 20.0 g. A trace solution (0.1mL/L) (FeSOc7HzO,5g; ZnSOe7Hz0, 1.75g; MnSOcH20,0.076 g; CuSOc5H20,O.l g) was added. The pH was adjusted to 5.5 using KOH or HC1. A suspension of spores was made after 14 days of growth of the fungus on 4 % MEA (malt extract agar). The medium was inoculated with the spore suspension (1mL/100 mL) witha concentration of 4 X 106spores/mL. The inoculated media were incubated at 25 "C under continuous shaking for 72 h (corresponding to exponential phase of growth) or 96 h (corresponding to stationary phase of growth). The mycelia were harvested by centrifugation after the various times of incubation and washed three times in sterile KC1 of appropriate ionic strength (0,001, 0.01, or 0.1 M). Experiments were made on mycelium directly after the washing procedure and on mycelium starved for 2 days. The starved mycelium was washed as described above and then resuspended in 0.1 M KCl and incubated in 25 "C under continuous shaking. The mycelium was harvested by centrifugation after 2 days of incubation
Table 1. Extraction Procedures for Isolation of Fungal Metabolites extraction with CHzClz from solution desalting and extraction with di"ethy1ether from solution adsorption on activated carbon; leaching of adsorbent with CHzClz adsorption on active carbon; leaching of adsorbent with hexane adsorption on Al2O3; leaching of adsorbent with CH2C12 adsorption on AlzOs; leaching of adsorbent with hexane
without nutrients and washed twice in sterile KC1. The washed mycelium was employed in the acid-base titrations. Potentiometric Titrations. Potentiometric titrations were performed on starved and nonstarved mycelia from Trichodermaharzianum at three different ionic strengths (0.001,0.010, and 0.100 M KC1). About 50-100 mg (dry weight) of mycelium was suspended in the desiredmedium (0.001 or 0.1 M KCI) to a final volume of 50 mL. The dry weight was estimated after each titration. The pH of the suspension was adjusted to about 3.5 with HC1. A stream of NOwas bubbled through the solution to remove C02HC03 from the system. Aliquots of 0.05 mL of 0.1000 M KOH were carefully added, and pH was measured 10 min after each addition using a PHM 85 precision pH meter (Radiometer, Copenhagen) equipped with a combination glass electrode (GK2401C). The ionic strength was adjusted by the addition of an appropriate quantity of 1.00 M KCl to obtain a final concentration of 0.01 M KC1 after the pH had reached 9.5 in the system. The pH of the suspension was again lowered to 3.5 by the addition of 0.100 M HC1, and after an equilibration time of approximately 10 min, the titration was repeated (in 0.1 M KC1) as described above. In another set of experiments,titrations were performed on the solution after the removal of the mycelium to investigate the possible release of soluble compounds from the mycelium into the 0.1 M KCl solution. After a titration in 0.1 M KC1, the mycelium was harvested by centrifugation. The supernatant solution was filtered through a 0.8pm filter (Millipore, AAWP) to ensure that no mycelium remained in the solution. The filtered solution was titrated as described above. Characterization of Metabolites. The molecular weights of the compounds produced by fungi and released into the solution phase were studied by gel filtration chromatography performed on HPLC equipment (Waters) using a TSK2000SW column (7.5 X 300 mm) (18). The mobile phase was a 50 mM phosphate buffer at pH 6.8 with a flow rate of 0.5 mL/min. Polystyrene sulfonates of known molecularweight (MW 1600,4000,6500,and 16000) and phenylalanine (MW 169) were used as reference substances. UV detection was performed at 254 nm. A set of extractions and leachings were performed in order to isolate and further characterize the compounds released into solution by nonstarved mycelium of the fungus (Table 1). The mycelium was harvested by centrifugation (15000g; 10 min) after starvation overnight in 0.1 M KC1, and the supernatant was filtered through a 0.8-pm filter (Millipore, AAWP) to make sure that no hypha were left in the solution. The solution with metabolites produced by fungi were hydrolyzed in concentrated HC1, evacuated, sealed, and incubated at 105 "C overnight. The solution was evaporated, and the sample was resuspended in a small volume (1mL) of water and separated by two-dimensional thinlayer chromatography (TLC plate; cellulose) for the determination of amino acids. The mobile phases utilized were as follows: phase 1 (acidic): 2-propanol, butanone, Envlron. Sci. Technol., Vol. 28, No. 8, 1994
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2.5
2.5
2.0 1.5
2-o 1.5
1.0
p
0.5
v
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I
1.0 0.5
4
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-1.0 -1.5
-1.5
-2.0
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-2.0 2
3
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6 7 PH
8
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Figure 1. Charge balance (A-) of starved and nonstarved mycelia of T. harzianum as a function of pH at various ionic strength (KCI). Symbols: (0) 0.100 M, nonstarved; (0)0.010 M, nonstarved; (0) 0.001 M, nonstarved; (A)0.01 M, starved; (0) 0.001 M, starved.
and 1M HC1(12:3:5); phase 2 (basic): tert-amyl alcohol, butanone, acetone, methanol, water, and ammonia (25%) (10:4:2:1:3:1). The amino acids were developed by spraying the TLC plate with a ninhydrin solution.
Results and Discussion Potentiometric Titrations. Some examples of potentiometric titration curves for mycelium suspensions are given in Figure 1. There were no differences (in terms of acid capacity) between mycelia harvested in exponential and in stationary phases of growth. The titration curves of both starved and nonstarved mycelia resembled, in principle, those of heterogeneous weak acid moieties, i.e., titration of a weak acid against a strong base (19). An additional significant buffering was observed for the nonstarved mycelium. The acid capacity obtained for the nonstarved system was comparable to those determined (atthe same laboratory) for aquatic humic substances (20). The buffering capacity of the nonstarved system has been attributed to the presence of acidic moieties produced by the nonstarved mycelia as a result of possible metabolic activity. In the titrations of the starved fungal mycelium (starved in 0.1 M KC1 for 2 days), it was observed that the acid employed to lower the initial pH of the system to -3.5 was not quantitatively retrievable. This can be explained by assuming that the fungal mycelium surface when starved, sequesters protons (H+), Le., the surface is essentially basic in character. The potentiometric titrations of nonstarved fungal mycelium resulted in total acid capacities in the range of 450-790 mequiv/kg fungal mycelium (dry weight). These acid capacities represent the sum of three equivalence points observed around pH 4, 6, and 8. Adaptation of Gibbs-Donnan Approach To Describe Dissociation Properties. An adaptation of the unified physicochemical approach (21)was made. Plots of the charge balance term, A- (which is essentially the difference between base added and acid added), versus pH or versus the difference between pH and p(sa1t concentration), i.e., pH - pK (whereK denotes potassium) have been made as a function of ionic strength. 1504
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0
1
2
3
4 5 6 pH-pK+
7
8
9
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Figure 2. Charge balance (A-) of nonstarved mycelia of T. harzianum versus the difference between pH and p(salt concentration), pH pK 0.100 M; (0)0.010M; (0) 0.001 M. Symbols: (0)
-
A plot of A- versus pH illustrates the strong influence of ionic strength on the nonstarved mycelia system (Figure 1). However, a replot of the charge balance, A-, versus pH - pK yielded an essentially unique curve (Figure 2), indicating that the nonstarved mycelium allowed the transport of components into its cell wall structure and or its intracellular space (15). Plots of A- versus pH of the external solution (Figure 1)showed an insensitivity to ionic strength changes in the starved system, suggesting that the diffusion of components into and from the mycelium domain was resisted. The differences in the effects of bulk electrolyte concentration on the dissociation properties of the starved and nonstarved systems may be related to the different degrees of metabolic activities. A complete understanding is yet to be found. Determination of Point of Zero Charge. The point of zero charge, pzc, has provisionally been defined in this paper to represent the pH at which the charge balance, A-, expressed by eq 2, has a value of zero. For the nonstarved fungal mycelium, the shape of A- versus pH made the estimation of pzc impossible (Figure 1). The absence of any distinguishable pzc has been attributed to the production of metabolites in the nonstarved system. For the starved fungal mycelium, the pzc was determined to be in the pH range 8.75-9.0 (Figure 1). Characteristics of Metabolites. Titrations on the solution after the removal of nonstarved fungal mycelia from the potassium chloride solution resulted in a total acid capacity of 110-140 mequiv. The estimated acid capacity was the total sum of two equivalence points at pH around 5 and 8. Extraction of the metabolites into an organic solvent was not successful with the present choice of extractants (Table 1) due to their hydrophilic nature. The analysis performed by thin layer chromatography revealed the presence of all of the 20 common protein-forming amino acids. This strongly indicates that the compounds are polypeptides. The molecular weight distribution revealed that the mixture consisted of at least four different UV-absorbing compounds (Figure 3). Furthermore, the molecularweight determinations indicated that the possible polypeptides were of intermediate molecular size. The reference
amounts of nutrients, one should consider both the presence of exudates and the possibility of the metal ion diffusing into the domain of the mycelia. Studies with such objectives are in progress. Acknowledgments
We are grateful to Dr J. Schniirer, Department of Microbiology,Swedish Agricultural University, Uppsala, Sweden for contributing the fungal strain. This work has been financially supported by the SwedishNatural Science Research Council and the Swedish Environmental Protection Agency. a
Time
b
Gelflltratlon chromatogramof the fungalmetabolites. Intensity (absorbance 254 nm) versus retention time. Retention time for polystyrene sulfonates (MW 6500) (a)and phenyl alanln (MW 169) (b) are indlcated. Flgure 3.
compounds (polystyrene sulfonates)used as standards are not the most suitable ones for studying polypeptides. However, it could be concluded that the weight of the molecules was below 5000.
Conclusions The results of the dissociation properties of starved and nonstarved fungal mycelia have demonstrated significant differences. Whereas the results for starved mycelium indicated the presence of a basic surface (illustrated by the sequestering of protons), those for the nonstarved mycelium exhibited the presence of a spectrum of heterogeneous acidic moieties. Mycelia harvested in exponential or stationary phase showed negligible differences and, even though there could be differences in the cell wall composition during the growth of the fungi, these differences did not influence the results of the potentiometric titrations. The fact that two equivalence points were observed during the titration of the supernatant solution resulting from the filtration of the nonstarved mycelium instead of the three observed in the nonstarved mycelium suspension suggests that the mycelium surface accounts for one acid while the additional two acids are attributable to metabolites produced by the nonstarved mycelium. Since these metabolites were produced by the fungus after the nutrients had been removed, they may either be related to reserve metabolism or due to nutritional stress of the fungus. Most of the microorganisms in a natural environment, e.g., soil, are under stress due to nutrient limitations. If fungi produce this compounds with significant acid capacities in soil, they would influence the mobility of trace metals both through the formation of surface complexes and by the release of complexing metabolites into the local microenvironment. Further studies to characterize the compound(s) and metal complexing properties are presently in progress. The novelty of this study is the application of a relatively simple experimental procedure, i.e., potentiometric titrations, to obtain significant information that may facilitate a comprehensive description of the chemical properties of fungi. For example, in describing the metal accumulation of a fungal mycelium under situations of insignificant
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