Filter centrifugation as a sampling method for miniaturization of extracellular fungal enzyme activity measurements in solid media

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Methodological Advances

Filter centrifugation as a sampling method for miniaturizationof extracellular fungal enzyme activity measurements in solidmedia

J. HEINONSALOa, G. KABIERSCHa,*, R.M. NIEMIb, S. SIMPANENb,c, H. ILVESNIEMId,M. HOFRICHTERe, A. HATAKKAa, K.T. STEFFENa

aViikki Biocenter, Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, P.O. Box 56,

FIN-00014 University of Helsinki, Helsinki, FinlandbFinnish Environment Institute, Research Programme for Biodiversity, P.O. Box 140, FIN-00251 Helsinki, FinlandcDepartment of Environmental Sciences, University of Helsinki, Niemenkatu 73, 15140 Lahti, FinlanddFinnish Forest Research Institute, Vantaa Research Station, P.O. Box 18, FIN-01301 Vantaa, FinlandeDepartment of Environmental Biotechnology, International Graduate School of Zittau, Markt 23, 02763 Zittau, Germany

a r t i c l e i n f o

Article history:

Received 16 December 2010

Revision received 12 May 2011

Accepted 20 July 2011

Available online 21 September 2011

Corresponding editor:

Petr Baldrian

Keywords:

Enzyme extraction

Fungi

Peroxidase

Sampling method

Screening

* Corresponding author. Tel.: þ358 9 191 59 3E-mail address: [email protected]

1754-5048/$ e see front matter ª 2011 Elsevdoi:10.1016/j.funeco.2011.07.008

a b s t r a c t

A novel sampling method to evaluate extracellular fungal enzyme activities was developed

and the validity tested for agar media. The method is based on centrifugation of small agar

pieces taken from growing fungal solid-state cultures. Centrifuge tubes that allow spinning

liquid out from small samples containing, for example, the hyphal front of a growing

mycelium are essential for the protocol. Centrifugation recovers a liquid phase from the

samples, which contains soluble material including many enzymes. The recovery of two

added model enzymes, namely laccase and manganese peroxidase (MnP), from agar media

was sufficient (ranging from 50 % to 75 %) but the addition of humic material into agar

decreased the observed MnP activity significantly to approx. 25 % of the stock solution. Using

growing cultures, the presence of humus as well as Scots pine sawdust on Hagem’s agar

plates induced the production of laccase and peroxidase in certain fungi, which indicates

that themethod is suitable for screening enzyme activities on different growthmedia or with

variable additives or growth conditions. The use of the presented sampling method for

functional enzyme fingerprinting of different fungi may be a promising tool for investigating

the behaviour and ecological role of forest soil fungi. This method also allows obtaining

spatial data from very small and defined areas of solid fungal cultures, e.g. frommicrocosms.

ª 2011 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction dilution of enzymes below the detection limit. As a conse-

Analysis of enzymatic activities in samples obtained from

solid media or solid environmental samples usually requires

the addition of water and/or buffer. This may lead to the

18; fax: þ358 9 191 59 32(G. Kabiersch).ier Ltd and The British M

quence, additional laborious and costly working steps to

concentrate the sample may be required. A common way to

extract enzymes from solid media is to add buffer (e.g. 50 mM

sodium acetate, pH 5.0) or water, soak and shake the samples

2.

ycological Society. All rights reserved.

262 J. Heinonsalo et al.

for a certain time and then filter the suspensions through filter

paper. The enzyme activities are afterwards measured in the

filtrates (�Snajdr & Baldrian 2006; Steffen et al. 2007; Elisashvili

et al. 2008; Isikhuemhen et al. 2009). As a consequence, many

current methods use relatively large samples (several grams)

for extraction (Dinis et al. 2009; Valent�ın et al. 2010). Further-

more, destructive harvesting for enzyme activity analysis

increases the need of replicates (e.g. culture flasks, bottles,

etc), especially when the effects ofmultiple factors on enzyme

production are screened.

To improve enzyme recovery, it is an advantage if the

amount of required steps is minimized, since each phase in

processing the samples usually results in a loss of activity.

Reduction in working effort, preparation costs and analysis

methods, as well as lowering the number of replicate growth

units, are important for practical and financial reasons. One

approach is to increase measurement sensitivity by using

fluorogenic substrates and measurement in suspensions

without extraction (e.g. Veps€al€ainen et al. 2001, 2004).

Here we describe a method where small pieces of agar

media with or without supplements of natural organic matter

can be sampled and analysed for extracellular enzyme activ-

ities. This facilitates studies on the impact of different ingre-

dients and growth conditions on enzyme production and

secretion, and the comparison of enzyme patterns of different

fungal strains. At optimal performance, this method does not

need any additional liquid for the extraction of enzymes.

Consequently, even low enzyme activities can be detected

allowing the determination of enzyme fingerprints and

spatially representative data. The aim of the study was to

describe the method, test its robustness and suitability for

screening experiments and discuss its potential application in

ecological studies on fungi.

Table 1 e The fungal strains and their ecology, strain and cult

Name Ecology

Irpex lacteus White-rot

Phanerochaete velutina White-rot

Physisporinus rivulosus White-rot

Sphaerobolus stellatus White-rot

Piptoporus betulinus Brown-rot

Postia caesia Brown-rot

Serpula lacrymans Brown-rot

Agrocybe praecox Litter-decomposing

Collybia dryophila Litter-decomposing

Hypholoma fasciculare Litter-decomposing

Stropharia aeruginosa Litter-decomposing

Stropharia hornemannii Litter-decomposing

Stropharia rugosoannulata Litter-decomposing

Cenococcum geophilum Ectomycorrhizal

Lactarius rufus Ectomycorrhizal

Piloderma olivaceum Ectomycorrhizal

Suillus bovinus Ectomycorrhizal

Suillus variegatus Ectomycorrhizal

Cadophora finlandica Root-endophytic

Meliniomyces variabilis Root-endophytic

Ophiostoma abietinum Root-endophytic

Phialocephala fortinii Root-endophytic

Rhizoscyphus ericae aggr. Root-endophytic

a FBCC¼ Fungal Biotechnology Culture Collection at University of Helsin

Materials and methods

Organisms, growth media and enzyme preparations

The strains used in the experiments are listed in Table 1 and

are currently deposited in Fungal Biotechnology Culture

Collection (FBCC) of the University of Helsinki, Finland (fbcc@

helsinki.fi).

Cultures were routinely grown at room temperature

(þ22 �C) on Petri dishes containing Hagem’s agar (HA; (Modess

1941)) with or without added humus or sawdust. HA contains

per litre: 0.5 g KH2PO4, 0.4 g NH4Cl, 0.5 g MgSO4� 7H2O, 0.5 ml

FeCl3 (1 % aqueous solution), 5 g glucose, 5 g malt extract, 15 g

agar. The pH was adjusted to 4.5e4.8. Humus was obtained

from Finnish boreal Scots pine (Pinus sylvestris) dominated

forest in M€ammil€a at Hyyti€al€a forest research station of the

University of Helsinki (for detailed site description, see

Ilvesniemi et al. 2000), sieved through a 4 mm mesh and dried

at room temperature before being autoclaved twice at 121 �Cfor 15 min. Sawdust was obtained from dry, debarked Scots

pine logs. The sawdust was dried and autoclaved like humus.

Humus and sawdust plates were prepared by adding 1 ml

suspensions (containing approx. 340 and 85 mg solidmaterial,

respectively) on top of freshly poured but not yet solidified

agar plates. In this way, the humus and sawdust were incor-

porated into the top layer of the agar and could come into

immediate contact with the growing fungus.

In another experiment, special agar plates were prepared

with spent coffee ground (CG), an organic waste product. It

contains caffeine, tannins and polyphenols (Fan et al. 2000),

which may have an impact on production of lignin-degrading

enzymes. The CG medium contained 9.5 g l�1 (20 g l�1 wet

ure collection number information

Strain Collection number

CCB-196 FBCCa 384

T244i FBCC 941

T241i FBCC 397

PO203 FBCC 253

T35 FBCC 703

T91 FBCC 757

Md1144/VTT FBCC 1009

TM 70.84 FBCC 476

K209 FBCC 626

CCBAS 281 FBCC 1034

K47 FBCC 521

K122 FBCC 565

11372 FBCC 475

JH101 FBCC 1388

JH84 FBCC 1389

JH33 FBCC 1391

JH95 FBCC 1392

JH35 FBCC 1390

JH163 FBCC 1393

JH31 FBCC 1394

JH142 FBCC 1395

JH103 FBCC 1396

JH135 FBCC 1397

ki.

Filter centrifugation as a sampling method 263

weight) autoclaved, spent coffee ground that was obtained

after preparing espresso from Puro� Fairtrade espresso beans

(Miko Koffie NV, Turnhout, Belgium), and 15 g l�1 agar.

Laccase (34.7 Umg�1 protein from Trametes versicolor) and

manganese peroxidase (MnP) (22 Umg�1 protein from Nem-

atoloma frowardii) preparations used in the tests were obtained

from Jena Bios GmbH (Jena, Germany).

Enzyme sampling

A circular agar plug (diameter 8 mm, volume approx. 250 ml)

from the growing hyphal front (age of themyceliumwas 1e2 d

for quickly growing species and 1e6 d for slowly growing

species) was cut using a cork borer. The plug was placed into

a Costar� Spin-X� CLS8162 (Corning Inc., NY, USA) centrifuge

tube filter containing a cellulose acetate membrane with

a pore size of 0.45 mm and then centrifuged at þ20 �C and

15 700�g for 30 min; in centrifugal force tests, plugs were

treated in the same way but at different rotation centrifugal

force (200, 2700, 7900 and 15 700�g). Approximately 150e200 ml

of solution was obtained after spinning and the whole volume

was used immediately for enzyme assays.

Enzyme assays

To assess the activity of laccase and peroxidase in a coupled

assay, 2,20-azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS)

was used as substrate and the resulting colour change was

measured spectrophotometrically at a wavelength of 420 nm

(Hofrichter et al. 1998). Briefly, 0.99 ml of the reaction solution

contained: 50 mM sodium malonate (pH 4.5), 0.5 mM MnSO4,

1 mMABTS, sample. 10 ml H2O2wasaddedafter 20 s togive1 ml

final reaction volume and a final concentration of 0.1 mM, and

ABTS oxidation was followed for another 20 s. Peroxidase

activity was corrected through the value of laccase activity.

To evaluate the suitability of the centrifugation sampling

method for functional enzyme activity fingerprinting, the

ZymProfiler� method (Veps€al€ainen et al. 2001, 2004) was also

used. In brief, using fluorogenic substrate analogues, the

activities of a-glucosidase, b-glucosidase, b-xylosidase, cello-

biosidase, N-acetylglucosaminidase, phosphomonoesterase,

phosphodiesterase, arylsulphatase, leucine-aminopeptidase

and alanine-aminopeptidase were measured. Substrate and

4-methylumbelliferone (MUF) standard solutions were freeze-

dried in 96-well plates and the latter stored at �20 �C. Enzyme

activities were measured with four replicates using a substrate

concentration of 500 mM, except for N-acetylglucosaminidase

where 200 mM substrate was used. Standards were measured

with three replicates at MUF concentrations ranging from

0.5 mM to 100 mM. No standard curve was used for aminopepti-

dases and only the intensity of the fluorescence signal was

analysed. The samples were diluted with MUB (modified

universal buffer (Tabatabai 1994)) at pH 4.0 so that the final

dilutionswere1:100and1:1000.MUBstocksolutioncontainsper

litre: tris(hydroxymethyl)aminomethane 12.1 g, maleic acid

11.6 g, citric acid 14.0 g, boric acid 6.3 g, sodium hydroxide

(1 mol l�1) 488 ml, deionizedwater added tomakeup to 1000 ml.

For theworkingbuffer, pHof 200 ml stock solutionwasadjusted

with 0.1 mol l�1 hydrochloric acid or sodium hydroxide, and

deionizedwaterwasadded toobtainafinal volumeof1000 ml.A

volume of 200 ml of the diluted samples was added into the

substrate and standard plates. The plates were incubated at

þ20 �C for 3 hr, 6 hr, 24 hr and 30 hr. Fluorescence values were

measured with Wallac 1420 Victor2 (PerkinElmer, Inc., USA)

multilabel plate reader using excitation at l¼ 355 nm and

emission at l¼ 460.

Testing the sampling method

Three experiments were performed to test the reliability of

tube centrifugation as a sampling method for enzyme assays.

Laccase and MnP were used as model enzymes due to their

simple and rapid analysis procedure in the laboratory.

In the first experiment, different growth media and

different supplements were tested to estimate the recovery of

a known amount of added enzyme from a stock solution

injected into the agar plugs (20 ml). After injection, the agar

with the added enzyme was incubated for 1 hr at room

temperature. Then the samplewas centrifuged at 15 700�g for

30 min before beingmeasured for enzyme activities. Theword

recovery is used in this study to indicate the detected enzyme

activity after addition of a measured and known amount of

tested enzyme.

In the second experiment, we evaluated the effect of the

centrifugal force (from 200 to 15 700�g) on extractable enzyme

activities from living fungal agar cultures. The aim was to

estimate whether a high centrifugal force may result in the

partial disruption of fungal cells, which would liberate e

against the scope of the method e intracellular enzymes into

the measurement solutions. A sonication treatment was also

applied to see if sonication (30 min) in combination with

centrifugation (15 700�g) could release intracellular or cell

wall-bound enzymes to the supernatant thus increasing

oxidative enzyme activities.

In the third experiment, the effect of the liquid volume on

the recovery of added enzyme activities was analysed. The

same quantities of model enzymes were added, without agar,

into the centrifugation tubes but diluted to different volumes

(50, 150 and 300 ml). The aim was to evaluate whether

a possible retaining of enzymes in the centrifugation tube is

volume-dependent and whether the results could be affected

by the moisture content of the samples.

Culture tests

A set of three different experiments was performed to test the

suitability of the method for: (i) the screening fungal culture

collections for laccase and peroxidase producers; (ii) spatial

distribution of enzyme activities; and (iii) functional enzyme

activity fingerprinting.

In the first experiment, a set of 23 different fungal strains

was studied for their laccase and peroxidase production.

These strains comprised white-rot, brown-rot, litter-

decomposing, ectomycorrhizal and root-endophytic fungi,

thus covering the major eco-physiological groups of fungi

found in boreal forest soils (Table 1). The strains were grown

on HA and on HA agar plates supplemented with humus or

sawdust. The samples were taken from the hyphal front

growing on the agar media and the liquid was recovered by

centrifugation at 15 700�g for 30 min. In addition to absolute

Fig 1 e The effect of growth media on activity of added

laccase and MnP in the centrifugation assay. Stock solution

activity describes the activity before addition to agar and

before extraction; extracted stock shows the activity of

stock solution after centrifugation treatment in the

absence of agar medium. Different letters above the

standard deviation bars indicate statistically significant

(P £ 0.05) differences between treatments (N[ 3).

264 J. Heinonsalo et al.

activities, the results are presented as corrected values using

the correction coefficient obtained from the recovery tests.

In the second experiment, the litter-decomposer Agrocybe

praecoxwas grown on CG agar. On four different days, samples

were taken from different parts of plates representing

different ages of the mycelium. Laccase and peroxidase

activities were determined in the liquids obtained after

centrifugation.

In the third experiment, two fungal strains of different

eco-physiological groups (a litter-decomposing and an ecto-

mycorrhizal fungus), A. praecox and Piloderma olivaceum, pre-

grown on HA or HA supplemented with Mn(II) (as

MnCl2� 4H2O 100 mg l�1), humus or sawdust, were tested for

the production/secretion of 12 different enzymes that are

important in carbon, nitrogen, sulfur and phosphorus utili-

zation from organic sources. Sampling was performed as

described for the first experiment.

Statistical analyses

The data were analysed using a one-way ANOVA following

Tukey’s HSD post hoc-test using P¼ 0.05 as limit for statistical

significance. Homogeneity of variances and the normality of

the data were tested and in case the requirements of ANOVA

were not met even after log or square root transformations,

the non-parametric ManneWhitney test was used. The

statistical analyses were carried out using SPSS for Windows

version 16.0 software (SPSS Inc. USA).

Results

Verification of the sampling method

Known amounts of laccase and MnP were added to different

agar media, left to settle for 1 hr at room temperature before

centrifugation sampling. In the case of laccase, enzyme

activity in the agar media was generally and significantly

(P� 0.05) lower than the added enzyme activity (25 % to 45 %

less) but no differences were found between the agar media

(Fig 1). In the case of MnP,mediawith humus addition showed

significantly lower enzyme activity in relation to the added

stock solution (Fig 1). However, there were no significant

differences (P> 0.05), probably due to the large variation

(25e75 %). The correction coefficient of enzyme activity (100

divided by the average activity percentage) for laccase on HA,

HAwith humus and HAwith sawdust were 1.84, 1.35 and 1.62,

respectively. The coefficients for MnP were 1.66, 4.08 and 1.84,

respectively.

The effect of the centrifugal forces on laccase and peroxi-

dase activity was studied using living cultures of A. praecox

and Collybia dryophila (Fig 2). In general, a force of 15 700�g

gave the highest activity, although the difference was in most

cases not significant (P> 0.05). Only the peroxidase activity of

A. praecox increased with increasing force. Sonication did not

further increase the detectable enzyme activities, so it seems

that a partial lysis of hyphae did not occur at these centrifugal

forces and intracellular enzymes (e.g. some laccase isoen-

zymes) were not liberated into the solution; on the contrary,

enzyme activities always decreased after the sonication

treatment (Fig 2). The volume of the enzyme solution did not

have any significant effect on the percentage of enzyme

activity indicating that the filter does not retain comparatively

more enzymes when small volumes are used (Fig 3).

Culture tests

Laccase (Table 2A) and peroxidase (Table 2B) activities were

measured for a set of 23 basidiomycetous fungi and corrected

by the correction coefficients obtained from Fig 1. In the case

of laccase, higher activities weremeasured when themedium

contained humus or sawdust, sometimes the activities were

several hundred-times higher (e.g. in case of A. praecox).

However, the effect of additives differed clearly between the

species. In the case of peroxidase, the same trend was

observed with considerable enhancement of activities on

plates with organic additives. When the data were corrected

using the coefficients, they suggest low but significant

peroxidase activities also for some ectomycorrhizal and

root-endophytic ascomycetous fungi (Table 2B). Whether

these activities are really based on MnP enzyme proteins or

may be the result of other (bio)chemical processes slowly

oxidizing Mn(II) or even just assay artefacts will have to be

clarified in additional studies.

In the second experiment, the spatial distribution of lac-

case and peroxidase activities of A. praecox was determined

(Fig 4). Agar plugs were taken on different days and from

different areas of the plate. The use of the filter centrifugation

method for single agar plugs allowed visualizing the spatial

distribution of laccase and peroxidase (Fig 4A). Based on

a growth curve (data not shown), the actual age of the myce-

lium at the sampling time point was calculated and plotted

together with themeasured enzyme activities to obtain a time

Fig 3 e The effect of sample volume on laccase and MnP

activity. Different letters above the standard deviation bars

indicate statistically significant (P £ 0.05) differences

between treatments (N[ 3). Separate statistical tests are

performed for different enzymes.

Fig 2 e The effect of centrifugal forces on laccase and

peroxidase activity. The test was performed using living

cultures of the litter-decomposing fungi A. praecox and C.

dryophila growing on HA medium. Sonic means that the

sample was ultrasonicated for 30 min before

centrifugation. Different letters above the standard

deviation bars indicate statistically significant (P £ 0.05)

differences between treatments (N[ 3). Separate statistical

tests are performed for different fungi and enzymes.

Filter centrifugation as a sampling method 265

course of enzyme activities (Fig 4B). High laccase activities

were found in the growing part of the mycelium. Laccase

activity appeared to be stable before its activity decreased in

the area of around 11-d-old mycelium. Peroxidase seemed to

be secreted later than laccase and after hyphae had success-

fully established on the growth substratum. Also peroxidase

activity appeared to remain stable up to 10 d in agar plates

before measurable activity started to decrease.

In the third experiment, the sampling method was used to

analyse the activities of 12 enzymes from two fungal strains,

A. praecox and P. olivaceum; the species represent two different

eco-physiological fungal groups in soil-litter-decomposing

and ectomycorrhizal fungi respectively. Clear differences in

the enzyme fingerprint between studied species were evident

(Table 3). Mn(II) addition stimulated most enzymes in both

species, humus particularly induced activities in A. praecox

and sawdust in P. olivaceum. A. praecox did not show any

activity of arylsulfatase nor of a-glucosidase. P. olivaceum did

not show any activities of leucine- and alanine-

aminopeptidase, nor of peroxidase and hardly any activity of

a-glucosidase. Also, the level of enzyme activities differed

substantially between the species.

Discussion

Sampling method

The application of centrifuge tube filters as a method for

fine-scale sampling of fungal enzymes proved to be useful in

Petri dish experiments. During centrifugation, aqueous and

soluble compounds including enzymes were recovered. The

percentage detected activity of the model enzymes (laccase

and MnP) was sufficient, although that of MnP was probably

affected by humus (Fig 1). This could be due to adsorption of

the enzyme or an interference of humic ingredients with the

enzyme activity measurement. The inhibitory effect of HA on

laccase has been very well demonstrated by Zavarzina et al.

(2004) and was considered to be due to the functional

groups, molecular weight and hydrophobicity of the HA

preparations (Zavarzina et al. 2004). Furthermore, in our

particular test, a definite activity of enzyme was added by

injection to the agar. So it is possible that the injection created

a hole in the agar and only part of the enzyme added was

absorbed into the agar.

Veps€al€ainen et al. (2001) concluded that soil extracts cannot

be used to assess the enzyme activities of bulk soil due to very

low protein recovery. In our case, although so far not estab-

lished with authentic soil but with soil-like organic additives,

the apparent enzyme activity was inmost cases between 50 %

and 75 % of that added, which is quite high compared to

previous studies. The agar media used in our study differ in

the adsorption properties fromnatural samples but they serve

as suitable model to mimic the fate of enzymes in complex

solid materials. Especially the addition of humus resulted in

low enzyme activities. It is uncertain whether this is due to

protein absorption, enzyme inactivation or the interference of

humic substances with the enzyme assays. To determine the

Table 2 e The production of laccase (2A) peroxidase (2B) on HAwith and without added sterilized boreal forest humus and Scots pine sawdust by a selection of white-rot,brown-rot, litter-decomposing, ectomycorrhizal and root-endophytic fungi. The values that are corrected using correction factors obtained in tests presented in Fig 1 arealso presented. The factors are for laccase on HA, HA with humus and HA with sawdust 1.84, 1.35 and 1.62, and for MnP 1.66, 4.08 and 1.84, respectively

HA HAþhumus HAþ sawdust

Measuredaverage

stdev Correctedaverage

stdev Measuredaverage

stdev Correctedaverage

stdev Measuredaverage

stdev Correctedaverage

stdev

A. Laccase U/cm2

Irpex lacteus (WR) 1.0 0.7 1.8 1.4 0.0 0.0 0.0 0.0 7.6 6.6 12.3 10.7

Phanerochaete velutina (WR) 2.8 2.8 5.2 5.2 0.0 0.0 0.0 0.0 12.0 3.7 19.4 6.0

Physisporinus rivulosus (WR) 68.3 3.7 125.9 6.9 679.8 69.6 919.4 94.2 272.2 128.7 440.4 208.2

Sphaerobolus stellatus (WR) 28.8 5.4 53.1 10.0 124.8 112.4 168.8 152.1 63.5 35.1 102.8 56.7

Piptoporus betulinus (BR) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Postia caesia (BR) 7.9 7.5 14.5 13.9 2.0 2.2 2.7 2.9 0.3 0.5 0.5 0.8

Serpula lacrymans (BR) 1.0 1.8 1.9 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Agrocybe praecox (LDF) 0.6 1.0 1.1 1.9 256.0 85.5 346.2 115.6 1.1 1.9 1.8 3.1

Collybia dryophila (LDF) 253.7 52.6 468.0 97.0 307.1 57.3 415.3 77.6 412.1 46.1 666.8 74.7

Hypholoma fasciculare (LDF) 42.1 13.0 77.7 24.0 59.2 11.0 80.1 14.9 24.5 10.3 39.6 16.7

Stropharia aeruginosa (LDF) 1.2 1.1 2.2 2.1 28.3 43.9 38.3 59.4 19.1 2.5 30.9 4.0

Stropharia hornemannii (LDF) 8.2 1.4 15.1 2.5 326.6 45.4 441.7 61.4 114.5 18.3 185.3 29.7

Stropharia rugosoannulata (LDF) 0.0 0.0 0.0 0.0 4.9 8.4 6.7 11.3 2.1 3.1 3.4 5.0

Cenococcum geophilum (ECM) 9.9 2.6 18.4 4.7 33.9 1.9 45.9 2.6 10.3 1.0 16.6 1.7

Lactarius rufus (ECM) 38.0 3.4 70.0 6.3 0.0 0.0 0.0 0.0 131.5 4.0 212.8 6.5

Piloderma olivaceum (ECM) 20.7 7.8 38.1 14.5 100.3 8.8 135.7 12.0 146.6 66.2 237.1 107.2

Suillus bovinus (ECM) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Suillus variegatus (ECM) 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.2 0.0 0.0 0.0 0.0

Cadophora finlandica (ENDO) 4.8 2.9 8.9 5.3 0.3 0.5 0.4 0.6 4.8 7.5 7.8 12.1

Meliniomyces variabilis (ENDO) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 1.9 2.1 3.1

Ophiostoma abietinum (ENDO) 0.0 0.0 0.0 0.0 0.2 0.3 0.3 0.5 0.0 0.0 0.0 0.0

Phialocephala fortinii (ENDO) 1.3 2.3 2.4 4.2 0.5 0.9 0.7 1.3 4.5 1.4 7.3 2.3

Rhizoscyphus ericae aggr. (ENDO) 0.1 0.2 0.2 0.3 0.7 1.0 0.9 1.4 0.1 0.2 0.2 0.3

B. Peroxidase U/cm2

Irpex lacteus (WR) 3.4 3.3 5.6 5.5 42.1 13.1 171.5 53.4 45.9 34.6 84.4 63.5

Phanerochaete velutina (WR) 199.0 44.5 330.1 73.9 0.0 0.0 0.0 0.0 153.3 211.2 281.6 387.9

Physisporinus rivulosus (WR) 92.9 22.8 154.0 37.8 123.8 13.2 504.6 53.9 51.3 36.8 94.2 67.6

Sphaerobolus stellatus (WR) 77.2 24.5 128.0 40.6 195.6 99.5 797.3 405.5 113.0 37.7 207.6 69.3

Piptoporus betulinus (BR) 6.5 11.3 10.8 18.7 2.0 3.4 8.0 13.9 0.0 0.0 0.0 0.0

Postia caesia (BR) 3.6 3.8 6.1 6.4 9.2 3.7 37.3 15.2 7.5 2.7 13.8 5.0

Serpula lacrymans (BR) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1

Agrocybe praecox (LDF) 1.4 1.1 2.3 1.9 59.0 20.3 240.5 82.8 3.7 3.2 6.8 5.8

Collybia dryophila (LDF) 0.0 0.0 0.0 0.0 20.2 13.3 82.3 54.3 1359.5 485.8 2496.8 892.2

Hypholoma fasciculare (LDF) 505.0 415.5 837.5 689.2 4.3 2.6 17.7 10.5 239.8 185.8 440.5 341.2

Stropharia aeruginosa (LDF) 82.2 83.7 136.3 138.9 725.2 862.5 2956.2 3515.8 223.4 57.4 410.2 105.4

Stropharia hornemannii (LDF) 32.1 28.5 53.2 47.3 234.6 33.3 956.2 135.7 182.1 93.7 334.4 172.1

Stropharia rugosoannulata (LDF) 13.2 6.0 21.9 9.9 171.1 38.6 697.4 157.4 168.1 48.3 308.7 88.6

266

J.Heinonsa

loet

al.

Cen

ococcu

mgeop

hilum

(ECM)

0.3

0.5

0.5

0.8

4.3

4.0

17.5

16.3

0.0

0.0

0.0

0.0

Lactariusrufus(ECM)

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Pilod

ermaolivaceum

(ECM)

4.5

4.6

7.5

7.7

14.9

5.8

60.6

23.5

0.4

0.7

0.8

1.4

Suillusbov

inus(ECM)

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.5

0.8

0.9

1.5

Suillusvariegatus(ECM)

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.1

0.1

0.1

Cadop

horafinlandica(ENDO)

4.6

4.0

7.6

6.6

7.7

4.7

31.2

19.0

6.8

2.6

12.5

4.8

Meliniomyces

variabilis

(ENDO)

6.7

3.8

11.2

6.3

2.1

1.3

8.6

5.3

6.5

1.2

11.9

2.2

Ophiostom

aabietinum

(ENDO)

0.2

0.3

0.3

0.5

2.7

3.4

11.1

13.9

0.0

0.0

0.0

0.0

Phialocephala

fortinii(ENDO)

0.7

1.2

1.1

2.0

1.8

2.6

7.2

10.5

19.6

9.4

36.0

17.2

Rhizoscyphusericaeaggr.

(ENDO)

6.9

0.8

11.4

1.3

36.1

1.8

147.1

7.5

2.7

0.4

4.9

0.7

Fig 4 e Growth of Agrocybe praecox, and laccase and

peroxidase activity on agar based on spent coffee ground.

(A) Sampling was performed at four different times (7, 11,

18 and 30 d after inoculation) from three different parts of

the plate. Enzyme activities are expressed as mUmlL1 for

the investigated agar plug with a surface area of 50 mm2:

laccase, black figures in white squares; peroxidase, white

figures in black squares. The enzyme activities represent

the actual values measured in this plate. (B) Time course of

enzyme activities. Age of the mycelium on the sampling

time point was calculated according to a growth curve (not

shown) and plotted against the measured enzyme

activities. Error bars show the standard deviation of

enzyme activities (N[ 3).

Filter centrifugation as a sampling method 267

quantities of total protein in soil solutions, several commer-

cial kits are available. However, when the kits were compared,

none of the kits tested (e.g. Bradford reagent kit, Coomassie�

dye binding protein assay reagent, QuantiPro� BCA assay,

FluoroProfile� protein quantification kit) could provide a reli-

able indicator of the soil solution protein content due to

significant interference with high MW humic substances also

present in the solution (Roberts & Jones 2008). For that reason

we did not measure the total protein in the tests performed.

If the enzyme of interest is available in sufficient amounts

(commercial product or own preparations), the recovery

Table 3 e The summary of the 12 enzyme activities analysed (semiquantitative from no activity[L to strong activity[DDD). The fungi were grown on Hagem’s agar without supplements or supplemented with Mn II, humus or Scots pinesawdust

Enzyme activity A. praecox P. croceum Control

Mn II Humus Sawdust Hagem’s Mn II Humus Sawdust Hagem’s Hagem’s

Arylsulphatase � � � � � � � � �a-glucosidase � � � � � � � � �b-glucosidase þ � � � þ þ þþþ � �Cellobiosidase � � � � � � � � �b-xylosidase þ � � � � � þþþ � �Phosphomonoesterase þþ � � � þþ � þþþ � �Phosphodiesterase þ � � � � � þþþ � �N-acetylglucosamidase þþ � � � þþþ � � � �Leucine-aminopeptidase � � � � � � � � �Alanine-aminopeptidase � � � � � � � � �Laccase þþþ þþþ � � � þ þ � �Peroxidase þþþ þ � � � � � � �

268 J. Heinonsalo et al.

percentage can be calculated to obtain a correction coefficient

that can be later used to adjust results of strain screening

experiments. In our case, the correction coefficient for MnP in

agar plates supplemented with humus was 4.1, which signif-

icantly affected the results obtained. Despite the reduced

enzyme activities observed after artificial supplementation of

the agar with model enzymes (probably caused by the organic

additives/ingredients), the lack of a dilution effect in this

sampling method results in good sensitivity and improved

detection of enzymatic activities even at very low levels.

In the centrifugal force test, the aim was to verify that the

fungal hyphae do not lyse and release intracellular enzymes

to the sample solution. In three out of four tests, there was no

significant (P> 0.05) difference between the highest and

lowest centrifugal force, which demonstrates that the cells do

not break due to the centrifugal force. In the case of A. praecox

peroxidase the highest force yielded a higher activity than the

lower centrifugal forces (Fig 2) indicating a correlation

between centrifugal force and recoverable peroxidase activity.

Sonication decreased the activities both of laccase and

peroxidase suggesting that ultrasound waves have damaging

effects on the enzyme proteins. This phenomenon has been

known for decades and is a general problem of intracellular

enzyme preparation using sonication for cell disruption

(Coakley et al. 1973).

Different amounts of added enzymes did not have any

effect on the activities in the volumetric test, which means

that also in case of small volumes (around 50 ml) enzymes are

as efficiently recovered from the filters as in the case of larger

solution volumes (up to 300 ml). This is important if the

method is used to detect activities in small fine-scale samples

with no water (or other buffer/extractant) additions and

variable moisture content.

Culture tests

The filter centrifugation method proved to be useful in

detecting potential laccase and peroxidase producers in pure

cultures. It also facilitated the use of different solid or liquid

supplements in simple Petri dishes, which makes it easier to

screen growth media for optimization of enzyme production.

Fungi producing high amounts of enzymes in liquid cultures

did so also on agar plates (data not shown).

The spatial distribution of laccase and peroxidase activity

determined at several points during the cultivation of A.

praecox on agar plates also reflected the temporal develop-

ment of its colony. Traditionally, extracellular enzyme activ-

ities have been localized and visualized by staining agar plates

(White & Boddy 1992; Hiscox et al. 2010). In a study of Dinis

et al. (2009), enzyme production of four fungi during growth

on wheat straw was investigated. At each sampling time the

whole culture was harvested. In our experiment, samples at

different time points and from different areas were just taken

from one set of agar plates (three replicate plates). Estimation

of themycelium age based on a growth curve allowed plotting

of the enzyme activities as a time course of measured enzyme

activities during solid-state cultivation (Fig 4B). It was neither

necessary to stain the plates nor to apply laborious extraction

steps to determine and quantify enzyme activities. Further-

more the results may give hints about which part of the

mycelium actively produces enzymes at a certain time point.

Enzyme activity fingerprints for two different types of fungi

were obtained using the centrifugation sampling method. Two

species, A. praecox and P. olivaceum, had clearly different func-

tional profiles with different levels of activities. A. praecox

produced all envisaged enzymes except arylsulfatase and

a-glucosidase. P. olivaceum produced all others except a-gluco-

sidase, leucine- and alanine-aminopeptidase and peroxidase.

Enzyme production by P. olivaceum and A. praecox was strongly

stimulated by addition of sawdust and humus, respectively.

This kind of functional profiling may be useful to identify and

segregate functional groups of fungi, which in turn could be

used to highlight roles of cryptic species in forest soil

ecosystems.

Applicability of the sampling method

The method presented here facilitates studies on fungal

ecology using small, fine-scale sampling from fungal cultures

growing on different agar media. The recovery of model

enzymes was reasonably good from agar media. Thus, the

method allows screening for a variety of enzymes and also

Filter centrifugation as a sampling method 269

facilitates investigation of the effect of various supplements

on growth and enzyme production. Even the conversion of

colourless toxic additives might be studied, if subsequent

analysing tools for small volumes become available.

Further studies are needed to test the suitability of the

method for soil samples, since low recoveries with several

extraction methods were previously reported for soil samples

(Veps€al€ainen et al. 2001). The possibility of taking small-scale

samples may allow the detection of spatial and temporal

variation in enzyme activities in different kinds of micro- and

mesocosms where fungal hyphae can be harvested and

samples taken with precision. Our proposed method may be

applicable for the use of small amounts of soil, or even wood,

to give sufficient amounts of enzyme-containing liquid easily

utilisable for further analysis.

Acknowledgement

This research was funded by Academy of Finland (project

number 1212915 and 130984 for J.H., project number 206085 for

H.I., and project number 1133022 for G.K.) and EnSTe Graduate

School (for G.K.).

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