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A community-level concept of controls on decomposition processes: Decomposition of barley straw by Phanerochaete chrysosporium or Phlebia radiata in pure or mixed culture
Soil
003&0717CWOO164-2 . _
Liiol.Lfiochem. Vol. 27. No. 2, pp. 173179, 1995 Copyright 0 1995 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0038-0717/959.50+ 0.00
A COMMUNITY-LEVEL CONCEPT OF CONTROLS ON DECOMPOSITION PROCESSES: DECOMPOSITION OF BARLEY STRAW BY PHANEROCHAETE CHRYSOSPORIUM OR PHLEBIA RADIATA IN PURE OR MIXED CULTURE R. A. JANZEN,’ J. F. DORMAAR’ and W. B. MCGILL’* ‘Department of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada T6G 2E3 and 2Land Resources Science Section, Agriculture and Agri-Food Canada, Research Centre, Box 3000 Main, Lethbridge, Alberta, Canada Tl J 4Bl (Accepted I1 July 1994) Summary-We tested the hypothesis that interactions among populations in communities of decomposer microbes integrate physicochemical and population-level factors to control decomposition. To this end, we measured the relationship between inoculation with Phanerochaete chrysosporium and Phlebia radiata and the evolution of CO, from barley straw by decomposer communities. At 45” C, 80% as much CO, was evolved fr’om straw inoculated with P. chrysosporiumin pure culture as from mixed cultures containing P. chrysosporium or P. radiata with indigenous barley straw and compost microflora. P. radiata in pure culture evolvetrl less CO, than all other tested inocula. At 20”C, P. radiata evolved 3-fold more CO, in pure culture on barley straw over 42 days than did other tested inocula. Adding P. radiata to straw in co-culture with Trichoderma harzianum or indigenous barley straw microflora did not increase CO, evolution, and by inference decomposition rate, compared to microcosms without added P. radiata. We conclude that community-level controls on decomposition dynamics integrate physicochemical and population-level controls, and therefore might facilitate extension to the environment.
INTRODUCMON
Our concern in this study is control on decomposition, which we treat under two categories. First, as used here, physicochemical control includes factors such as temperature., pox status, water content, substrate composition or soil clay content; and second, population -level control pertains to temperature optimum, lignolytic capability, etc. We set out to test the hypothesis that community-level controls integrate physicochemical and population- or geneticlevel c6ntrols during interactions among populations within communities of decomposer microbes. This is not a new hypothesis; in articulating and testing it we aim to address explicitly a concept that has been addressed implicitly or indirectly in previous reports. Investigations of controls on the decomposition of lignocellulosic materials have emphasized either physicochemical or genetically-determined aspects of the decomposition process. For example, the dynamics of decomposition have been related to the chemical composition of substrates (Herman et al., 1977; Janzen et al., 1988; Neely et al., 1991), to the physical structure of lignocellulosic materials (Juma and McGill, 1986) or to the genetically-derived po*Author for correspondence.
tential of microorganisms to degrade lignocellulose (Agosin et al., 1985). Within this conceptual framework, regulation of lignolytic activity becomes a primary control on the rate of decomposition. Studies of white-rot fungi, Class Basidiomycetes, document that substrate composition, (Dill et al., 1987), temperature (Agosin et al., 1985) and the capabilities and requirements of populations (Valmaseda et al., 1991) control the rate of CO, evolution from straw by white-rot fungi in pure culture. This evidence supports the conclusion that physicochemical and population level factors control the function of basidiomycetes in pure culture. We propose, however, that implicit in the above studies is the concept that decomposition processes are also controlled at a hierarchical level higher than physicochemical and population-level controls. Studies of cereal straw decomposition have used mixed cultures also. Bowen (1990) measured the rate of CO2 evolution when unidentified basidiomycetes isolated from wheat straw decomposing in soil were inoculated onto wheat straw in pure cultures or with cellulolytic fungi. Halsall (1993) measured CO2 evolution and nitrogenase activity from co-cultures of Cyathus stercoreus, a lignolytic basidiomycete, with free-living N,-fixing bacteria (Beijerinckia indica B 15 or Azospiriflwn sp. DN64) and cellulolytic fungi growing on straw. Both Bowen (1990) and 173
R. A. Janzen et al.
174
Barley straw (Hordeum vuigare L. var. Galt; mature plant material collected from the field after harvest) was ground (< 2 mm) and 5.0 g (air dry) added to 250 ml conical flasks and autoclaved (twice for 15 min at 121” C). This sterile straw was then inoculated to produce microcosms in one of three ways: (i) with a 9-mm disc of fungal colony margin; (ii) with 0.1 g of air dry barley straw; or (iii) 0.1 g barley straw + 0.1 g compost, and moistened with 20ml of mineral medium. Each 1OOOml of medium contained 200 mg MgSO, .7H,O, 100 mg NaCl, 20 mg CaCl,, 2 mg Na,MoO, .2H,O, 10 mg MnSO,.H,O, 1OOOmg (NH4)2S04 and 328 mg FeNaEDTA. After sterilizing the basal salts component of the medium, separately sterilized solutions were added such that 1OOOml of the complete medium also contained 100 mmol KH,PO, (pH adjusted to 7.0 with NaOH), 5 mg biotin, 10 mg p-aminobenzoate, 10 mg thiamin, 2.5 g glucose and 2.5 g malic acid. Flasks were periodically weighed and water added to maintain water content. The flasks were covered with aluminum foil caps and kept in darkness at 20 or 45” C.
Halsall (1993) reported that the influence of added basidiomycetes on decomposition depended on the type and numbers of populations in the co-cultures. This evidence stimulates the question: do communitylevel interactions among populations, which have specific requirements and capabilities, control decomposition dynamics in mixed cultures? The focus of our study is not lignin decomposition per se; rather, it is the relationship between inoculation with potentially lignolytic fungi and the evolution of CO2 from barley straw by decomposer communities. We used two white-rot basidiomycetes, Phanerochaete chrysosporium and Phlebia radiata. The physiology, biochemistry, genetics and molecular biology of P. chrysosporium have been characterized (Kirk et al., 1986). The peroxidase production pattern of P. radiata is similar to that of P. chrysosporium and the peroxidases produced by the two fungi are immunologically coreactive (Kantelinen et al., 1988).
We report here on the evolution of CO2 at 20 and 45” C by P. chrysosporium and P. radiata on barley straw in pure culture, in culture with Trichoderma harzianum or in culture with indigenous barley straw microflora. Our results are consistent with evidence deduced from the literature, that interactions among populations integrate physicochemical and population-level factors to control CO, evolution from barley straw by the tested microbial communities.
Experimental design
Three sets of experiments were conducted, two of which were at 20” C, and one at 45” C (Table 1). The first two experiments compared respiration from microcosms of: (i) P. chrysosporium alone or in combination with either T. harzianum or indigenous straw microflora; or (ii) P. radiata alone or in combination with either T. harzianum or indigenous straw microflora, and were conducted at 20” C to represent the temperature of surface soil during late-summer in a temperate climatic zone. The third experiment was conducted at 45” C and designed to compare respiration from microcosms of P. chrysosporium or P. radiata alone or in combination with indigenous straw and compost microflora at a temperature at the lower range of temperature in the composting process.
MATERIALS AND METHODS
Microorganisms and inoculation
Pure cultures of fungi were maintained on 2% malt extract agar. Phanerochaete chrysosporium Burds (UAMH No. 4521) and Trichoderma harziunum (UAMH No. 4162) were obtained from the University of Alberta Microfungus Herbarium in Edmonton, Alberta. Phlebia radiata Fr. was isolated from barley straw in Edmonton, Alberta and identified at Biosystematics Research Centre in Ottawa, Ontario. Cultures of indigenous barley straw microflora were obtained as non-autoclaved barley straw and those of compost microflora as non-autoclaved compost derived from cattle manure.
CO2 analysis Evolution of CO, in all three experiments was measured with a Varian 3400 GC using a pre-column H,O trap (magnesium perchlorate-Carlo Erba,
Table I. Experimental conditions and treatments used in the three experiments P. ehrysosporium P. radiala
Expt
Conditions
Expl
20” C; 42 days; 3 replicates
Combination
20” C; 42 days; 3 replicates
EXPI
45°C; 19 days; 3 replicates
TlVatmeflts P. radiata P. radiara
P. chrysosporium +
Ind*
T. har:ianum T. harzianum
P. chrysosporium
P. chrysosporium +
Ind
T. harzianum +
Ind
T. har:ianum
Ind
Ind
CO, Extractable N & enzyme activities
CO2
P. chrysosporium P. radiara
+
Ind
P. radiata
Measurements
*Ind = indigenous microflora of barley straw. tlndd = indigenous microflora of barley straw and compost
CO,
+
lndd
+
lnddt
Community-level controls on decomposition
18 mesh granules) and a Porapak N column. The column, injector polrt and thermal conductivity detector were at 50, 50 and 100” C, respectively. The carrier gas, helium, was set at 30ml min-‘. Every 2 or 3 d.ays during incubation the flasks containing the str,aw decomposition cultures were sealed with No. 57 SubaSealTM stoppers. An aliquot of gas (0.5 ml) was, removed from the flask after 15 min, analyzed for CO2 concentration, and designated initial concentration. The final CO2 concentration was measured in a second 0.5 ml aliquot after about 3 h, at which time the SubaSeal stoppers were removed and the foil caps replaced. The calculated values of C02-C accumulated during the assay periods were integrated to determine total C02-C evolved during the incubation. CHCI,/K2SObextractable
N
Partially-decomposed barley straw residue from the P. radiata experiment was fumigated overnight with CHCI, and extracted with 40 ml of 500 mM K,SO, (Brookes et al., 1985). The K,SO,-extract was acidified with l.Oml 6 M HCl, evaporated in a stream of NH,-free air, digested with concentrated H,SO,-30%H,O, (Thomas et al., 1967) and total N determined by steam distillation (Bremner, 1965). At day 42, both fumigated or non-fumigated samples were extracted with K,SO,. There was little difference in extracted N between the two extractions. Consequently, the results reported are the total N extracted from the fumigated residues and include both microbial and “free” N. N content of the :rtraw was measured using a Carlo Erba automated Dumas system. Enzyme activity
Enzymes were extracted from the barley straw residue of the P. ,*adiata experiment according to the method of Wood and Goodenough (1977). Citrate buffer (40.0 ml 50 mM; pH 6.0) was added to the partially decomposed residue and shaken at 250 rev min-’ for 1 h. The slurry was squeezed through muslin, centrifuged at 10,OOOg for 15 min, and dialyzed (Spectra/Par 6000-8000 MW cutoff) for 72 h at 4” C against two changes of deionized water. Aliquots on non-dialyzed citrate-extract were retained for reducing sugar determination by the Somogyi-Nelson method (Somogyi, 1952). Avicelase,
carboxymethylcellulase
and xylanase.
Avicel (MERCK Art. 2331), carboxymethyl cellulose (Na-salt, SIGMA C-8758) and xylan (ICN Pharmaceuticals 103298) were used as substrates to assay avicelase, carboxymethylcellulase (CMCase) and xylanase activity, re,spectively, in the dialyzed citrateextract. The modified assay of Rouau and Odier (1986) used 4.0 ml of a 1% (w/v) suspension of avicel, CMC or xylan in 50 mM Na-acetate (pH 5.0) plus 0.1 ml citrate-extract at 37” C with continuous shaking for 2 h. Enzyme activity was measured as pro-
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duction of reducing sugars per unit time determined by the Somogyi-Nelson method. Lactase. The assay of Bollag and Leonowicz (1984) was modified. The total assay volume of 8.6 ml consisted of 7.0 ml 100 mM citric acid-Na,HPO, buffer (pH 5.0), 1.0 ml citrate-extract and 0.6 ml 100 mM syringaldazine in ethanol. Enzyme activity was measured as the increase in A5z5per unit time. Lignin peroxidase. The assay of Kirk et al. (1986) was followed. The total assay volume of 3.0 ml used 2mM veratryl alcohol (purified by dissolution in CHCl,, partition against 1 M NaOH and rotary evaporation of organic phase to remove CHCl,), 4OOmM H,02, 50 mM Na tartrate (pH 2.5) and 0.1 ml citrate-extract. Lignin peroxidase activity was measured as production of veratrylaldehyde by the increase in A3,0 per unit time. Lignin peroxidase (crude extract of lignin peroxidase from P. chrysosporium purchased from Tienzyme Inc., State College, Pa) was used to assess the extraction and assay procedures. Exogenous lignin peroxidase was added to sterile nutrient solution and to sterile buffers (50 mM Na citrate buffer at pH 4.5 to 6.0; 25 mM Na citrate buffer pH 4.5; 50mM Na citrate buffer pH 4.5; 50mM Na citrate + Na succinate buffer pH 4.5; 25 mM Na succinate buffer pH 4.5; and 50mM Na succinate buffer pH 4.5) stored for several hours and assayed for activity. The exogenous lignin peroxidase was added to autoclaved barley straw, moistened with nutrient solution, immediately extracted with 50mM citrate buffer (pH 6.0) dialyzed against MILLI-Q water for 48 h and assayed for activity. Statistical analysis
Non-linear (hyperbolic) regression (Izaurralde et al., 1986) was used to compare the amounts of C02-C evolved from straw at 20” C in the P. chrysosporium experiment by P. chrysosporium, T. harzianum and P. chrysosporium with T. harzianum. RESULTS Evolution of CO, by P. chrysosporium and P. radiata from straw P. radiata experiment. This experiment was carried out twice; the first time CO*-C evolution was measured, but enzyme activities and extractable N were not. The relative differences in CO,-C evolution between treatments measured the first time the experiment was carried out are similar to those shown in Fig. 1. Therefore, only data from the second time are presented. After 42 days at 20” C (Fig. l.), evolution of CO,C by P. radiata in pure culture was at least )-fold higher than by any other tested culture, and was 7-fold higher than by T. harzianum. When P. radiata was mixed with indigenous straw microorganisms, however, respiration did not exceed that of the indigenous microorganisms alone, even though P. radiata in pure culture was about 3.5-fold
R. A. Janzen et al.
176
200 Y 0” 0
f3ooo
8 600
?i
400
200
0 0
IO
20
30
40
TIME (days) Fig. 1. Accumulated evolved CO,-C from cultures growing on barley straw at 20°C. Ind, indigenous barley straw microorganisms; PC, P. chrysosporium; Pr, P. radiata; Th, T. harzianum. (Error bars indicate standard deviation, n = 3.)
more active than the indigenous community. No differences, according to assessment of standard deviation from the means, were observed among the other tested pure and mixed cultures. P. chrysosporium experiment. During 19 days, at 20” C, P. chrysosporium evolved less CO& as determined by hyperbolic regression (Izaurralde et al., 1986) than did T. harzianum or T. harzianum with P. chrysosporium (Table 2). A major difference among these isolates was in the high value of K, which is the days of incubation required to achieve C,,,/2, and may be related to the lag phase. In addition, the predicted maximum amount respired was also least in the P. chrysosporium microcosms after 40 days. The amount of C02-C evolved from straw inoculated with T. harziunum was not different from that from straw inoculated with T. harzianum and P. chrysosporium.
In contrast to respiration from microcosms containing only P. radiata, those containing only P. chrysosporium evolved less CO,-C during 42 days than did the indigenous community. However, as was observed in the P. radiata experiment, mixing P. chrysosporium with the indigenous community made, no difference to evolution of CO*-C by the indigenous straw microorganisms. Combination experiment. After 19 days at 45”C, P. chrysosporium evolved SO-fold more CO, than did P. radiuta (Fig. 2). P. chrysosporium evolved 25% less CO, than did P. chrysosporium or P. radiata with microflora or barley straw and compost. The amount of CO,-C evolved by P. rudiuta with microflora of barley straw and compost was not different, according to assessment of standard deviations, from that by P. chrysosporium with microflora of barley straw and compost. Fruiting bodies formed in the microcosms inoculated with P. chrysosporium or P. radiata with microflora of barley straw and compost, but not in flasks containing only added P. chrysosporium or P. radiata. We infer that the microflora from barley or compost survived, and may have predominated, in the microcosms with mixed cultures. Biochemical characteristics of the decomposition systems in the P. radiata experiment
The amount of CHCl, /K, SO,-extractable N (Fig. 3) and reducing sugar (Fig. 4) in the P. radiata culture was double that of the other treatments. The other tested cultures were similar to each other with respect to extractable N and reducing sugar content. The measured activity of avicelase tended to be lowest and that of xylanase the highest in all cultures, while estimated CMCase activity was intermediate (Fig. 5). Lactase activity (data not shown) was found in trace amounts in cultures
600 Y, 00
Table 2. Comoarison bv non-linear reeression of hvaerbolic lines ,. . + d)] describing . *d/(K evolved CO,-C accumulatedfrom cultures at 20” C growing on barley straw
[C,=C,.
Inoculum P. chrysosporium T. harzianum P. chrysosporium + T. harrianum %“.
C m*i*
K
R*
C,
P
55.0 51.1
13.8 0.5
0.914 0.967
40.9 50.5
:
48.5
1.6
0.842
46.6
b
7predicted maximum accumulated CO,-C (mg); K, days of
incubation required to achieve C&/2; and C,, predicted CO,-C accumulated after 40 days (mg). tRegression lines followed by the same letter are not significantly different.
400
f3 3
200
Fig. 2. Accumulated evolved CO& from cultures growing on barley straw at 45” C. Indd, indigenous microorganisms from barley straw and compost in combination. All other symbols as in Fig. I. (Error bars indicate standard deviation, n = 3.)
Community-level controls on decomposition
177
3.0 2.5
0.0 ,I,,
h
Th+Pr
r
Ind
Ind+Pr
Fig. 3. N extracted by 5OOmM K,S04 from CHCl,fumigated barley straw residue in the P. radiuta experiment. (Abbreviations and error bars as described in Fig. 1.)
Fig. 4. Reducing sugars extracted @H 6.0) from barley straw residue by 5OmM citrate buffer. (Abbreviations and error bars as described in Fig. 1.)
containing indigenous barley straw microflora, but no activity was detected in other cultures. No lignin peroxidase activity was detected in any cultures. Furthermore, no activity of exogenous lignin peroxidase (data not shown) was recovered from sterile barley straw, from sterile nutrient solution or from 50 mM citrate buffer at the tested pH values. We confirmed the assay was successful wen purchased lignin peroxidase was added immediately to the assay solution. Subsequent work with purchased lignin peroxidase, however, showed that the purchased enzyme was inactivated under the conditions of the citrate extraction procedure. The purchased peroxidase remained active in succinate buffer, but not in citrate. Thus, problems with the enzyme extraction, and not with the enzyme assay, prevented estimation of lignin peroxidase activity in the barle:y straw microcosms. The experimental observation of no estimated extractable ligninase activity, therefore, does not mean that lignin peroxidase was absent from the decomposition system.
during 19 days as did P. radiata (about 500mg) during 19 days at 7” C below its published optimum. Temperature thus controlled the activity of these two fungi in pure culture, but their contrasting temperature optima demonstrate how decomposition might be independent of such a physicochemical control if two such populations were able to function independently in a community. The higher amounts of CHCl,/K, SO,-extractable N associated with P. rudibta in pure culture compared to the other inocula was also associated with higher amounts of CO,-C evolved by P. radiata. The N obtained by CHCl, fumigation followed by K*SO, extraction includes that from the added medium, the soluble component of straw, and the microbial biomass. The 20 ml of medium added to the straw contained 0.85 mg N g-’ straw, and the straw contained 5.7mg N g-l. The N extracted ranged from 1.1 to 1.3 mg g-l straw in cultures containing T. harzianum or indigenous microflora, but was 3.0 mg g-’ in the pure culture of P. radiutu. N extracted in excess of that in the added medium indicates N was released from straw during decomposition, and the amount of N extracted was highest in the culture which evolved the most CO*-C.
DISCUSSION
Our study adds to the evidence in support of the hypothesis that community-level controls on decomposition are hierarchically higher than are physicochemical! or population-level controls. P. radiutu has superior capability to evolve CO, from straw at 2O”C, but this capability is not expressed in co-cultures with T. harzianum or with indigenous barley straw microflora. Temperature was the physicochemical control manipulated in our eKperimenta1 design. Agosin et al. (1985) reported opl:imal temperatures, under the conditions of their study, of 39” C for P. chrysosporium and 26” C fi3r P. radiata. The responses of P. chrysosporium and P. radiata to the temperatures imposed in our experiment are consistent with published optima. Furthermore, at a temperature of 7” C above its published optimum P. chrysosporium evolved similar amount of CO*-C (about 400 mg)
0.4
0.3 0.2 0.1 0.0
Fig. 5. Activity of avicelase, carboxymethylcellulase and xylanase in dialyzed citrate buffer-extract of barley straw residue. (Abbreviations and error bars as described in Fig. 1.)
178
R. A. Janzen et al.
Higher amounts of reducing sugar after 40 days, associated with P. radiuta in pure culture compared to the other inocula also correlate to the higher amount of CO,-C evolved. This should be interpreted with caution because a one-time sample may not be characteristic of the entire study. A higher concentration of reducing sugars could explain the inability of P. radiata to compete in co-culture with the other tested inocula-that is, the other inocula might have been more efficient in scavenging C from solution. Alternatively, reducing sugars may have accumulated in the aging culture as non-C nutrients became limiting for growth, but residual enzymes continued to hydrolyze the barley residues. Further work is warranted to understand the ecological significance of the apparent correlation in the tested cultures between concentration of reducing sugar and the amount of CO,-C evolved. In contrast, potential activities of xylanase, carboxymethylcellulase and avicelase did not correlate to respiration. This observation is consistent with enzymatic activities measured during the decomposition of wheat straw by Trametes versicolor and Pleurotus ostreatus in pure cultures (Valmaseda et al., 1991). The assays of the selected enzymes in our study were poor indicators of decomposer activities. Attempts to modify microbial decomposition of straw normally aim to accelerate selected system functions. Our results, however, suggest it might ’ be fruitful to ask: can the rate of COZ evolution be inhibited to decrease the rate of decomposition? Co-cultures of P. radiata (maximum rate > 2000 pg CO& h-‘) and indigenous barley straw microflora or T. harzianum (maximum rate for both < 1000 pg C02-C h-‘) however, exhibited respiration rates characteristic of the less rapid decomposer population. Bruehl and Lai (1966) reasoned that degree of possession of substrate determines dominance. Thus, if T. harzianum and indigenous barley straw microflora were able more rapidly to colonize the barley straw, P. radiata might have been prevented from becoming established. Alternatively, T. harziunum is an agent for biological control of some bacterial and fungal phytopathogens (Chet, 1987). Therefore, repression of the decomposition potential of P. radiata in the co-cultures may derive from the hyperparasitic capability of T. harzianum or of similar microbes in the indigenous barley straw microflora. In contrast, however, T. harzianum and Trametes versicolor are compatible in co-culture (Freitag and Morrell, 1992). Greater understanding of control mechanisms and enhanced predictive capability are therefore essential for developing microbiology-based technology to accelerate decomposition, or to slow CO, evolution and sequester C in soil. Such understanding and predictive capability may arise from testing the hypothesis that interactions among microbial populations are the highest hierarchical level of control on plant residue decomposition. For example, Herman et al. (1977) measured -
the rate of decomposition of root material from three species of grass suspended in continuously aerated water inoculated with a suspension of the soil from which the roots were obtained. They concluded that decomposition was invers:ly related to [(C/N) (%lignin)] (% carbohydrate-*), but that “the effect of the soil is postulated to be. as great or greater than the effect of changes in the type of organic substrate”. Herman et al. (1977) did not relate explicitly their “soil effect” hypothesis to controls on the function of decomposer communities. Janzen et al. (1988) related incorporation of 14Cand “N into active soil organic matter to the C-to-N ratio and stage of maturity of labeled plant materials, and to the clay and organic matter content of amended soil samples. They did not address the possible relationship between microbial ecology and the marked substrate- and soil-specific humification dynamics reported in their study. We conclude that community-level controls on decomposition dynamics integrate physicochemical and population-level controls, and therefore might facilitate extension to the environment. Acknowledgements-Financial support was provided by a Natural Sciences and Engineering Research Council of Canada (NSERC) Postgraduate Scholarship to R. A. Janzen, and by operating grants from the Western Grains Research Foundation and NSERC to W. B. McGill.
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003&0717CWOO164-2 . _
Liiol.Lfiochem. Vol. 27. No. 2, pp. 173179, 1995 Copyright 0 1995 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0038-0717/959.50+ 0.00
A COMMUNITY-LEVEL CONCEPT OF CONTROLS ON DECOMPOSITION PROCESSES: DECOMPOSITION OF BARLEY STRAW BY PHANEROCHAETE CHRYSOSPORIUM OR PHLEBIA RADIATA IN PURE OR MIXED CULTURE R. A. JANZEN,’ J. F. DORMAAR’ and W. B. MCGILL’* ‘Department of Renewable Resources, University of Alberta, Edmonton, Alberta, Canada T6G 2E3 and 2Land Resources Science Section, Agriculture and Agri-Food Canada, Research Centre, Box 3000 Main, Lethbridge, Alberta, Canada Tl J 4Bl (Accepted I1 July 1994) Summary-We tested the hypothesis that interactions among populations in communities of decomposer microbes integrate physicochemical and population-level factors to control decomposition. To this end, we measured the relationship between inoculation with Phanerochaete chrysosporium and Phlebia radiata and the evolution of CO, from barley straw by decomposer communities. At 45” C, 80% as much CO, was evolved fr’om straw inoculated with P. chrysosporiumin pure culture as from mixed cultures containing P. chrysosporium or P. radiata with indigenous barley straw and compost microflora. P. radiata in pure culture evolvetrl less CO, than all other tested inocula. At 20”C, P. radiata evolved 3-fold more CO, in pure culture on barley straw over 42 days than did other tested inocula. Adding P. radiata to straw in co-culture with Trichoderma harzianum or indigenous barley straw microflora did not increase CO, evolution, and by inference decomposition rate, compared to microcosms without added P. radiata. We conclude that community-level controls on decomposition dynamics integrate physicochemical and population-level controls, and therefore might facilitate extension to the environment.
INTRODUCMON
Our concern in this study is control on decomposition, which we treat under two categories. First, as used here, physicochemical control includes factors such as temperature., pox status, water content, substrate composition or soil clay content; and second, population -level control pertains to temperature optimum, lignolytic capability, etc. We set out to test the hypothesis that community-level controls integrate physicochemical and population- or geneticlevel c6ntrols during interactions among populations within communities of decomposer microbes. This is not a new hypothesis; in articulating and testing it we aim to address explicitly a concept that has been addressed implicitly or indirectly in previous reports. Investigations of controls on the decomposition of lignocellulosic materials have emphasized either physicochemical or genetically-determined aspects of the decomposition process. For example, the dynamics of decomposition have been related to the chemical composition of substrates (Herman et al., 1977; Janzen et al., 1988; Neely et al., 1991), to the physical structure of lignocellulosic materials (Juma and McGill, 1986) or to the genetically-derived po*Author for correspondence.
tential of microorganisms to degrade lignocellulose (Agosin et al., 1985). Within this conceptual framework, regulation of lignolytic activity becomes a primary control on the rate of decomposition. Studies of white-rot fungi, Class Basidiomycetes, document that substrate composition, (Dill et al., 1987), temperature (Agosin et al., 1985) and the capabilities and requirements of populations (Valmaseda et al., 1991) control the rate of CO, evolution from straw by white-rot fungi in pure culture. This evidence supports the conclusion that physicochemical and population level factors control the function of basidiomycetes in pure culture. We propose, however, that implicit in the above studies is the concept that decomposition processes are also controlled at a hierarchical level higher than physicochemical and population-level controls. Studies of cereal straw decomposition have used mixed cultures also. Bowen (1990) measured the rate of CO2 evolution when unidentified basidiomycetes isolated from wheat straw decomposing in soil were inoculated onto wheat straw in pure cultures or with cellulolytic fungi. Halsall (1993) measured CO2 evolution and nitrogenase activity from co-cultures of Cyathus stercoreus, a lignolytic basidiomycete, with free-living N,-fixing bacteria (Beijerinckia indica B 15 or Azospiriflwn sp. DN64) and cellulolytic fungi growing on straw. Both Bowen (1990) and 173
R. A. Janzen et al.
174
Barley straw (Hordeum vuigare L. var. Galt; mature plant material collected from the field after harvest) was ground (< 2 mm) and 5.0 g (air dry) added to 250 ml conical flasks and autoclaved (twice for 15 min at 121” C). This sterile straw was then inoculated to produce microcosms in one of three ways: (i) with a 9-mm disc of fungal colony margin; (ii) with 0.1 g of air dry barley straw; or (iii) 0.1 g barley straw + 0.1 g compost, and moistened with 20ml of mineral medium. Each 1OOOml of medium contained 200 mg MgSO, .7H,O, 100 mg NaCl, 20 mg CaCl,, 2 mg Na,MoO, .2H,O, 10 mg MnSO,.H,O, 1OOOmg (NH4)2S04 and 328 mg FeNaEDTA. After sterilizing the basal salts component of the medium, separately sterilized solutions were added such that 1OOOml of the complete medium also contained 100 mmol KH,PO, (pH adjusted to 7.0 with NaOH), 5 mg biotin, 10 mg p-aminobenzoate, 10 mg thiamin, 2.5 g glucose and 2.5 g malic acid. Flasks were periodically weighed and water added to maintain water content. The flasks were covered with aluminum foil caps and kept in darkness at 20 or 45” C.
Halsall (1993) reported that the influence of added basidiomycetes on decomposition depended on the type and numbers of populations in the co-cultures. This evidence stimulates the question: do communitylevel interactions among populations, which have specific requirements and capabilities, control decomposition dynamics in mixed cultures? The focus of our study is not lignin decomposition per se; rather, it is the relationship between inoculation with potentially lignolytic fungi and the evolution of CO2 from barley straw by decomposer communities. We used two white-rot basidiomycetes, Phanerochaete chrysosporium and Phlebia radiata. The physiology, biochemistry, genetics and molecular biology of P. chrysosporium have been characterized (Kirk et al., 1986). The peroxidase production pattern of P. radiata is similar to that of P. chrysosporium and the peroxidases produced by the two fungi are immunologically coreactive (Kantelinen et al., 1988).
We report here on the evolution of CO2 at 20 and 45” C by P. chrysosporium and P. radiata on barley straw in pure culture, in culture with Trichoderma harzianum or in culture with indigenous barley straw microflora. Our results are consistent with evidence deduced from the literature, that interactions among populations integrate physicochemical and population-level factors to control CO, evolution from barley straw by the tested microbial communities.
Experimental design
Three sets of experiments were conducted, two of which were at 20” C, and one at 45” C (Table 1). The first two experiments compared respiration from microcosms of: (i) P. chrysosporium alone or in combination with either T. harzianum or indigenous straw microflora; or (ii) P. radiata alone or in combination with either T. harzianum or indigenous straw microflora, and were conducted at 20” C to represent the temperature of surface soil during late-summer in a temperate climatic zone. The third experiment was conducted at 45” C and designed to compare respiration from microcosms of P. chrysosporium or P. radiata alone or in combination with indigenous straw and compost microflora at a temperature at the lower range of temperature in the composting process.
MATERIALS AND METHODS
Microorganisms and inoculation
Pure cultures of fungi were maintained on 2% malt extract agar. Phanerochaete chrysosporium Burds (UAMH No. 4521) and Trichoderma harziunum (UAMH No. 4162) were obtained from the University of Alberta Microfungus Herbarium in Edmonton, Alberta. Phlebia radiata Fr. was isolated from barley straw in Edmonton, Alberta and identified at Biosystematics Research Centre in Ottawa, Ontario. Cultures of indigenous barley straw microflora were obtained as non-autoclaved barley straw and those of compost microflora as non-autoclaved compost derived from cattle manure.
CO2 analysis Evolution of CO, in all three experiments was measured with a Varian 3400 GC using a pre-column H,O trap (magnesium perchlorate-Carlo Erba,
Table I. Experimental conditions and treatments used in the three experiments P. ehrysosporium P. radiala
Expt
Conditions
Expl
20” C; 42 days; 3 replicates
Combination
20” C; 42 days; 3 replicates
EXPI
45°C; 19 days; 3 replicates
TlVatmeflts P. radiata P. radiara
P. chrysosporium +
Ind*
T. har:ianum T. harzianum
P. chrysosporium
P. chrysosporium +
Ind
T. harzianum +
Ind
T. har:ianum
Ind
Ind
CO, Extractable N & enzyme activities
CO2
P. chrysosporium P. radiara
+
Ind
P. radiata
Measurements
*Ind = indigenous microflora of barley straw. tlndd = indigenous microflora of barley straw and compost
CO,
+
lndd
+
lnddt
Community-level controls on decomposition
18 mesh granules) and a Porapak N column. The column, injector polrt and thermal conductivity detector were at 50, 50 and 100” C, respectively. The carrier gas, helium, was set at 30ml min-‘. Every 2 or 3 d.ays during incubation the flasks containing the str,aw decomposition cultures were sealed with No. 57 SubaSealTM stoppers. An aliquot of gas (0.5 ml) was, removed from the flask after 15 min, analyzed for CO2 concentration, and designated initial concentration. The final CO2 concentration was measured in a second 0.5 ml aliquot after about 3 h, at which time the SubaSeal stoppers were removed and the foil caps replaced. The calculated values of C02-C accumulated during the assay periods were integrated to determine total C02-C evolved during the incubation. CHCI,/K2SObextractable
N
Partially-decomposed barley straw residue from the P. radiata experiment was fumigated overnight with CHCI, and extracted with 40 ml of 500 mM K,SO, (Brookes et al., 1985). The K,SO,-extract was acidified with l.Oml 6 M HCl, evaporated in a stream of NH,-free air, digested with concentrated H,SO,-30%H,O, (Thomas et al., 1967) and total N determined by steam distillation (Bremner, 1965). At day 42, both fumigated or non-fumigated samples were extracted with K,SO,. There was little difference in extracted N between the two extractions. Consequently, the results reported are the total N extracted from the fumigated residues and include both microbial and “free” N. N content of the :rtraw was measured using a Carlo Erba automated Dumas system. Enzyme activity
Enzymes were extracted from the barley straw residue of the P. ,*adiata experiment according to the method of Wood and Goodenough (1977). Citrate buffer (40.0 ml 50 mM; pH 6.0) was added to the partially decomposed residue and shaken at 250 rev min-’ for 1 h. The slurry was squeezed through muslin, centrifuged at 10,OOOg for 15 min, and dialyzed (Spectra/Par 6000-8000 MW cutoff) for 72 h at 4” C against two changes of deionized water. Aliquots on non-dialyzed citrate-extract were retained for reducing sugar determination by the Somogyi-Nelson method (Somogyi, 1952). Avicelase,
carboxymethylcellulase
and xylanase.
Avicel (MERCK Art. 2331), carboxymethyl cellulose (Na-salt, SIGMA C-8758) and xylan (ICN Pharmaceuticals 103298) were used as substrates to assay avicelase, carboxymethylcellulase (CMCase) and xylanase activity, re,spectively, in the dialyzed citrateextract. The modified assay of Rouau and Odier (1986) used 4.0 ml of a 1% (w/v) suspension of avicel, CMC or xylan in 50 mM Na-acetate (pH 5.0) plus 0.1 ml citrate-extract at 37” C with continuous shaking for 2 h. Enzyme activity was measured as pro-
175
duction of reducing sugars per unit time determined by the Somogyi-Nelson method. Lactase. The assay of Bollag and Leonowicz (1984) was modified. The total assay volume of 8.6 ml consisted of 7.0 ml 100 mM citric acid-Na,HPO, buffer (pH 5.0), 1.0 ml citrate-extract and 0.6 ml 100 mM syringaldazine in ethanol. Enzyme activity was measured as the increase in A5z5per unit time. Lignin peroxidase. The assay of Kirk et al. (1986) was followed. The total assay volume of 3.0 ml used 2mM veratryl alcohol (purified by dissolution in CHCl,, partition against 1 M NaOH and rotary evaporation of organic phase to remove CHCl,), 4OOmM H,02, 50 mM Na tartrate (pH 2.5) and 0.1 ml citrate-extract. Lignin peroxidase activity was measured as production of veratrylaldehyde by the increase in A3,0 per unit time. Lignin peroxidase (crude extract of lignin peroxidase from P. chrysosporium purchased from Tienzyme Inc., State College, Pa) was used to assess the extraction and assay procedures. Exogenous lignin peroxidase was added to sterile nutrient solution and to sterile buffers (50 mM Na citrate buffer at pH 4.5 to 6.0; 25 mM Na citrate buffer pH 4.5; 50mM Na citrate buffer pH 4.5; 50mM Na citrate + Na succinate buffer pH 4.5; 25 mM Na succinate buffer pH 4.5; and 50mM Na succinate buffer pH 4.5) stored for several hours and assayed for activity. The exogenous lignin peroxidase was added to autoclaved barley straw, moistened with nutrient solution, immediately extracted with 50mM citrate buffer (pH 6.0) dialyzed against MILLI-Q water for 48 h and assayed for activity. Statistical analysis
Non-linear (hyperbolic) regression (Izaurralde et al., 1986) was used to compare the amounts of C02-C evolved from straw at 20” C in the P. chrysosporium experiment by P. chrysosporium, T. harzianum and P. chrysosporium with T. harzianum. RESULTS Evolution of CO, by P. chrysosporium and P. radiata from straw P. radiata experiment. This experiment was carried out twice; the first time CO*-C evolution was measured, but enzyme activities and extractable N were not. The relative differences in CO,-C evolution between treatments measured the first time the experiment was carried out are similar to those shown in Fig. 1. Therefore, only data from the second time are presented. After 42 days at 20” C (Fig. l.), evolution of CO,C by P. radiata in pure culture was at least )-fold higher than by any other tested culture, and was 7-fold higher than by T. harzianum. When P. radiata was mixed with indigenous straw microorganisms, however, respiration did not exceed that of the indigenous microorganisms alone, even though P. radiata in pure culture was about 3.5-fold
R. A. Janzen et al.
176
200 Y 0” 0
f3ooo
8 600
?i
400
200
0 0
IO
20
30
40
TIME (days) Fig. 1. Accumulated evolved CO,-C from cultures growing on barley straw at 20°C. Ind, indigenous barley straw microorganisms; PC, P. chrysosporium; Pr, P. radiata; Th, T. harzianum. (Error bars indicate standard deviation, n = 3.)
more active than the indigenous community. No differences, according to assessment of standard deviation from the means, were observed among the other tested pure and mixed cultures. P. chrysosporium experiment. During 19 days, at 20” C, P. chrysosporium evolved less CO& as determined by hyperbolic regression (Izaurralde et al., 1986) than did T. harzianum or T. harzianum with P. chrysosporium (Table 2). A major difference among these isolates was in the high value of K, which is the days of incubation required to achieve C,,,/2, and may be related to the lag phase. In addition, the predicted maximum amount respired was also least in the P. chrysosporium microcosms after 40 days. The amount of C02-C evolved from straw inoculated with T. harziunum was not different from that from straw inoculated with T. harzianum and P. chrysosporium.
In contrast to respiration from microcosms containing only P. radiata, those containing only P. chrysosporium evolved less CO,-C during 42 days than did the indigenous community. However, as was observed in the P. radiata experiment, mixing P. chrysosporium with the indigenous community made, no difference to evolution of CO*-C by the indigenous straw microorganisms. Combination experiment. After 19 days at 45”C, P. chrysosporium evolved SO-fold more CO, than did P. radiuta (Fig. 2). P. chrysosporium evolved 25% less CO, than did P. chrysosporium or P. radiata with microflora or barley straw and compost. The amount of CO,-C evolved by P. rudiuta with microflora of barley straw and compost was not different, according to assessment of standard deviations, from that by P. chrysosporium with microflora of barley straw and compost. Fruiting bodies formed in the microcosms inoculated with P. chrysosporium or P. radiata with microflora of barley straw and compost, but not in flasks containing only added P. chrysosporium or P. radiata. We infer that the microflora from barley or compost survived, and may have predominated, in the microcosms with mixed cultures. Biochemical characteristics of the decomposition systems in the P. radiata experiment
The amount of CHCl, /K, SO,-extractable N (Fig. 3) and reducing sugar (Fig. 4) in the P. radiata culture was double that of the other treatments. The other tested cultures were similar to each other with respect to extractable N and reducing sugar content. The measured activity of avicelase tended to be lowest and that of xylanase the highest in all cultures, while estimated CMCase activity was intermediate (Fig. 5). Lactase activity (data not shown) was found in trace amounts in cultures
600 Y, 00
Table 2. Comoarison bv non-linear reeression of hvaerbolic lines ,. . + d)] describing . *d/(K evolved CO,-C accumulatedfrom cultures at 20” C growing on barley straw
[C,=C,.
Inoculum P. chrysosporium T. harzianum P. chrysosporium + T. harrianum %“.
C m*i*
K
R*
C,
P
55.0 51.1
13.8 0.5
0.914 0.967
40.9 50.5
:
48.5
1.6
0.842
46.6
b
7predicted maximum accumulated CO,-C (mg); K, days of
incubation required to achieve C&/2; and C,, predicted CO,-C accumulated after 40 days (mg). tRegression lines followed by the same letter are not significantly different.
400
f3 3
200
Fig. 2. Accumulated evolved CO& from cultures growing on barley straw at 45” C. Indd, indigenous microorganisms from barley straw and compost in combination. All other symbols as in Fig. I. (Error bars indicate standard deviation, n = 3.)
Community-level controls on decomposition
177
3.0 2.5
0.0 ,I,,
h
Th+Pr
r
Ind
Ind+Pr
Fig. 3. N extracted by 5OOmM K,S04 from CHCl,fumigated barley straw residue in the P. radiuta experiment. (Abbreviations and error bars as described in Fig. 1.)
Fig. 4. Reducing sugars extracted @H 6.0) from barley straw residue by 5OmM citrate buffer. (Abbreviations and error bars as described in Fig. 1.)
containing indigenous barley straw microflora, but no activity was detected in other cultures. No lignin peroxidase activity was detected in any cultures. Furthermore, no activity of exogenous lignin peroxidase (data not shown) was recovered from sterile barley straw, from sterile nutrient solution or from 50 mM citrate buffer at the tested pH values. We confirmed the assay was successful wen purchased lignin peroxidase was added immediately to the assay solution. Subsequent work with purchased lignin peroxidase, however, showed that the purchased enzyme was inactivated under the conditions of the citrate extraction procedure. The purchased peroxidase remained active in succinate buffer, but not in citrate. Thus, problems with the enzyme extraction, and not with the enzyme assay, prevented estimation of lignin peroxidase activity in the barle:y straw microcosms. The experimental observation of no estimated extractable ligninase activity, therefore, does not mean that lignin peroxidase was absent from the decomposition system.
during 19 days as did P. radiata (about 500mg) during 19 days at 7” C below its published optimum. Temperature thus controlled the activity of these two fungi in pure culture, but their contrasting temperature optima demonstrate how decomposition might be independent of such a physicochemical control if two such populations were able to function independently in a community. The higher amounts of CHCl,/K, SO,-extractable N associated with P. rudibta in pure culture compared to the other inocula was also associated with higher amounts of CO,-C evolved by P. radiata. The N obtained by CHCl, fumigation followed by K*SO, extraction includes that from the added medium, the soluble component of straw, and the microbial biomass. The 20 ml of medium added to the straw contained 0.85 mg N g-’ straw, and the straw contained 5.7mg N g-l. The N extracted ranged from 1.1 to 1.3 mg g-l straw in cultures containing T. harzianum or indigenous microflora, but was 3.0 mg g-’ in the pure culture of P. radiutu. N extracted in excess of that in the added medium indicates N was released from straw during decomposition, and the amount of N extracted was highest in the culture which evolved the most CO*-C.
DISCUSSION
Our study adds to the evidence in support of the hypothesis that community-level controls on decomposition are hierarchically higher than are physicochemical! or population-level controls. P. radiutu has superior capability to evolve CO, from straw at 2O”C, but this capability is not expressed in co-cultures with T. harzianum or with indigenous barley straw microflora. Temperature was the physicochemical control manipulated in our eKperimenta1 design. Agosin et al. (1985) reported opl:imal temperatures, under the conditions of their study, of 39” C for P. chrysosporium and 26” C fi3r P. radiata. The responses of P. chrysosporium and P. radiata to the temperatures imposed in our experiment are consistent with published optima. Furthermore, at a temperature of 7” C above its published optimum P. chrysosporium evolved similar amount of CO*-C (about 400 mg)
0.4
0.3 0.2 0.1 0.0
Fig. 5. Activity of avicelase, carboxymethylcellulase and xylanase in dialyzed citrate buffer-extract of barley straw residue. (Abbreviations and error bars as described in Fig. 1.)
178
R. A. Janzen et al.
Higher amounts of reducing sugar after 40 days, associated with P. radiuta in pure culture compared to the other inocula also correlate to the higher amount of CO,-C evolved. This should be interpreted with caution because a one-time sample may not be characteristic of the entire study. A higher concentration of reducing sugars could explain the inability of P. radiata to compete in co-culture with the other tested inocula-that is, the other inocula might have been more efficient in scavenging C from solution. Alternatively, reducing sugars may have accumulated in the aging culture as non-C nutrients became limiting for growth, but residual enzymes continued to hydrolyze the barley residues. Further work is warranted to understand the ecological significance of the apparent correlation in the tested cultures between concentration of reducing sugar and the amount of CO,-C evolved. In contrast, potential activities of xylanase, carboxymethylcellulase and avicelase did not correlate to respiration. This observation is consistent with enzymatic activities measured during the decomposition of wheat straw by Trametes versicolor and Pleurotus ostreatus in pure cultures (Valmaseda et al., 1991). The assays of the selected enzymes in our study were poor indicators of decomposer activities. Attempts to modify microbial decomposition of straw normally aim to accelerate selected system functions. Our results, however, suggest it might ’ be fruitful to ask: can the rate of COZ evolution be inhibited to decrease the rate of decomposition? Co-cultures of P. radiata (maximum rate > 2000 pg CO& h-‘) and indigenous barley straw microflora or T. harzianum (maximum rate for both < 1000 pg C02-C h-‘) however, exhibited respiration rates characteristic of the less rapid decomposer population. Bruehl and Lai (1966) reasoned that degree of possession of substrate determines dominance. Thus, if T. harzianum and indigenous barley straw microflora were able more rapidly to colonize the barley straw, P. radiata might have been prevented from becoming established. Alternatively, T. harziunum is an agent for biological control of some bacterial and fungal phytopathogens (Chet, 1987). Therefore, repression of the decomposition potential of P. radiata in the co-cultures may derive from the hyperparasitic capability of T. harzianum or of similar microbes in the indigenous barley straw microflora. In contrast, however, T. harzianum and Trametes versicolor are compatible in co-culture (Freitag and Morrell, 1992). Greater understanding of control mechanisms and enhanced predictive capability are therefore essential for developing microbiology-based technology to accelerate decomposition, or to slow CO, evolution and sequester C in soil. Such understanding and predictive capability may arise from testing the hypothesis that interactions among microbial populations are the highest hierarchical level of control on plant residue decomposition. For example, Herman et al. (1977) measured -
the rate of decomposition of root material from three species of grass suspended in continuously aerated water inoculated with a suspension of the soil from which the roots were obtained. They concluded that decomposition was invers:ly related to [(C/N) (%lignin)] (% carbohydrate-*), but that “the effect of the soil is postulated to be. as great or greater than the effect of changes in the type of organic substrate”. Herman et al. (1977) did not relate explicitly their “soil effect” hypothesis to controls on the function of decomposer communities. Janzen et al. (1988) related incorporation of 14Cand “N into active soil organic matter to the C-to-N ratio and stage of maturity of labeled plant materials, and to the clay and organic matter content of amended soil samples. They did not address the possible relationship between microbial ecology and the marked substrate- and soil-specific humification dynamics reported in their study. We conclude that community-level controls on decomposition dynamics integrate physicochemical and population-level controls, and therefore might facilitate extension to the environment. Acknowledgements-Financial support was provided by a Natural Sciences and Engineering Research Council of Canada (NSERC) Postgraduate Scholarship to R. A. Janzen, and by operating grants from the Western Grains Research Foundation and NSERC to W. B. McGill.
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