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Mineralization of organic compounds at low concentrations by filamentous fungi
793
Mycol. Res. 94 (6):793-798 (1990) Printed in Great Britain
Mineralization of organic compounds at low concentrations by filamentous fungi
K. M. SCOW, D. LI, V. B. MANILAL A N D M. ALEXANDER Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York 14853, U.S.A.
Mineralization of organic compounds at low concentrations by filamentous hngi. Mycological Research 94 ( 6 ) :793-798 (1990). The mineralization of low concentrations of phenol and glucose by Penicillium sp., Fusarium oxysporum, and Rhiwcfonia sp. was measured in liquid culture. At concentrations of 1.0 to 500 ng per ml, the rate constant of mineralization of phenol by Penicillium sp. decreased with a lowering of the initial substrate concentration. The lower the inoculum density, the lower was the maximum rate of mineralization of 3 pg of phenol ml-l and the longer the acclimation period. The curves of mineralization of phenol by Penicilliurn sp. and of low concentrations of glucose by F. oxysporum and Rhiwctonia sp. were usually fitted best by the logistic and not the firstorder model of the Monod family of kinetic models. The logistic model also provided the best fit to data depicting mineralization of phenol by Penicillium sp. inoculated into sterile sand. The parameters estimated by the logistic model, however, were not always consistent with the measurements. Thus, this form of the logistic model derived from the Monod equation, originally developed for bacteria, may not be appropriate for describing the biodegradation of organic compounds by filamentous fungi. Key words: Biodegradation, Kinetics, Phenol, Penicillium, Fusarium oxysporum, Rhiwctonia.
Fungi metabolize a wide variety of organic compounds, both natural and xenobiotic. Many species of fungi degrade phenolics (Henderson, 1956), petroleum constituents (Fedorak ef al., 1984), polyaromatic hydrocarbons (Bumpus, 1989) and other aromatic compounds (Cemigila, 1981). Usually, studies of biodegradation by fungi are conducted with pure cultures incubated with high concentrations of test compounds. Pollutants in natural waters and soils often are present at low concentrations and, as with bacteria, extrapolation of results from studies of the biodegradation of high substrate concentrations may be inappropriate for the low levels in soils and surface waters (Alexander, 1985). Many fungi are adapted to grow or survive when the level of organic nutrients is low or when under starvation conditions (Hawker, 1957; Tribe & Mabadeje, 1972), and they exploit many niches in soils (Cooke & Rayner, 1984)' which are usually deficient in readily available carbon (Lockwood & Filonow, 1981). The kinetics of fungal growth are strongly influenced by the conditions of growth and vary with the species. Cubic and logarithmic kinetics are considered to describe the growth of many fungi in liquid culture (Prosser, 1982). The rate of increase in hyphal length and in the radii of colonies growing on the surface of solid media is linear (Trinci, 1971), but it is likely that the kinetics would be different in a three-dimensional porous matrix such as soil. When subshate concentrations are too low to support growth of bacteria, first-order kinetics, in which there is no increase in the enzyme concentration and the rate of substrate disappearance is a function of the concentration remaining, usually describe the kinetics of
biodegradation (Simkins & Alexander, 1984). If all the organic substrate is available for metabolism, it is reasonable to assume that first-order theory should be applicable to fungi as well as bacteria at substrate concentrations too low to support growth. The purpose of this study was to test whether kinetic models developed for bacteria also describe biodegradation of low concentrations ( <3 wg ml-l) of simple organic compounds by several species of fungi in sterile buffer, sand and soil.
MATERIALS A N D METHODS Media The phthalate buffer-salts solution contained (1-' of deionized, distilled water) 2.0 g potassium hydrogen phthalate, 200 mg NaOH, 200 mg KH,PO,, 40 mg NH4C1, 10 mg MgSO, . 7 H,O, 10 mg CaCI, - 2 H,O, 0.5 mg FeC1, - 6 H,O and The pH was adjusted to 4.7 with 0.5 mg MnSO;H,O. NaOH. The phosphate buffer-salts solution consisted of (I-' of deionized, distilled water) 125 mg KH,PO,, 60 mg Na,HPO,, 40 mg NH,Cl, 10 mg MgSO, .7H,O, 10 mg CaCl, .2 H,O, 0.5 mg FeCI, - 6 H,O and 0.5 mg MnSO, .H,O. The final pH was 6-2. All glassware was soaked overnight in concentrated sulphuric acid, followed by three rinses in doubly distilled water. These solutions were supplemented with a labelled or an unlabelled organic substrate or both. [U-'4C]Phenol (87 mCi mmol-') was obtained from
Mineralization of organic compounds Amersham Corp., Arlington Heights, Ill., and [U-14C]glucose moist material for 7 d at 29', and then streaking subsamples (348 mCi mmol-') was purchased from New England Nuclear on plates containing 1.5 % Bacto-agar and 0.3% Trypticase Corp., Boston, Mass. Reagent-grade phenol and glucose were soy broth (Beckton Dickinson and Co., Cockeysville, Md). purchased from Mallinckrodt Inc., Paris, Ky. Measurement of mineralization Fungi
Penicilliurn sp. was isolated from Mardin silt loam that had been amended with 50 pg phenol, 1.0 mg streptomycin and 25 pg erythromycin per g and incubated at 23 OC. After 7 d, a small amount of soil was added to 100 ml phosphate bufferl and the salts solution amended with 50 pg ~ h e n o ml-', suspension was incubated in 250 ml Erlenmeyer flasks without shaking for 7 d at 23'. Dilutions were ~ l a t e don a medium containing (I-') phthalate buffer-salts, 50 mg phenol, 1 g streptomycin, 25 mg erythromycin and 15 g Bacto agar. The culture of Penicillium sp. was purified and shown to grow with phenol but not phthalate as C source. Fusarium oxysporum was a stock culture, and Rhizoctonia sp. 84-128 was obtained from Dr R. P. Korf (Dept of Plant Pathology, Cornell University). F. oxysporum and Rhizoctonia sp. use glucose but not phthalate as a C source. Inocula of Penicillium sp., F. oxysporurn and Rhizoctonia sp. were prepared by growing the fungi for 72 h at 29f 2' in unshaken 250 ml Erlenmeyer flasks containing 100 ml phthalate buffer-salts solution supplemented with 50 pg phenol or glucose ml-'. Penicillium sp. was collected by centrifugation, and the hyphae were washed with buffer and centrifuged three times. F. oxysporum and Rhizoctonia sp. were separated from spent media by passage through 0.2 pm polycarbonate filters (Nuclepore Corp., Pleasanton, Calif.) and the mycelia were washed three times with sterile buffer. The hyphae were then suspended in 5 ml buffer and ground in a 15 ml sterile Broeck tissue grinder (Fisher, Rochester, N.Y.) for 2-3 min to give a uniform suspension. This inoculurn was then diluted and dispensed in 250 ml biometer flasks (Bellco Glass, Inc., Vineland, N.J.) (Bartha & Pramer, 1965). A portion of the inoculum was collected on a 0.2 pm Nuclepore filter, dried for 3 h at 90' and weighed. Inoculum densities were calculated based on the measured yield of biomass of Penicillium sp. grown in solutions containing 50 pg phenol ml-l, so that experiments could be performed'to test conditions of both non-growth and growth. In an experiment conducted with unfragmented mycelia, Penicillium sp. and F. oxysporum were grown in phthalate buffer-salts solution containing 50 pg phenol or glucose ml-', respectively. At 72 h, two large clumps of hyphae of each species were removed with a sterile loop, gently washed in three rinses of sterile buffer and then placed in 50 ml phthalate buffer-salts solution containing 10 ng radiolabelled phenol or glucose ml-'. Inoculated sand and soil
Sand was combusted for 12 h at 500' to remove organic carbon, washed successively in tap water and double distilled water and then autoclaved moist for 2 h. Mardin silt loam was sterilized with 2.5 Mrad of gamma-irradiation. The sterility of the sand and soil was confirmed by incubating portions of the
The main compartment of the biometer flasks contained 50 ml of phthalate buffer-salts solution or 50 g sterile sand or soil amended with phenol or glucose. Substrate was added in enough distilled water to bring the soil to a moisture level of 70% of $bar (equivalent to 24.5 % moisture w/w) or the sand to 25% moisture. The substrate was thoroughly mixed into the soil with a spatula and each flask was immediately sealed with two silicon stoppers, one of which was fitted with a stainless-steel cannula for removal of NaOH. The incubation temperature was 23'f 2'. All flasks received similar amounts of 14C-labelled compounds (40000-150000 dpm per flask), but the final substrate concentration was varied by adding different amounts of unlabelled compounds. The concentrations of substrate in sand and soil are expressed in terms of the levels in the added buffer solution. Duplicate flasks were used for each treatment. Evolved 14C0, was trapped in 2-5 ml 0.5 N-NaOH contained in the sidearm of the biometer flask. At certain time intervals, all the NaOH was removed and replaced with fresh base. The NaOH that was removed was placed in 7 ml scintillation mini-vials (Kimble, Toledo, Ohio) containing 3.5 ml liquiscint scintillation cocktail (National Diagnostics, Inc., Somerville, N.J.), and the radioactivity was counted with a liquid scintillation counter (model LS 7500, Beckman Instruments, Inc., I ~ i n eCalif.). , The values thus obtained were corrected for background radioactivity in the NaOH. Data analysis
Non-linear-regression analyses were performed on all curves of substrate disappearance. The Monod family of models, shown in Table I, was adapted for non-linear regression analysis in the MARQFIT program (Simkins & Alexander,
Table I. Models for mineralization kinetics derived from the Monod equation Model
Equation*
Zero order
dS -a= kl
Michaelis-Menten
-$
=k,s/(~.+n
First order
dS
-a = k,S
Logistic
-$ = k.S(S~,+X,,-n
Monod with growth
-3 = [urn,,S(S,,+ X,,- 31/K,
Logarithmic
-dS -
dS
+S
a - UCL,,,(S" + X"- 3
' The parameter S is substrate concentration; k,, k, and k, are rate constants for mineralization; K, is the half-saturation constant; S,, is initial is initial population density; p,. is the maximum substrate concentration; X,, specific growth rate.
K. M. Scow, D. Li, V. B. Manila1 and M. Alexander 1984). This program fits data by minimizing the least squares of the differences between the data and the model curve using the Marquardt method (Bard, 1974). Fits of the non-linearregression models were compared using an F-test (Snedecor & Cochran, 1967) to determine the model of best fit at the 95 % confidence level or higher (P < 0.05) (Robinson, 1985). Sensitivity analyses were conducted for several concentrations of substrates with respect to each initial parameter estimate to make sure that the selection of poor initial estimates did not lead to convergence of the models with an incorrect set of parameter estimates. The maximum rate of mineralization for each curve was also independently determined from a plot of the actual data representing the rate of mineralization.
RESULTS Penicillium sp. ( 0 . 5 4 p g ~ . w .ml-') was incubated in phthalate-salts solution containing 500, 250, 125, 75 and 1.0 ng phenol ml-l. With the exception of the lowest concentration, the shapes of the curves depicting mineralization were concave-down after an initial phase of acceleration (Fig. I). The rate of mineralization of 1.0 ng phenol ml-' was almost linear after about 120 h. The maximum rate of mineralization decreased on a percentage basis as the initial phenol concentration decreased. The maximum rate on a concentration basis was 1.8, 0.74, 0.24, 0.12 and 0.000 82 ng ml-' h-' at 500, 250, 125, 75 and 1.0 ng ml-', respectively. Approximately 60 % of the substrate C had been mineralized at 750 h at all phenol concentrations except the lowest, at which concentration the mineralization curve did not reach an asymptote even after 750 h (data not shown).
Fig. 1. Mineralization of different concentrations of phenol b y Penicillium sp. The initial concentrations of phenol were 1.0 (a), 75 125 (A),250 (0) and 500 (0) ng ml-l. Curves represent averages of duplicate flasks.
(v),
The information in Fig. 1has been cited in a recent review of the kinetics of biodegradation (Alexander & Scow, 1989). At an initial concentration of 3.0 pg phenol ml-' and inoculum densities of 11, 1.4, 0.14 and 0.014 pg ml-', the duration of the acclimation period preceding the onset of detectable mineralization by Penicillium sp. increased with every decrease in inoculum density (Fig. 2). In medium receiving 0.0014 ug mycelia ml-' the acclimation phase was only slightly longer than that in medium receiving 0.014 pg ml-'. The maximum rate of mineralization declined with each decrease in inoculum size; the values were 53, 36, 19, 15 and 8.0 ng ml-' h-' for 11, 1.4, 0.14, 0.014 and 0.0014 pg biomass ml-', respectively. A greater number of clumps of hyphae were visible in the flasks receiving higher inoculum densities. Furthermore, the lower the inoculum density, the greater was the size of the individual clumps. These differences in size were not quantified. The rates of mineralization of 10 and 500 ng glucose ml-' by Rhizocfonia sp. and of 500 ng ml-' by F. oxysporum increased after a short period of acceleration (Fig. 3). The subsequent rate declined during the test period. At 10-1000 ug ml-l, the rates were higher on a percentage basis, the percentage of glucose mineralized was greater and the mineralization curves were sigmoidal (data not shown). Of the models of the Monod family, the logistic model provided the best fit (P < 0.05) to curves depicting the mineralization of phenol and glucose by the three fungi. The initial periods of acceleration in the rates of mineralization evident in each curve did not permit good fits by the firstorder model. The values of the parameters estimated by the logistic model for the activity of Penicillium sp. on five concentrations of phenol and for F. oxysporum and Rhizocfonia sp. are given in Table 2. In the study of Penicillium sp., in which the same inoculum size was added to all flasks, the estimate of the initial population density (X,), averaged for the duplicate flasks, was 17, 11, 9.0, 4.9 and 0.095 ng ml-' for initial phenol concentrations of 500, 250, 125, 75 and 1.0 ng ml-', respectively. The estimate of Xo increased substantially as the initial substrate concentration increased from 1.0 to 75 ng phenol ml-' for Penicillium sp. and from 10 to 500 ng glucose ml-' for F. oxysporum. Slight increases in values of X , estimated for Penicillium sp. at the higher Fig. 2. Mineralization of 3.0 ug phenol ml-' by 0.0014 (A),0.014 (01,0-14 (1) 1.4 .(0) and 11 ( 0 )pg dry wt of Penicilliurn sp. ml-'. Cunres represent averages of duplicate flasks.
Mineralization of organic compounds
796
Fig. 3, Mineralization of 500 ng glucose ml-l by F. oxysporum
(0)Fig.
and 500 (a) and 10 (0) ng glucose ml-' by Rhiwcfonia sp. Curves represent averages of duplicate flasks.
Time (h)
Time (h)
Table 2. Parameter estimates from fits of logistic model to curves depicting mineralization of phenol and glucose
Substrate -
Initial concn (ng ml-l)
K, (X
h)
5, (ng ml-')
-
Penicilli~lmsp.
Phenol
1.0' 75 125 250 500
F. oxysporum
Glucose
500
Rhiwcfonia sp.
Glucose
10 500
4. Mineralization of 10 ng phenol and glucose ml-' by unfragmented hyphae of Penicillium sp. (top) and F. oxysporum (bottom). respectively.
1.80 0.57 1.43 1.43 1.35 1.66 2.16 2.23 3.52 2.27 3.39 2.47 3.94 3.77 3.77 3.95
0.036 0.154 5.41 4.43 11.9 6.31 11.9 975 5.60 2 87 158 180 0.568 0.655 36.5 44.7
The two sets of values are results from duplicate flasks.
concentrations of phenol were evident; however, in some cases there was not good agreement between the estimated Xo values for duplicate flasks containing the same concentration of phenol. Since the actual inoculum of Penicillium sp. contained 0.54 pg biomass ml-', the model predicted that only 0.9-3.1% of the added inoculum was active in mineralizing phenol in media containing 75-500 ng phenol ml-', and only 0.02% of the inoculum added to medium containing 1.0 ng ml-'. The logistic model also provided the best fit (data not shown) to the curves of mineralization of phenol by the different inoculum densities of Penicillium sp.
shown in Fig. 2, and the model estimates of initial population density were not always proportional to the amount of inoculum added. An experiment was conducted using hyphae of Penicillium sp. and F. oxysporum that were not fragmented in preparing the inoculum to test whether the initial period of acceleration observed at low concentrations of substrate resulted from growth of the fragments. The slow rate of mineralization before the active phase of biodegradation was almost abolished in media inoculated with unfragmented hyphae (Fig. 4). This initial phase was previously noted to be long when fragmented mycelia were used as inocula. The first-order model provided a significantly better fit (P < 0.05) to these data than did the logistic model or any other models of the Monod family. The existence of a linear relationship between a timedependent process and the square-root of time suggests that the rate of a reaction is controlled by diffusion (Crank, 1975; Hamaker, 1972; Weber & Gould, 1966).Transformation of the data in Fig. 4, as a function of the square root of time, yielded a long linear plot after an initial acceleration phase (Fig. 5). The transformed data for Penicillium sp. showed greater deviation from the straight line than those for F. oxysporum. Correlation coefficients (r2) were determined for linear regressions performed with the logarithmic transformation of the mineralization data, i.e. the first-order model, and with the untransformed data plotted as a function of the square root of time. The r2 values were 0963 and 0.928 for the logarithmic transformation and 0.992 and 0.973 for the transformation as a square root of time for F. oxysporum and Penicillium sp., respectively. To determine how a porous matrix affects the kinetics, the mineralization of 30 and 3000 ng phenol ml-' by Penicillium sp. (0.10 ~g ml-', D.w.) was measured following inoculation
K. M. Scow, D. Li, V. B. Manila1 and M. Alexander Fig. 5. Mineralization of 10 ng phenol and glucose ml-', respectively, Table 3. Parameter estimates from fits of logistic model to curves of by unfragmented hyphae of Penicillium sp. (0) and F. oxyspomm (0)mineralization of phenol in buffer and sand plotted as a Function of the square root of time. Curves represent Phenol concn k4 X0 averages of duplicate flasks. Environment (ng ml-l) (X h) (ng mlkl) Buffer
30" 3000
Sand
30 3000
3.89 5.29 17.43 16.77 13.60 14.99 7.77 10.87
The two sets of values are results from duplicate flasks.
0
2
4
6
Time (h) Fig. 6. Mineralization of 30 ( 0 )and 300 (m) ng ~henolmi-' by Penicillium sp. inoculated into sterile buffer, sand and soil. Curves
represent averages of duplicate flasks.
thereafter was rapid in sand and buffer (Fig. 6). The substrate was extensively degraded in both sand and buffer. The shapes of the curves of mineralization were similar to those observed in Fig. 1. Of the models of the Monod family, the logistic model provided the best fit to the data depicting mineralization of both concentrations of phenol in buffer and sand (Table 3). The rate of mineralization (k,) of 30 ng ml-' in solution was much lower than the rate at 3000 ng ml-'. On the other hand, the rate constant of mineralization of the lower concentration of phenol in sand was more than twice that in buffer, whereas the rate of mineralization of the higher concentration in sand was approximately half that in buffer. When Penicillium sp. was inoculated into sterile soil amended with 30 and 3000 ng phenol g soil-', a rapid phase of mineralization was evident after about 40 h. Thereafter, the rate of mineralization was slow. At 150 h, when the experiment was terminated, the extent of mineralization in soil was much lower than in sand or buffer. Penicillium sp. presumably grew on the organic carbon in the sterilized soil, as evidenced by the presence of fungal mycelia throughout the soil after 80 h at both concentrations of phenol. By 120 h, the surface of the soil was covered with hyphae bearing spores.
DISCUSSION
Time (h)
of sterile phthalate-salts buffer, sand and soil. Little of the substrate was mineralized in the first 40 h, but the rate
The data indicate that kinetic models formulated for bacteria may not provide meaningful fits to mineralization data for these fungi. The model that would be expected to fit at low substrate concentrations, the first-order model, only fitted data depicting mineralization by hyphae derived from inocula that had not been fragmented. As the initial substrate concentration decreased, the rate of mineralization declined by a greater amount than that which would be predicted by the assumption that the rate is linearly related to substrate concentration. The model of the Monod family that usually fitted best the curves of mineralization in liquid and sand cultures inoculated with suspensions of hyphae was the logistic equation, which, as derived, should apply only to growth conditions at substrate concentrations less than K, and at a low population density (Simkins & Alexander, 1984). The model fitted the curves regardless of initial substrate concentration or population density, however, and the estimates of initial population density were not always consistent with measured data. Under all conditions, there appeared to be growth of the
Mineralization of organic compounds fragmented hyphae, even when the substrate was present at a concentration too low t o support appreciable increases in biomass. Growth may have been supported by carbon leaking from damaged hyphae o r b y dissolved organic carbon present as contaminants in buffer (Schmidt & Alexander, 1985). Use of spore suspensions instead of fragmented hyphae as inocula may eliminate some growth C i the reserves of carbon contained in the spores are low. When the data for mineralization of phenol and glucose at 10 ng ml-' were plotted as a function of the square root of time, a straight-line relationship was evident, particularly for F. oxysporurn. Such a relationship suggests a diffusion limitation (Crank, 1975). Diffusion of substrates t o the insides of hyphal masses might b e substantially slower than in solutions containing suspensions of hyphal fragments or bacterial cells, but further studies are required t o determine whether the rate of mineralization of low concentrations of substrate by nongrowing fungi are indeed limited b y diffusion of the substrate t o the organisms. Differences were evident in the kinetics of mineralization of 30 n g phenol ml-' in sand and in liquid medium. In fact, the rate of mineralization was greater in sand than in liquid. This stimulation did not result- from growth of the fungus o n organic carbon associated with the sand because the sand had been combusted. In the sand, the fungus may have exploited a greater volume of space than in liquid because it could have become attached to the solid surfaces and extended its hyphae from the sand grains. Consequently, the degree of contact between the substrate and hyphae could have been greater in sand than in liquid culture, in which the hyphae formed dense clumps. Data o n the mineralization of test chemicals in sterile soil inoculated with individual species probably cannot b e used t o predict patterns of mineralization in natural soil because the high concentrations of carbon that were available t o the pure culture would not be available in the presence of other species. The percentage of phenol that had been mineralized was much lower in sterile soil than in sand o r liquid medium inoculated with Penicillium sp. The extent of mineralization of phenol may also b e low in non-sterile soil (Scow et a!., 1986). Kinetic models are frequently used t o describe biodegradation in soil and other environments without considering the identities o r characteristics of the microbial populations carrying out the transformation. The results of the present investigation indicate that it may not b e appropriate t o assume that kinetic models developed to describe biodegradation of organic chemicals b y bacteria will describe biodegradation b y fungi. Therefore, further study is needed of the kinetics of biodegradation of fungi in the environments in which these organisms are important in destroying synthetic compounds.
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Mycol. Res. 94 (6):793-798 (1990) Printed in Great Britain
Mineralization of organic compounds at low concentrations by filamentous fungi
K. M. SCOW, D. LI, V. B. MANILAL A N D M. ALEXANDER Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York 14853, U.S.A.
Mineralization of organic compounds at low concentrations by filamentous hngi. Mycological Research 94 ( 6 ) :793-798 (1990). The mineralization of low concentrations of phenol and glucose by Penicillium sp., Fusarium oxysporum, and Rhiwcfonia sp. was measured in liquid culture. At concentrations of 1.0 to 500 ng per ml, the rate constant of mineralization of phenol by Penicillium sp. decreased with a lowering of the initial substrate concentration. The lower the inoculum density, the lower was the maximum rate of mineralization of 3 pg of phenol ml-l and the longer the acclimation period. The curves of mineralization of phenol by Penicilliurn sp. and of low concentrations of glucose by F. oxysporum and Rhiwctonia sp. were usually fitted best by the logistic and not the firstorder model of the Monod family of kinetic models. The logistic model also provided the best fit to data depicting mineralization of phenol by Penicillium sp. inoculated into sterile sand. The parameters estimated by the logistic model, however, were not always consistent with the measurements. Thus, this form of the logistic model derived from the Monod equation, originally developed for bacteria, may not be appropriate for describing the biodegradation of organic compounds by filamentous fungi. Key words: Biodegradation, Kinetics, Phenol, Penicillium, Fusarium oxysporum, Rhiwctonia.
Fungi metabolize a wide variety of organic compounds, both natural and xenobiotic. Many species of fungi degrade phenolics (Henderson, 1956), petroleum constituents (Fedorak ef al., 1984), polyaromatic hydrocarbons (Bumpus, 1989) and other aromatic compounds (Cemigila, 1981). Usually, studies of biodegradation by fungi are conducted with pure cultures incubated with high concentrations of test compounds. Pollutants in natural waters and soils often are present at low concentrations and, as with bacteria, extrapolation of results from studies of the biodegradation of high substrate concentrations may be inappropriate for the low levels in soils and surface waters (Alexander, 1985). Many fungi are adapted to grow or survive when the level of organic nutrients is low or when under starvation conditions (Hawker, 1957; Tribe & Mabadeje, 1972), and they exploit many niches in soils (Cooke & Rayner, 1984)' which are usually deficient in readily available carbon (Lockwood & Filonow, 1981). The kinetics of fungal growth are strongly influenced by the conditions of growth and vary with the species. Cubic and logarithmic kinetics are considered to describe the growth of many fungi in liquid culture (Prosser, 1982). The rate of increase in hyphal length and in the radii of colonies growing on the surface of solid media is linear (Trinci, 1971), but it is likely that the kinetics would be different in a three-dimensional porous matrix such as soil. When subshate concentrations are too low to support growth of bacteria, first-order kinetics, in which there is no increase in the enzyme concentration and the rate of substrate disappearance is a function of the concentration remaining, usually describe the kinetics of
biodegradation (Simkins & Alexander, 1984). If all the organic substrate is available for metabolism, it is reasonable to assume that first-order theory should be applicable to fungi as well as bacteria at substrate concentrations too low to support growth. The purpose of this study was to test whether kinetic models developed for bacteria also describe biodegradation of low concentrations ( <3 wg ml-l) of simple organic compounds by several species of fungi in sterile buffer, sand and soil.
MATERIALS A N D METHODS Media The phthalate buffer-salts solution contained (1-' of deionized, distilled water) 2.0 g potassium hydrogen phthalate, 200 mg NaOH, 200 mg KH,PO,, 40 mg NH4C1, 10 mg MgSO, . 7 H,O, 10 mg CaCI, - 2 H,O, 0.5 mg FeC1, - 6 H,O and The pH was adjusted to 4.7 with 0.5 mg MnSO;H,O. NaOH. The phosphate buffer-salts solution consisted of (I-' of deionized, distilled water) 125 mg KH,PO,, 60 mg Na,HPO,, 40 mg NH,Cl, 10 mg MgSO, .7H,O, 10 mg CaCl, .2 H,O, 0.5 mg FeCI, - 6 H,O and 0.5 mg MnSO, .H,O. The final pH was 6-2. All glassware was soaked overnight in concentrated sulphuric acid, followed by three rinses in doubly distilled water. These solutions were supplemented with a labelled or an unlabelled organic substrate or both. [U-'4C]Phenol (87 mCi mmol-') was obtained from
Mineralization of organic compounds Amersham Corp., Arlington Heights, Ill., and [U-14C]glucose moist material for 7 d at 29', and then streaking subsamples (348 mCi mmol-') was purchased from New England Nuclear on plates containing 1.5 % Bacto-agar and 0.3% Trypticase Corp., Boston, Mass. Reagent-grade phenol and glucose were soy broth (Beckton Dickinson and Co., Cockeysville, Md). purchased from Mallinckrodt Inc., Paris, Ky. Measurement of mineralization Fungi
Penicilliurn sp. was isolated from Mardin silt loam that had been amended with 50 pg phenol, 1.0 mg streptomycin and 25 pg erythromycin per g and incubated at 23 OC. After 7 d, a small amount of soil was added to 100 ml phosphate bufferl and the salts solution amended with 50 pg ~ h e n o ml-', suspension was incubated in 250 ml Erlenmeyer flasks without shaking for 7 d at 23'. Dilutions were ~ l a t e don a medium containing (I-') phthalate buffer-salts, 50 mg phenol, 1 g streptomycin, 25 mg erythromycin and 15 g Bacto agar. The culture of Penicillium sp. was purified and shown to grow with phenol but not phthalate as C source. Fusarium oxysporum was a stock culture, and Rhizoctonia sp. 84-128 was obtained from Dr R. P. Korf (Dept of Plant Pathology, Cornell University). F. oxysporum and Rhizoctonia sp. use glucose but not phthalate as a C source. Inocula of Penicillium sp., F. oxysporurn and Rhizoctonia sp. were prepared by growing the fungi for 72 h at 29f 2' in unshaken 250 ml Erlenmeyer flasks containing 100 ml phthalate buffer-salts solution supplemented with 50 pg phenol or glucose ml-'. Penicillium sp. was collected by centrifugation, and the hyphae were washed with buffer and centrifuged three times. F. oxysporum and Rhizoctonia sp. were separated from spent media by passage through 0.2 pm polycarbonate filters (Nuclepore Corp., Pleasanton, Calif.) and the mycelia were washed three times with sterile buffer. The hyphae were then suspended in 5 ml buffer and ground in a 15 ml sterile Broeck tissue grinder (Fisher, Rochester, N.Y.) for 2-3 min to give a uniform suspension. This inoculurn was then diluted and dispensed in 250 ml biometer flasks (Bellco Glass, Inc., Vineland, N.J.) (Bartha & Pramer, 1965). A portion of the inoculum was collected on a 0.2 pm Nuclepore filter, dried for 3 h at 90' and weighed. Inoculum densities were calculated based on the measured yield of biomass of Penicillium sp. grown in solutions containing 50 pg phenol ml-l, so that experiments could be performed'to test conditions of both non-growth and growth. In an experiment conducted with unfragmented mycelia, Penicillium sp. and F. oxysporum were grown in phthalate buffer-salts solution containing 50 pg phenol or glucose ml-', respectively. At 72 h, two large clumps of hyphae of each species were removed with a sterile loop, gently washed in three rinses of sterile buffer and then placed in 50 ml phthalate buffer-salts solution containing 10 ng radiolabelled phenol or glucose ml-'. Inoculated sand and soil
Sand was combusted for 12 h at 500' to remove organic carbon, washed successively in tap water and double distilled water and then autoclaved moist for 2 h. Mardin silt loam was sterilized with 2.5 Mrad of gamma-irradiation. The sterility of the sand and soil was confirmed by incubating portions of the
The main compartment of the biometer flasks contained 50 ml of phthalate buffer-salts solution or 50 g sterile sand or soil amended with phenol or glucose. Substrate was added in enough distilled water to bring the soil to a moisture level of 70% of $bar (equivalent to 24.5 % moisture w/w) or the sand to 25% moisture. The substrate was thoroughly mixed into the soil with a spatula and each flask was immediately sealed with two silicon stoppers, one of which was fitted with a stainless-steel cannula for removal of NaOH. The incubation temperature was 23'f 2'. All flasks received similar amounts of 14C-labelled compounds (40000-150000 dpm per flask), but the final substrate concentration was varied by adding different amounts of unlabelled compounds. The concentrations of substrate in sand and soil are expressed in terms of the levels in the added buffer solution. Duplicate flasks were used for each treatment. Evolved 14C0, was trapped in 2-5 ml 0.5 N-NaOH contained in the sidearm of the biometer flask. At certain time intervals, all the NaOH was removed and replaced with fresh base. The NaOH that was removed was placed in 7 ml scintillation mini-vials (Kimble, Toledo, Ohio) containing 3.5 ml liquiscint scintillation cocktail (National Diagnostics, Inc., Somerville, N.J.), and the radioactivity was counted with a liquid scintillation counter (model LS 7500, Beckman Instruments, Inc., I ~ i n eCalif.). , The values thus obtained were corrected for background radioactivity in the NaOH. Data analysis
Non-linear-regression analyses were performed on all curves of substrate disappearance. The Monod family of models, shown in Table I, was adapted for non-linear regression analysis in the MARQFIT program (Simkins & Alexander,
Table I. Models for mineralization kinetics derived from the Monod equation Model
Equation*
Zero order
dS -a= kl
Michaelis-Menten
-$
=k,s/(~.+n
First order
dS
-a = k,S
Logistic
-$ = k.S(S~,+X,,-n
Monod with growth
-3 = [urn,,S(S,,+ X,,- 31/K,
Logarithmic
-dS -
dS
+S
a - UCL,,,(S" + X"- 3
' The parameter S is substrate concentration; k,, k, and k, are rate constants for mineralization; K, is the half-saturation constant; S,, is initial is initial population density; p,. is the maximum substrate concentration; X,, specific growth rate.
K. M. Scow, D. Li, V. B. Manila1 and M. Alexander 1984). This program fits data by minimizing the least squares of the differences between the data and the model curve using the Marquardt method (Bard, 1974). Fits of the non-linearregression models were compared using an F-test (Snedecor & Cochran, 1967) to determine the model of best fit at the 95 % confidence level or higher (P < 0.05) (Robinson, 1985). Sensitivity analyses were conducted for several concentrations of substrates with respect to each initial parameter estimate to make sure that the selection of poor initial estimates did not lead to convergence of the models with an incorrect set of parameter estimates. The maximum rate of mineralization for each curve was also independently determined from a plot of the actual data representing the rate of mineralization.
RESULTS Penicillium sp. ( 0 . 5 4 p g ~ . w .ml-') was incubated in phthalate-salts solution containing 500, 250, 125, 75 and 1.0 ng phenol ml-l. With the exception of the lowest concentration, the shapes of the curves depicting mineralization were concave-down after an initial phase of acceleration (Fig. I). The rate of mineralization of 1.0 ng phenol ml-' was almost linear after about 120 h. The maximum rate of mineralization decreased on a percentage basis as the initial phenol concentration decreased. The maximum rate on a concentration basis was 1.8, 0.74, 0.24, 0.12 and 0.000 82 ng ml-' h-' at 500, 250, 125, 75 and 1.0 ng ml-', respectively. Approximately 60 % of the substrate C had been mineralized at 750 h at all phenol concentrations except the lowest, at which concentration the mineralization curve did not reach an asymptote even after 750 h (data not shown).
Fig. 1. Mineralization of different concentrations of phenol b y Penicillium sp. The initial concentrations of phenol were 1.0 (a), 75 125 (A),250 (0) and 500 (0) ng ml-l. Curves represent averages of duplicate flasks.
(v),
The information in Fig. 1has been cited in a recent review of the kinetics of biodegradation (Alexander & Scow, 1989). At an initial concentration of 3.0 pg phenol ml-' and inoculum densities of 11, 1.4, 0.14 and 0.014 pg ml-', the duration of the acclimation period preceding the onset of detectable mineralization by Penicillium sp. increased with every decrease in inoculum density (Fig. 2). In medium receiving 0.0014 ug mycelia ml-' the acclimation phase was only slightly longer than that in medium receiving 0.014 pg ml-'. The maximum rate of mineralization declined with each decrease in inoculum size; the values were 53, 36, 19, 15 and 8.0 ng ml-' h-' for 11, 1.4, 0.14, 0.014 and 0.0014 pg biomass ml-', respectively. A greater number of clumps of hyphae were visible in the flasks receiving higher inoculum densities. Furthermore, the lower the inoculum density, the greater was the size of the individual clumps. These differences in size were not quantified. The rates of mineralization of 10 and 500 ng glucose ml-' by Rhizocfonia sp. and of 500 ng ml-' by F. oxysporum increased after a short period of acceleration (Fig. 3). The subsequent rate declined during the test period. At 10-1000 ug ml-l, the rates were higher on a percentage basis, the percentage of glucose mineralized was greater and the mineralization curves were sigmoidal (data not shown). Of the models of the Monod family, the logistic model provided the best fit (P < 0.05) to curves depicting the mineralization of phenol and glucose by the three fungi. The initial periods of acceleration in the rates of mineralization evident in each curve did not permit good fits by the firstorder model. The values of the parameters estimated by the logistic model for the activity of Penicillium sp. on five concentrations of phenol and for F. oxysporum and Rhizocfonia sp. are given in Table 2. In the study of Penicillium sp., in which the same inoculum size was added to all flasks, the estimate of the initial population density (X,), averaged for the duplicate flasks, was 17, 11, 9.0, 4.9 and 0.095 ng ml-' for initial phenol concentrations of 500, 250, 125, 75 and 1.0 ng ml-', respectively. The estimate of Xo increased substantially as the initial substrate concentration increased from 1.0 to 75 ng phenol ml-' for Penicillium sp. and from 10 to 500 ng glucose ml-' for F. oxysporum. Slight increases in values of X , estimated for Penicillium sp. at the higher Fig. 2. Mineralization of 3.0 ug phenol ml-' by 0.0014 (A),0.014 (01,0-14 (1) 1.4 .(0) and 11 ( 0 )pg dry wt of Penicilliurn sp. ml-'. Cunres represent averages of duplicate flasks.
Mineralization of organic compounds
796
Fig. 3, Mineralization of 500 ng glucose ml-l by F. oxysporum
(0)Fig.
and 500 (a) and 10 (0) ng glucose ml-' by Rhiwcfonia sp. Curves represent averages of duplicate flasks.
Time (h)
Time (h)
Table 2. Parameter estimates from fits of logistic model to curves depicting mineralization of phenol and glucose
Substrate -
Initial concn (ng ml-l)
K, (X
h)
5, (ng ml-')
-
Penicilli~lmsp.
Phenol
1.0' 75 125 250 500
F. oxysporum
Glucose
500
Rhiwcfonia sp.
Glucose
10 500
4. Mineralization of 10 ng phenol and glucose ml-' by unfragmented hyphae of Penicillium sp. (top) and F. oxysporum (bottom). respectively.
1.80 0.57 1.43 1.43 1.35 1.66 2.16 2.23 3.52 2.27 3.39 2.47 3.94 3.77 3.77 3.95
0.036 0.154 5.41 4.43 11.9 6.31 11.9 975 5.60 2 87 158 180 0.568 0.655 36.5 44.7
The two sets of values are results from duplicate flasks.
concentrations of phenol were evident; however, in some cases there was not good agreement between the estimated Xo values for duplicate flasks containing the same concentration of phenol. Since the actual inoculum of Penicillium sp. contained 0.54 pg biomass ml-', the model predicted that only 0.9-3.1% of the added inoculum was active in mineralizing phenol in media containing 75-500 ng phenol ml-', and only 0.02% of the inoculum added to medium containing 1.0 ng ml-'. The logistic model also provided the best fit (data not shown) to the curves of mineralization of phenol by the different inoculum densities of Penicillium sp.
shown in Fig. 2, and the model estimates of initial population density were not always proportional to the amount of inoculum added. An experiment was conducted using hyphae of Penicillium sp. and F. oxysporum that were not fragmented in preparing the inoculum to test whether the initial period of acceleration observed at low concentrations of substrate resulted from growth of the fragments. The slow rate of mineralization before the active phase of biodegradation was almost abolished in media inoculated with unfragmented hyphae (Fig. 4). This initial phase was previously noted to be long when fragmented mycelia were used as inocula. The first-order model provided a significantly better fit (P < 0.05) to these data than did the logistic model or any other models of the Monod family. The existence of a linear relationship between a timedependent process and the square-root of time suggests that the rate of a reaction is controlled by diffusion (Crank, 1975; Hamaker, 1972; Weber & Gould, 1966).Transformation of the data in Fig. 4, as a function of the square root of time, yielded a long linear plot after an initial acceleration phase (Fig. 5). The transformed data for Penicillium sp. showed greater deviation from the straight line than those for F. oxysporum. Correlation coefficients (r2) were determined for linear regressions performed with the logarithmic transformation of the mineralization data, i.e. the first-order model, and with the untransformed data plotted as a function of the square root of time. The r2 values were 0963 and 0.928 for the logarithmic transformation and 0.992 and 0.973 for the transformation as a square root of time for F. oxysporum and Penicillium sp., respectively. To determine how a porous matrix affects the kinetics, the mineralization of 30 and 3000 ng phenol ml-' by Penicillium sp. (0.10 ~g ml-', D.w.) was measured following inoculation
K. M. Scow, D. Li, V. B. Manila1 and M. Alexander Fig. 5. Mineralization of 10 ng phenol and glucose ml-', respectively, Table 3. Parameter estimates from fits of logistic model to curves of by unfragmented hyphae of Penicillium sp. (0) and F. oxyspomm (0)mineralization of phenol in buffer and sand plotted as a Function of the square root of time. Curves represent Phenol concn k4 X0 averages of duplicate flasks. Environment (ng ml-l) (X h) (ng mlkl) Buffer
30" 3000
Sand
30 3000
3.89 5.29 17.43 16.77 13.60 14.99 7.77 10.87
The two sets of values are results from duplicate flasks.
0
2
4
6
Time (h) Fig. 6. Mineralization of 30 ( 0 )and 300 (m) ng ~henolmi-' by Penicillium sp. inoculated into sterile buffer, sand and soil. Curves
represent averages of duplicate flasks.
thereafter was rapid in sand and buffer (Fig. 6). The substrate was extensively degraded in both sand and buffer. The shapes of the curves of mineralization were similar to those observed in Fig. 1. Of the models of the Monod family, the logistic model provided the best fit to the data depicting mineralization of both concentrations of phenol in buffer and sand (Table 3). The rate of mineralization (k,) of 30 ng ml-' in solution was much lower than the rate at 3000 ng ml-'. On the other hand, the rate constant of mineralization of the lower concentration of phenol in sand was more than twice that in buffer, whereas the rate of mineralization of the higher concentration in sand was approximately half that in buffer. When Penicillium sp. was inoculated into sterile soil amended with 30 and 3000 ng phenol g soil-', a rapid phase of mineralization was evident after about 40 h. Thereafter, the rate of mineralization was slow. At 150 h, when the experiment was terminated, the extent of mineralization in soil was much lower than in sand or buffer. Penicillium sp. presumably grew on the organic carbon in the sterilized soil, as evidenced by the presence of fungal mycelia throughout the soil after 80 h at both concentrations of phenol. By 120 h, the surface of the soil was covered with hyphae bearing spores.
DISCUSSION
Time (h)
of sterile phthalate-salts buffer, sand and soil. Little of the substrate was mineralized in the first 40 h, but the rate
The data indicate that kinetic models formulated for bacteria may not provide meaningful fits to mineralization data for these fungi. The model that would be expected to fit at low substrate concentrations, the first-order model, only fitted data depicting mineralization by hyphae derived from inocula that had not been fragmented. As the initial substrate concentration decreased, the rate of mineralization declined by a greater amount than that which would be predicted by the assumption that the rate is linearly related to substrate concentration. The model of the Monod family that usually fitted best the curves of mineralization in liquid and sand cultures inoculated with suspensions of hyphae was the logistic equation, which, as derived, should apply only to growth conditions at substrate concentrations less than K, and at a low population density (Simkins & Alexander, 1984). The model fitted the curves regardless of initial substrate concentration or population density, however, and the estimates of initial population density were not always consistent with measured data. Under all conditions, there appeared to be growth of the
Mineralization of organic compounds fragmented hyphae, even when the substrate was present at a concentration too low t o support appreciable increases in biomass. Growth may have been supported by carbon leaking from damaged hyphae o r b y dissolved organic carbon present as contaminants in buffer (Schmidt & Alexander, 1985). Use of spore suspensions instead of fragmented hyphae as inocula may eliminate some growth C i the reserves of carbon contained in the spores are low. When the data for mineralization of phenol and glucose at 10 ng ml-' were plotted as a function of the square root of time, a straight-line relationship was evident, particularly for F. oxysporurn. Such a relationship suggests a diffusion limitation (Crank, 1975). Diffusion of substrates t o the insides of hyphal masses might b e substantially slower than in solutions containing suspensions of hyphal fragments or bacterial cells, but further studies are required t o determine whether the rate of mineralization of low concentrations of substrate by nongrowing fungi are indeed limited b y diffusion of the substrate t o the organisms. Differences were evident in the kinetics of mineralization of 30 n g phenol ml-' in sand and in liquid medium. In fact, the rate of mineralization was greater in sand than in liquid. This stimulation did not result- from growth of the fungus o n organic carbon associated with the sand because the sand had been combusted. In the sand, the fungus may have exploited a greater volume of space than in liquid because it could have become attached to the solid surfaces and extended its hyphae from the sand grains. Consequently, the degree of contact between the substrate and hyphae could have been greater in sand than in liquid culture, in which the hyphae formed dense clumps. Data o n the mineralization of test chemicals in sterile soil inoculated with individual species probably cannot b e used t o predict patterns of mineralization in natural soil because the high concentrations of carbon that were available t o the pure culture would not be available in the presence of other species. The percentage of phenol that had been mineralized was much lower in sterile soil than in sand o r liquid medium inoculated with Penicillium sp. The extent of mineralization of phenol may also b e low in non-sterile soil (Scow et a!., 1986). Kinetic models are frequently used t o describe biodegradation in soil and other environments without considering the identities o r characteristics of the microbial populations carrying out the transformation. The results of the present investigation indicate that it may not b e appropriate t o assume that kinetic models developed to describe biodegradation of organic chemicals b y bacteria will describe biodegradation b y fungi. Therefore, further study is needed of the kinetics of biodegradation of fungi in the environments in which these organisms are important in destroying synthetic compounds.
798
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