Comparative examination of the olive mill wastewater biodegradation process by various wood-rot macrofungi.

Olive mill wastewater (OMW) constitutes a major cause of environmental pollution in olive-oil producing regions. Sixty wood-rot macrofungi assigned in 43 species were evaluated for their efficacy to colonize solidified OMW media at initially established optimal growth temperatures. Subsequently eight strains of the following species were qualified: Abortiporus biennis, Ganoderma carnosum, Hapalopilus croceus, Hericium erinaceus, Irpex lacteus, Phanerochaete chrysosporium, Pleurotus djamor, and P. pulmonarius. Fungal growth in OMW (25%v/v in water) resulted in marked reduction of total phenolic content, which was significantly correlated with the effluent's decolorization. A. biennis was the best performing strain (it decreased phenolics by 92% and color by 64%) followed by P. djamor and I. lacteus. Increase of plant seeds germination was less pronounced evidencing that phenolics are only partly responsible for OMW's phytotoxicity. Laccase production was highly correlated with all three biodegradation parameters for H. croceus, Ph. chrysosporium, and Pleurotus spp., and so were manganese-independent and manganese dependent peroxidases for A. biennis and I. lacteus. Monitoring of enzymes with respect to biomass production indicated that Pleurotus spp., H. croceus, and Ph. chrysosporium shared common patterns for all three activities. Moreover, generation of enzymes at the early biodegradation stages enhanced the efficiency of OMW treatment.

1. Introduction

Three-phase centrifugal olive mills are widespread in most olive-oil producing countries and their operation results in the production of olive oil, solid pomace, and olive mill wastewater (OMW). The disposal of the latter creates major environmental problems since it is rich in organic compounds and contains large amounts of polyphenolics in high (and therefore) toxic concentrations [1, 2]. In addition, the olive mills seasonal operation, small size, and scattered distribution hinder the effective management of the effluent [3].

Among the large variety of methodologies adopted for the treatment of OMW [4-7], implementation of biological approaches is of particular importance. For example, the use of microorganisms could result in both the detoxification of the effluent and the generation of value-added products, for example, xanthan gum, citric acid, ethanol, antioxidants, polyhydroxyalkanoates, b-glucan, lipase, single cell oil, and edible biomass [8-18].

In particular, wood-rot macrofungi constitute a group of organisms equipped with a potent biochemical arsenal including one or more nonspecific groups of enzymes, which permits them to effectively decompose various lignocellulosic compounds [19-21]. The chemical affinity of the latter to a wide range of agroindustrial wastes (including OMW) led to their treatment with filamentous basidiomycetes originally isolated from woody substrates. Therefore, such organisms were examined either at a OMW pretreatment stage for decreasing the effluent's high content of phenolics and hence toxicity prior to its processing with other microorganisms [22, 23] or as the main/sole agents for OMW's remediation [24-27]. However, most pertinent investigations have focused on the exploitation of a few species only, for example, Phanerochaete chrysosporium, Ph. flavido-alba, Pleurotus ostreatus, and P. eryngii [28-32], and consequently a large number of other potentially useful macrofungi remain understudied or not examined at all.

The objective of the present work was the comparative evaluation of a wide variety of mushroom fungi for OMW biotreatment and the subsequent establishment of a battery of strains able to efficiently degrade OMW. The process was assessed by determining the activities of ligninolytic enzymes produced by qualified organisms at different time periods and through their association with concomitant OMW decolorization, reduction of phenolics, and decrease of plant toxicity.

2. Materials and Methods

2.1. Fungal Strains

Sixty wood-rot macrofungi belonging to 43 species of basidiomycetes (phylum Basidiomycota) were evaluated for the purposes of this study. Details of the strains identity are presented in Table 1. All the biological material is maintained in the fungal culture collection of the authors institution (AUA-LGAM).

Table 1

Details of the biological material evaluated for the purposes of this study.

Species	Geographic origin and host/substrate	Collection code
Abortiporus biennis	Czech Republic	ABC521
Abortiporus biennis	Greece	ABL436
Agrocybe cylindracea	China	ACL834
Auricularia mesenterica	Greece; Cupressus sp.	AML472
Coriolopsis trogii	Greece; Quercus sp.	CTL447
Daedalea quercina	Czech Republic	DQC528
Dichomitus squalens	Czech Republic	DSC750
Fistulina hepatica	Greece; Castanea sativa	FHL295
Flammulina velutipes	Commercial strain	FVS803
Fomitopsis pinicola	Greece; Abies cephalonica	FPL302
Ganoderma adspersum	Greece; Abies cephalonica	GAL401
Ganoderma carnosum	Greece	GCL642
Ganoderma carnosum	Greece; Olea sativa	GCL448
Ganoderma pfeifferi	Greece; Fagus sylvatica	GPL336
Ganoderma resinaceum	Greece; Morus alba	GRL334
Ganoderma resinaceum	Greece; Salix babylonica	GRL403
Ganoderma resinaceum	Czech Republic	GRC604
Grifola frondosa	Commercial strain	GFS805
Hapalopilus croceus	Czech Republic	HCC522
Hericium erinaceus	Greece	HEL801
Hericium erinaceus	Greece; Quercus pubescens	HEL802
Heterobasidion annosum	Greece; Abies cephalonica	HAL340
Hypsizygus ulmarius	Greece; Abies cephalonica	HUL417
Inocutis tamaricis	Greece; Tamarix hampeana	ITL314
Irpex lacteus	Czech Republic	ILC238
Laetiporus sulphureus	Greece; Castanea sativa	LSL331
Laetiporus sulphureus	Greece; Castanea sativa	LSL332
Lentinula edodes	Commercial strain	LES812
Neolentinus lepideus	Greece; Pinus nigra	NLL317
Omphalotus illudens	Greece; Quercus sp.	OIL347
Perenniporia fraxinea	Greece; Populus alba	PFL346
Phanerochaete chrysosporium	Greece	PHL322
Phanerochaete chrysosporium	Czech Republic	PHC571
Pholiota nameko	commercial strain	PNS806
Pleurotus abieticola	Russia	PAC854
Pleurotus citrinopileatus	Malaysia	PCC884
Pleurotus cornucopiae	Iran	PCL660
Pleurotus cystidiosus	USA; Populus deltoides	PCC897
Pleurotus cystidiosus subsp. abalonus	China	PAC891
Pleurotus djamor	Malaysia	PDC855
Pleurotus eryngii	Czech Republic	PEC847
Pleurotus eryngii var. eryngii	Greece; Eryngium sp.	PEL063
Pleurotus eryngii var. eryngii	Italy; Eryngium campestre	PEC810
Pleurotus eryngii subsp. tuoliensis	China	PEC856
Pleurotus flabellatus	Malaysia	PFC860
Pleurotus nebrodensis	Greece; Prangos ferulacea	PNL126
Pleurotus nebrodensis	Italy; Prangos ferulacea	PNC816
Pleurotus ostreatus	Italy	POC847
Pleurotus ostreatus	Czech Republic	POC843
Pleurotus ostreatus	Greece; Abies cephalonica	POL067
Pleurotus pulmonarius	Greece; Fagus sylvatica	PPL111
Pleurotus pulmonarius	France	PPC823
Pleurotus pulmonarius	Hong Kong	PSC757
Pleurotus tuber-regium	Papua New Guinea	PTC822
Stereum hirsutum	Czech Republic	SHC608
Trametes hirsuta	Czech Republic	THC610
Trametes ljubarskyi	Greece; Populus sp.	TLL473
Trametes versicolor	Greece	TVL469
Trametes versicolor	Czech Republic	TVC614
Tyromyces lacteus	Czech Republic	TLC616

2.2. Nutrient Media: Fungal Growth Substrates and Conditions

OMW was obtained from an olive-oil mill equipped with three-phase centrifugal decanters located in Kalamata (Peloponnese, S.W. Greece). Its composition and main physicochemical properties were previously assessed [2].

For the preparation of OMW-containing growth media, the effluent was adjusted at pH 6 by adding CaO and centrifuged for 20 min at 5000 g (5°C), and the supernatant was diluted or not with deionized water to prepare 25%, 50%, and 100% v/v OMW-based substrates, which were further heat-sterilized for 30 min (121°C, 1.1 atm). Fungal strains were subcultured on a 25% v/v OMW medium solidified with 1.7% w/v agar. Then, agar plugs (6 mm diam.) originating from the actively growing part of the fungal colonies were used to inoculate either solidified OMW media (for the purposes of mycelium growth rate determinations) or static liquid cultures (for OMW degradation experiments with selected strains).

2.3. Screening of Fungal Strains: Establishment of Temperature Optima and Measurements of Linear Growth Rates

The temperature optima for mycelium linear growth were assessed on potato dextrose agar (PDA; Conda) medium and over the range of 12–47°C with a 5°C graduation. Experiments were conducted in Petri dishes as previously described [33], and measurements were taken every 24 h for all species examined with the exception of Phanerochaete chrysosporium and Hapalopilus croceus strains whose growth was measured every 12 h until seven consecutive values were obtained for all strains.

Growth rates were determined by measuring the distance of the colony's front from the centre of the inoculum at four different points along two perpendicular lines [34]. Mycelium growth rates (k ) were calculated by fitting the linear growth function y = k x + c (where y is the distance covered by the hyphae and x is the respective time) and were expressed in mm d−1. All experiments were conducted in three replicates.

After establishing the optimal temperature for mycelium growth for each strain, all cultures were evaluated with respect to their growth rates on various OMW dilutions (25%, 50%, and 100% v/v OMW in water) at their optimal temperatures as previously determined, in the same way as described above. In addition, PDA was used as control. All mycelium growth rate experiments were conducted in three replicates.

2.4. Treatment of OMW by Qualified Fungal Strains: Assessment of Effluent's Selected Properties

The strains, which were qualified from the previous experiment, were further examined as regards their effect on OMW degradation process in liquid static cultures. The latter were performed in 250 mL Erlenmayer flasks containing 100 mL of 25% v/v OMW, at the optimal temperatures previously established for each strain, and lasted for a period from 20 to 30 days depending on the growth of each fungus, that is, Abortiporus biennis ABL436 (25 days), Ganoderma carnosum GCL448 (25 days), Hapalopilus croceus HCC522 (20 days), Hericium erinaceus HEL801 (25 days), Irpex lacteus ILC238 (25 days), Phanerochaete chrysosporium PHL322 (20 days), Pleurotus djamor PDC855 (30 days), and P. pulmonarius PPL111 (30 days). Four replicates were included for each treatment, while noninoculated substrates were also incubated in parallel and served as controls.

A destructive sampling process was adopted at five time points for dividing the entire cultivation at intervals of four, five, or six days (for growth periods of 20, 25, or 30 days, resp.), in order to determine pH, electric conductivity, biomass, total phenolics, decolorization, plant seed germination, and enzymes activity.

Measurements of pH and electric conductivity in OMW were performed by using a Corning EEL 12 pH meter and a Jenway 4010 conductivity meter, respectively. Biomass was harvested by filtration and its weight (mycelium dry weight) was determined by drying at 60°C until constant weight.

Total phenolics were analyzed by the Folin-Ciocalteu method [35], by measuring spectrophotometrically (at 760 nm) the formation of a blue complex as a result of the reduction of a phosphomolybdic-phosphotungstic reagent from the presence of phenolics. The concentration of total phenolics was determined against a syringic acid calibration curve (1 mg mL−1 syringic acid—initial concentration—resulted in an optical density of 0.377 at 760 nm).

Decolorization was estimated by measuring the absorbance of OMW samples at 525 nm using a U-2001 spectrophotometer (Hitachi Instruments Inc., USA).

The plant seed germination indexes were estimated on the basis of Zucconi et al. [36] protocol. Twenty-five cress seeds (Lepidium sativum L.) were placed on filter papers moistened with OMW (25%) and incubated in Petri dishes for three days at 25°C. The effect of OMW on germination was evaluated, and germination indexes (G.I.) were calculated as follows: G.I. = (% root elongation × % germination)/100 (control used: cress seeds moistened with water).

2.5. Determination of Enzymes Activities

Laccase (Lac, E.C. 1.10.3.2: benzenediol: oxygen oxidoreductase) activity was determined at 425 nm by oxidizing 0.4 mL ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) (1.5 mM) with 0.8 mL of OMW sample in 1.2 mL Na-tartrate buffer (0.1 M, pH 4.5) [37].

Manganese-independent peroxidase (MnIP) was measured at 590 nm by the oxidative coupling of 0.1 mL MBTH (3-methyl-2-benzothiazoline hydrazone) (1 mM) and 0.2 mL DMAB (3-dimethylaminobenzoic acid) (25 mM) in the presence of 0.01 mL H2O2 (10 mM) added to a solution consisting of 0.66 mL sample and 1 mL succinate-lactate buffer (0.1 M, pH 4.5), while background activity, determined as above in the absence of H2O2, was subtracted [32].

Manganese peroxidase (MnP, E.C. 1.11.1.13 (MnII): hydrogen peroxide oxidoreductase) activity was determined as described for manganese-independent peroxidase (MnIP) in the presence of 0.01 mL MnSO4 (20 mM), by subtracting MnIP activity [38]. For all enzymes determination, one activity unit was defined as the amount of enzyme transforming 1 μmol of substrate per minute.

2.6. Statistical Analysis

Analysis of variance (ANOVA) followed by Gabriel's multiple comparison tests (a < 0.05) was used to estimate statistical differences between treatment means through the use of SPSS (version 18) software. Standard deviations were calculated for all mean values, and regression analysis was carried out to evaluate relationships between variables at significance levels of 1% and 5%.

3. Results and Discussion

Data about the optimal conditions for the production of fungal biomass are available for a rather few species of macrofungi (i.e., mainly those exploited for edible mushroom production). The effect of temperature, in particular, is of fundamental importance in evaluating the biotechnological potential of such organisms [33, 34], and hence it was the first parameter which was assessed in the present study. The results of the comparative mycelium linear growth rate examination covering a wide temperature range (12–47°C) showed that most strains presented their optima at a narrow value area of 27–32°C (Table 2). Only Hypsizygus ulmarius, Omphalotus illudens, Pholiota nameko, and Pleurotus nebrodensis exhibited significantly higher growth rates at 22°C. On the other hand, Phanerochaete chrysosporium PHC571 was the only one presenting optimal growth at 37°C. It is noteworthy that almost all Pleurotus strains showed a growth (albeit limited) at 12°C; Pleurotus was the only genus among the 28 different genera evaluated demonstrating such a property. In contrast, Hapalopilus croceus and Phanerochaete chrysosporium were the only species showing hyphal development at the other temperature extreme (42°C). Furthermore, mycelium growth rates of almost all strains presented a gradual increase from lower temperatures to the optimal one and then a sharp decrease to the temperature of no growth; in fact, this effect was more pronounced for individuals demonstrating high growth values (Figure 1).

Table 2

Mycelium linear growth rates (K , mm day−1) for 60 strains of macrofungi established at different temperatures ranging from 12 to 47°C (ngd: no growth detected). Values are expressed as means ± standard deviation of means, n = 3. Lack of superscript letters commonly indicates statistically significant differences (Gabriel's t-test, P < 0.05) for comparisons of treatment means between different temperatures for each strain.

Strain	Temperature
	12	17	22	27	32	37	42	47
ABC521	ngd	4.06 ± 0.04d	7.46 ± 0.07c	11.47 ± 0.07a	10.17 ± 0.07b	1.47 ± 0.07e	ngd	ngd
ABL436	ngd	4.10 ± 0.18d	8.03 ± 0.03c	11.30 ± 0.08b	12.19 ± 0.29a	1.39 ± 0.08e	ngd	ngd
ACL834	ngd	1.64 ± 0.04c	3.12 ± 0.13b	4.02 ± 0.10a	3.06 ± 0.08b	ngd	ngd	ngd
AML472	ngd	ngd	6.24 ± 0.17b	6.97 ± 0.30a	5.62 ± 0.29c	ngd	ngd	ngd
CTL447	ngd	2.6 0± 0.08e	4.64 ± 0.15d	6.51 ± 0.13c	8.98 ± 0.15a	7.29 ± 0.02b	ngd	ngd
DQC528	ngd	3.94 ± 0.14e	7.03 ± 0.22c	7.81 ± 0.04b	11.14 ± 0.17a	4.34 ± 0.05d	ngd	ngd
DSC750	ngd	3.52 ± 0.13c	7.07 ± 0.04b	10.96 ± 0.17a	11.10 ± 0.12a	3.77 ± 0.04c	ngd	ngd
FHL295	ngd	0.78 ± 0.02b	2.10 ± 0.04a	1.91 ± 0.23a	0.66 ± 0.04b	ngd	ngd	ngd
FVS803	ngd	3.66 ± 0.15c	5.39 ± 0.03b	6.00 ± 0.14a	1.35 ± 0.11d	ngd	ngd	ngd
FPL302	ngd	2.34 ± 0.02d	5.55 ± 0.02c	7.61 ± 0.18b	7.94 ± 0.1a	ngd	ngd	ngd
GAL401	ngd	0.83 ± 0.15d	2.68 ± 0.35c	6.19 ± 0.25b	8.76 ± 0.23a	ngd	ngd	ngd
GCL642	ngd	3.39 ± 0.09d	5.95 ± 0.14b	7.28 ± 0.20a	4.21 ± 0.21c	ngd	ngd	ngd
GCL448	ngd	1.89 ± 0.17d	5.18 ± 0.15c	8.57 ± 0.18a	6.93 ± 0.10b	ngd	ngd	ngd
GPL336	ngd	1.12 ± 0.12c	2.64 ± 0.01b	4.21 ± 0.07a	0.45 ± 0.05d	ngd	ngd	ngd
GRL334	ngd	2.96 ± 0.07c	6.26 ± 0.10b	8.41 ± 0.32a	7.99 ± 0.14a	ngd	ngd	ngd
GRL403	ngd	2.43 ± 0.13d	6.43 ± 0.06c	8.96 ± 0.12a	8.29 ± 0.17b	ngd	ngd	ngd
GRC604	ngd	1.49 ± 0.11c	3.48 ± 0.11b	5.59 ± 0.02a	5.53 ± 0.07a	ngd	ngd	ngd
GRS805	ngd	0.41 ± 0.03b	3.28 ± 0.22a	3.63 ± 0.45a	ngd	ngd	ngd	ngd
HCC522	ngd	8.93 ± 0.03e	17.36 ± 0.77d	24.61 ± 0.47c	32.63 ± 0.58a	32.41 ± 0.47a	28.63 ± 0.12b
HEL801	ngd	6.89 ± 0.16c	9.69 ± 0.18b	13.17 ± 0.07a	12.25 ± 0.78a	ngd	ngd	ngd
HEL802	ngd	0.10 ± 0.05b	0.43 ± 0.06b	1.22 ± 0.27a	1.46 ± 0.11a	ngd	ngd	ngd
HAL340	ngd	2.79 ± 0.13c	6.46 ± 0.20b	8.29 ± 0.07a	8.48 ± 0.08a	ngd	ngd
HUL417	ngd	1.03 ± 0.13c	2.21 ± 0.04a	1.49 ± 0.31b	0.08 ± 0.03d	ngd	ngd	ngd
ITL314	ngd	0.46 ± 0.00d	2.92 ± 0.18c	4.44 ± 0.96b	4.96 ± 0.08a	3.05 ± 0.08c	ngd	ngd
ILC238	ngd	4.20 ± 0.06d	7.65 ± 0.24c	10.83 ± 0.24b	12.19 ± 0.27a	2.51 ± 0.10e	ngd	ngd
LSL331	ngd	3.78 ± 0.23c	5.79 ± 0.05b	7.92 ± 0.17a	7.84 ± 0.24a	ngd	ngd	ngd
LSL332	ngd	1.17 ± 0.24d	3.53 ± 0.16c	5.73 ± 0.17b	7.84 ± 0.28a	ngd	ngd	ngd
LES812	ngd	1.97 ± 0.16b	3.07 ± 0.16a	2.80 ± 0.09a	0.44 ± 0.05c	ngd	ngd	ngd
NLL317	ngd	1.05 ± 0.09c	3.03 ± 0.07b	4.35 ± 0.17a	4.17 ± 0.14a	ngd	ngd	ngd
OIL347	ngd	1.60 ± 0.06c	3.55 ± 0.08a	2.85 ± 0.13b	1.49 ± 0.09c	ngd	ngd
PFL346	ngd	1.37 ± 0.10d	3.08 ± 0.04c	4.95 ± 0.13a	3.67 ± 0.08b	ngd	ngd
PHL322	ngd	8.18 ± 0.23d	21.36 ± 0.24c	29.28 ± 0.20b	36.64 ± 0.91a	35.94 ± 0.66a	35.61 ± 0.20a	ngd
PHC571	ngd	9.89 ± 0.12f	22.98 ± 0.16e	30.46 ± 0.90c	34.23 ± 0.30b	40.30 ± 0.09a	26.21 ± 0.62d	ngd
PNS806	ngd	2.69 ± 0.22c	4.39 ± 0.29a	3.35 ± 0.20b	0.26 ± 0.09d	ngd	ngd	ngd
PAC891	0.22 ± 0.04b	0.34 ± 0.01b	1.17 ± 0.27a	1.47 ± 0.31a	0.38 ± 0.29b	ngd	ngd	ngd
PAC854	0.10 ± 0.02b	0.26 ± 0.11b	1.02 ± 0.23a	0.69 ± 0.13a	0.09 ± 0.05b	ngd	ngd	ngd
PCC884	0.47 ± 0.01c	1.58 ± 0.07b	2.62 ± 0.59ab	3.72 ± 0.95a	3.50 ± 0.37a	ngd	ngd	ngd
PCL660	0.85 ± 0.03c	1.05 ± 0.02c	3.34 ± 0.30b	5.07 ± 0.27a	3.59 ± 0.10b	ngd	ngd	ngd
PCC897	ngd	0.24 ± 0.08c	1.54 ± 0.45b	2.08 ± 0.25b	2.85 ± 0.10a	0.58 ± 0.07c	ngd	ngd
PDC855	0.40 ± 0.06c	0.90 ± 0.08c	4.17 ± 0.22b	7.09 ± 0.60a	6.33 ± 0.29a	ngd	ngd	ngd
PEC847	0.22 ± 0.02c	0.57 ± 0.10c	2.72 ± 0.33b	4.67 ± 0.18a	2.85 ± 0.01b	ngd	ngd	ngd
PEL063	ngd	0.38 ± 0.05d	1.60 ± 0.00c	2.96 ± 0.31a	2.51 ± 0.20b	ngd	ngd	ngd
PEC810	0.29 ± 0.04c	0.70 ± 0.07c	2.10 ± 0.10b	3.15 ± 0.37a	2.31 ± 0.14b	ngd	ngd	ngd
PEC856	0.60 ± 0.04c	1.28 ± 0.14b	4.16 ± 0.43a	4.54 ± 0.12a	0.41 ± 0.11c	ngd	ngd	ngd
PFC860	0.56 ± 0.06b	1.02 ± 0.11ab	1.32 ± 0.08a	1.15 ± 0.38a	0.09 ± 0.00c	ngd	ngd	ngd
PNL126	0.21 ± 0.06c	0.45 ± 0.04c	1.39 ± 0.24a	1.04 ± 0.11b	0.13 ± 0.02c	ngd	ngd	ngd
PNC816	0.22 ± 0.03bc	0.56 ± 0.08b	1.12 ± 0.42a	0.46 ± 0.06b	0.03 ± 0.02c	ngd	ngd	ngd
POC843	0.77 ± 0.31c	2.33 ± 0.13bc	4.42 ± 0.38ab	5.89 ± 0.20a	4.33 ± 0.06ab	ngd	ngd	ngd
POC847	0.68 ± 0.10cd	1.46 ± 0.24bc	6.43 ± 0.19a	7.28 ± 0.16a	1.80 ± 0.74b	0.26 ± 0.00d	ngd	ngd
POL067	1.33 ± 0.25d	2.64 ± 0.05c	7.26 ± 0.08a	7.36 ± 0.43a	4.00 ± 0.63b	ngd	ngd	ngd
PPL111	0.66 ± 0.08d	1.06 ± 0.35d	4.09 ± 0.25c	7.01 ± 0.15a	6.24 ± 0.26b	ngd	ngd	ngd
PPC823	0.70 ± 0.05b	0.91 ± 0.23b	3.37 ± 0.40a	3.36 ± 0.82a	0.94 ± 0.09b	ngd	ngd	ngd
PSC757	0.64 ± 0.22bc	1.07 ± 0.36ab	1.35 ± 0.08a	1.18 ± 0.14a	0.43 ± 0.01cd	ngd	ngd	ngd
PTC822	0.36 ± 0.07c	0.54 ± 0.10c	1.54 ± 0.47b	3.35 ± 0.50a	2.01 ± 0.02b	ngd	ngd	ngd
SHC608	ngd	7.73 ± 0.15c	12.28 ± 0.16a	12.29 ± 0.11a	8.37 ± 0.24b	0.21 ± 0.00d	ngd	ngd
THC610	ngd	3.76 ± 0.23e	6.90 ± 0.10c	8.47 ± 0.07b	9.66 ± 0.42a	5.44 ± 0.11d	ngd	ngd
TLL473	ngd	2.17 ± 0.06e	4.55 ± 0.05d	6.55 ± 0.10c	8.01 ± 0.16a	7.47 ± 0.05b	ngd	ngd
TVL469	ngd	4.60 ± 0.09d	6.89 ± 0.14c	8.49 ± 0.14b	8.88 ± 0.13a	0.38 ± 0.10e	ngd	ngd
TVC614	ngd	5.39 ± 0.49c	10.53 ± 0.18a	11.24 ± 0.23a	10.93 ± 0.58a	1.52 ± 0.16b	ngd	ngd
TLC616	ngd	5.55 ± 0.17d	8.01 ± 0.12c	9.29 ± 0.06b	10.25 ± 0.12a	0.48 ± 0.02e	ngd	ngd

Figure 1

Mycelium linear growth rates (K , mm day−1) on PDA as measured at different temperatures (°C) for selected indicative cases of macrofungi examined (i.e., A. biennis ABL436, H. croceus HCC522, I. lacteus ILC238, Ph. chrysosporium PHL322, and P. djamor PDC855).

In the next comparative evaluation test, the mycelium growth rates of all strains were measured at their optimal temperatures and on three different OMW concentrations (25%, 50%, and 100% OMW v/v, in water) plus on PDA (Table 3). As anticipated, mycelium growth on PDA was in all cases significantly higher than on OMW-based solidified media with the only exception of Ganoderma carnosum GCL448 growing on 25% OMW. Still, several other strains presented satisfactory growth on both 25% and 50% OMW, while some performed adequately on 100% OMW as well. Such noteworthy cases included (apart from GCL448) Abortiporus biennis, Coriolopsis trogii, Daedalea quercina, Ganoderma adspersum, G. resinaceum (GRL344 and GRL403), Hapalopilus croceus, Hericium erinaceus (HEL801 only), Irpex lacteus, Phanerochaete chrysosporium, Pleurotus djamor, P. pulmonarius, Stereum hirsutum, Trametes versicolor, and Tyromyces lacteus. On the other hand, practically no growth was detected on OMW-based media for Auricularia mesenterica, Fistulina hepatica, Grifola frondosa, Hericium erinaceus (HEL802 only), Hypsizygus ulmarius, Laetiporus sulphureus (LSL331 only), Lentinula edodes, Pholiota nameko, and Pleurotus abieticola. In some of the rather few cases in which a species was represented by more than one individual, results occasionally revealed much different behavior between certain conspecific strains, for example, in Ganoderma carnosum, Hericium erinaceus, and Laetiporus sulphureus. This observation is in accordance with previous findings reporting high intraspecific variability within Pleurotus spp. in the degradation of OMW phenolics [32], which advocates for a strain (rather than a species) dependent behavior.

Table 3

Mycelium linear growth rates (K , mm day−1) for 60 strains of macrofungi growing on PDA and on OMW-based substrates prepared in various dilutions in water (v/v); ngd: no growth detected. Values are expressed as means ± standard deviation of means, n = 3. Lack of common superscripts indicates statistically significant differences (Gabriel's t-test, P < 0.05) for comparisons of treatment means between different strains (numbers) and different substrates (lowercase letters).

Strain	Substrate
	PDA	25% OMW	50% OMW	100% OMW
ABC521	10.56 ± 0.095,a	6.31 ± 0.156,7,8,b	4.59 ± 0.086,7,8c	2.64 ± 0.053,4,d
ABL436	11.86 ± 0.174,a	9.03 ± 0.104,5,b	7.65 ± 0.281c	5.15 ± 0.051,d
ACL834	3.81 ± 0.3816,17,18,19,a	2.38 ± 0.1315,16,b	1.81 ± 0.1913,14,b	ngd
AML472	4.88 ± 1.0013,14,15,16,a	ngd	ngd	ngd
CTL447	9.20 ± 0.166a	6.84 ± 0.066,b	4.16 ± 0.107,8,9c	2.40 ± 0.134,5,d
DQC528	10.96 ± 0.105,a	6.04 ± 0.067,8,9,b	5.94 ± 0.083,4b	5.23 ± 0.181c
DSC750	11.19 ± 0.464,5,a	2.87 ± 0.0414,15,b	ngd	ngd
FHL295	1.88 ± 0.1224,25,26,27,a	ngd	ngd	ngd
FVS803	5.71 ± 0.3411,12,13,a	3.98 ± 0.1912,13,b	2.88 ± 0.2611c	ngd
FPL302	8.04 ± 0.147,8,9,a	0.40 ± 0.0022,23,24,b	ngd	ngd
GAL401	9.03 ± 0.206,7,a	6.05 ± 0.296,7,8,9,b	3.88 ± 0.189,c	2.51 ± 0.124,d
GCL642	6.20 ± 0.1911,12,a	2.44 ± 0.3315,16,b	1.70 ± 0.1313,14,c	ngd
GCL448	7.52 ± 0.189,10,a	6.62 ± 0.176,7,a	5.45 ± 0.624,5b	2.29 ± 0.094,5c
GPL336	2.57 ± 0.2322,23,24,25,a	1.68 ± 0.0616,17,18b	0.90 ± 0.0316,17,18,19c	0.54 ± 0.067,8,9,10,d
GRL344	7.73 ± 0.428,9,a	5.43 ± 0.919,10,b	4.83 ± 0.335,6,b	2.32 ± 0.494,5,c
GRL403	8.12 ± 0.037,8,9,a	5.87 ± 0.007,8,9,b	3.96 ± 0.068,9,c	2.14 ± 0.144,5,d
GRC604	4.62 ± 0.1014,15,16,17,a	3.57 ± 0.0813,14,b	2.59 ± 0.1811,12c	0.96 ± 0.156,7d
GFS805	3.24 ± 0.1917,18,19,20,a	ngd	ngd	ngd
HCC522	32.99 ± 0.571,a	12.12 ± 0.101,b	1.05 ± 0.0015,16,17,18c	ngd
HEL801	13.96 ± 0.072,a	8.27 ± 0.895,b	5.51 ± 0.214c	3.16 ± 0.122,3d
HEL802	1.50 ± 0.2825,26,27,28,a	ngd	ngd	ngd
HAL340	7.58 ± 0.048,9,10,a	3.62 ± 0.1313,14,b	2.08 ± 0.0912,13c	0.98 ± 0.036,7d
HUL417	1.87 ± 0.0224,25,26,27,a	ngd	ngd	ngd
ITL314	4.45 ± 0.0414,15,16,17,a	0.08 ± 0.0324,b	ngd	ngd
ILC238	13.42 ± 0.502,3,a	10.04 ± 0.052,3,b	6.80 ± 0.222,c	0.70 ± 0.007,8,9,d
LSL331	4.19 ± 0.7915,16,17,18,a	ngd	ngd	ngd
LSL332	6.53 ± 0.4810,11,a	2.86 ± 0.1414,15,b	2.71 ± 0.0811,12,b,c	1.96 ± 0.335,c
LES812	2.33 ± 0.2023,24,25,26,a	ngd	ngd	ngd
NLL317	3.95 ± 0.2216,17,18,19a	1.70 ± 0.3416,17,18b	ngd	ngd
OIL347	2.25 ± 0.0523,24,25,26,a	1.49 ± 0.0417,18,19,b	0.80 ± 0.0016,17,18,19,c	0.42 ± 0.088,9,10,d
PFL346	3.22 ± 0.0520,21,22,23,a	2.09 ± 0.0415,16,17,b	1.35 ± 0.0314,15,16,c	0.55 ± 0.037,8,9,10,d
PHL322	36.64 ± 0.921,a	12.57 ± 0.181,b	6.48 ± 0.032,3c	ngd
PHC571	34.23 ± 0.301a	10.83 ± 0.102,b	4.18 ± 0.156,7,8,9,c	ngd
PNS806	3.97 ± 0.3816,17,18,19,a	ngd	ngd	ngd
PAC891	2.74 ± 0.1121,22,23,24,a	0.44 ± 0.1622,23,24,b	ngd	ngd
PAC854	1.02 ± 0.12a	ngd	ngd	ngd
PCC884	4.69 ± 0.0513,14,15,16,a	2.56 ± 0.3215,b	2.06 ± 0.1714,15,16,17,b	0.23 ± 0.059,10,c
PCL660	2.55 ± 0.6822,23,24,25,a	0.06 ± 0.0122,23,24,b	ngd	ngd
PCC897	2.30 ± 0.2323,24,25,26,a	1.05 ± 0.1218,19,20,21b	0.58 ± 0.1617,18,19,20c	ngd
PDC855	5.38 ± 0.2812,13,14,a	4.19 ± 0.2911,12,13,b	3.09 ± 0.2710,11,c	0.78 ± 0.246,7,8,d
PEC847	3.98 ± 0.3816,17,18,19,a	0.87 ± 0.0620,21,22,23,b	0.32 ± 0.0319,20,21,c	ngd
PEL063	3.59 ± 0.2418,19,20,21,a	0.90 ± 0.0919,20,21,22,b	0.20 ± 0,0020,21,c	ngd
PEC810	3.76 ± 0.1217,18,19,20,a	1.30 ± 0,3417,18,19,20,b	0.42 ± 0.1318,19,20,c	ngd
PEC856	1.48 ± 0.0426,27,28,a	0.42 ± 0.2022,23,24,b	ngd	ngd
PFC860	1.03 ± 0.2827,28,a	0.51 ± 0.0221,22,23,24,b	0.32 ± 0.0319,20,21,b	ngd
PNL126	0.94 ± 0.0127,28,a	0.36 ± 0.0223,24,b	0.07 ± 0.0421,c	ngd
PNC816	0.64 ± 0.0428,a	0.15 ± 0.1022,23,24,b	ngd	ngd
POC843	3.45 ± 0.6919,20,21,22,a	1.74 ± 0.0616,17,18,b	0.47 ± 0.4118,19,20,c	ngd
POC847	5.10 ± 0.0312,13,14,a	1.76 ± 0.0716,17,18,b	1.15 ± 0.0518,19,20,21,c	ngd
POL067	6.64 ± 0.2210,11,a	2.39 ± 0.0515,16,b	0.75 ± 0.4816,17,18,19,c	0.07 ± 0.0310,c
PPL111	7.80 ± 0.218,9,a	2.74 ± 0.0515,b	1.67 ± 0.1613,14,15,c	ngd
PPC823	3.71 ± 0.2817,18,19,20,a	1.56 ± 0.1017,18,19b	1.06 ± 0.0915,16,17,18,c	0.20 ± 0.009,10,d
PSC757	0.80 ± 0.0627,28,a	0.50 ± 0.0622,23,24,b	0.30 ± 0.0019,20,21,c	ngd
PTC822	2.70 ± 0.2421,22,23,24,a	0.97 ± 0.1218,19,20,21b	ngd	ngd
SHC608	12.83 ± 0.143,a	5.81 ± 0.078,9,b	4.71 ± 0.036,7,c	3.61 ± 0.082,d
THC610	9.08 ± 0.066,7,a	5.78 ± 0.058,9,b	3.74 ± 0.199,10,c	2.62 ± 0.024,d
TLL473	7.40 ± 0.129,10,a	4.93 ± 0.0310,11,b	4.80 ± 0.0010,11,b	1.24 ± 0.106,c
TVL469	8.64 ± 0.186,7,8,a	5.61 ± 0.038,9,10,b	4.37 ± 0.036,7,8,9,c	3.61 ± 0.082,d
TVC614	8.98 ± 0.186,7,a	4.42 ± 0.0411,12,b	4.14 ± 0.057,8,9,b	3.37 ± 0.072,c
TLC616	13.41 ± 0.202,3,a	9.34 ± 0.103,4,b	6.77 ± 0.072,c	2.61 ± 0.064,d

Consequently, eight fungi (i.e., Abortiporus biennis ABL436, Ganoderma carnosum GCL448, Hapalopilus croceus HCC522, Hericium erinaceus HEL801, Irpex lacteus ILC238, Phanerochaete chrysosporium PHL322, Pleurotus djamor PDC855, and P. pulmonarius PPL111) were qualified for further study on the basis of the outcome of the previous experiment, on the effect their growth had on the decolorization of the OMW-based substrate (assessed by visual inspection, data not shown), and on the prospects that individual species present in their subsequent/future exploitation for the generation of value-added products (e.g., edible biomass from Pleurotus spp.).

In all cases, fungal growth resulted in pH decrease from an initial value of 5.65 to values of as low as 4.48 for I. lacteus, while for the rest of the strains it ranged from 5.00 to 5.50 (Table 4). However, differences in pH were less pronounced for Pleurotus spp., which is in accordance with previous pertinent results obtained after the use of P. ostreatus [39]. On the other hand, electric conductivity measurements demonstrated a gradual increase from 1.80 mS cm−1 in the initial material to 3.85–5.97 mS cm−1 in the biodegraded effluent (Table 4); this seems to be the general trend for conductivity values after microbial treatment of raw OMW [40].

Table 4

Biomass production by eight selected strains of macrofungi in OMW (25% v/v) substrates and pH, electric conductivity (E.C.), plant-seed germination index (G.I.), and biodegradation-related aspects as measured in five different time periods during fungal growth. T 1 to T 5 correspond to 4, 8, 12, 16, and 20 days for HCC522 and PHL322, to 5, 10, 15, 20, and 25 days for ABL436, GCL448, HEL801, and ILC238, and to 6, 12, 18, 24, and 30 days for PDC855 and PPL111. Noninoculated substrates served as controls. N.a.: not applicable. Values are expressed as means ± standard deviation of means, n = 4. Lack of common superscripts indicates statistically significant differences (Gabriel's t-test, P < 0.05) for comparisons of treatment means between different strains (capital letters) and different time periods (lowercase letters).

Parameter	Time	ABL436	GCL448	HCC522	HEL801	ILC238	PHL322	PDC855	PPL111	Control
pH	T 1	5.57 ± 0.06BCa	5.55 ± 0.10BCab	5.62 ± 0.00ABa	5.55 ± 0.01ABCa	5.44 ± 0.10Cb	5.65 ± 0.01Aa	5.64 ± 0.00Aa	5.60 ± 0.00Ba	5.65 ± 0.00a
	T 2	5.49 ± 0.10Cab	5.50 ± 0.00Cab	5.59 ± 0.00Ba	5.53 ± 0.02Ca	5.25 ± 0.05Dbc	5.65 ± 0.01Aa	5.60 ± 0.00Bb	5.50 ± 0.00Cb	5.65 ± 0.00a
	T 3	5.35 ± 0.05Bbc	5.43 ± 0.05ABb	5.53 ± 005Ab	5.52 ± 0.02Aa	5.07 ± 0.06Bc	5.40 ± 0.00Bb	5.50 ± 0.00Ac	5.44 ± 0.00Bc	5.65 ± 0.01a
	T 4	5.29 ± 0.12BCc	5.40 ± 0.05Bb	5.40 ± 0.00Bc	5.52 ± 0.01Aa	5.00 ± 0.05Dc	5.18 ± 0.01Cc	5.50 ± 0.00Ac	5.18 ± 0.01Cd	5.65 ± 0.00a
	T 5	5.20 ± 0.05Cc	5.30 ± 0.05Bb	5.13 ± 0.12Cd	5.50 ± 0.02Aa	4.48 ± 0.06Dc	5.00 ± 0.00CDd	5.48 ± 0.01Ac	5.14 ± 0.00Cd	5.64 ± 0.01a
E.C. (mS cm−1)	T 1	4.25 ± 0.03Ae	3.80 ± 0.10ABe	2.63 ± 0.01Ce	4.09 ± 0.04Ad	2.52 ± 0.04Ce	2.60 ± 0.03Ce	2.21 ± 0.01Ee	2.45 ± 0.06CDd	1.80 ± 0.00b
	T 2	4.47 ± 0.02Ad	4.09 ± 0.03Cd	3.27 ± 0.01Bd	4.21 ± 0.03Bd	3.05 ± 0.02Fd	3.03 ± 0.05Fd	2.75 ± 0.03Gd	2.89 ± 0.02Gc	1.82 ± 0.00b
	T 3	4.92 ± 0.04Ac	4.31 ± 0.07Bc	4.05 ± 0.03Cc	4.40 ± 0.04Bc	4.17 ± 0.02Cc	3.68 ± 0.05Ec	3.02 ± 0.04Fc	3.45 ± 0.10Fb	1.89 ± 0.02a
	T 4	5.27 ± 0.02Ab	4.53 ± 0.09BCb	4.87 ± 0.01Bb	4.62 ± 0.05Cb	4.84 ± 0.03Bb	4.19 ± 0.13Db	3.33 ± 0.06Eb	3.99 ± 0.0ab	1.94 ± 0.03a
	T 5	5.97 ± 0.05Aa	4.79 ± 0.07Da	5.67 ± 0.02Ba	4.85 ± 0.12Da	5.54 ± 0.01Ca	4.97 ± 0.05Da	3.85 ± 0.01Fa	4.23 ± 0.08Ea	1.95 ± 0.03a
Biomass (g)	T 1	0.05 ± 0.01Ac	0.02 ± 0.00Ec	0.03 ± 0.00BCe	0.02 ± 0.00Ec	0.04 ± 0.00ABd	0.03 ± 0.00CDe	0.02 ± 0.00DEc	0.02 ± 0.00DEd	n.a.
	T 2	0.15 ± 0.02Ab	0.03 ± 0.00Cbc	0.05 ± 0.00Cd	0.04 ± 0.01BCb	0.11 ± 0.01Bc	0.04 ± 0.00Cd	0.04 ± 0.00BCb	0.03 ± 0.01Ccd	n.a.
	T 3	0.2 ± 0.02Aa	0.04 ± 0.01Fb	0.09 ± 0.00Dc	0.06 ± 0.02EFb	0.14 ± 0.01Bb	0.07 ± 0.00Ec	0.11 ± 0.01Ca	0.04 ± 0.00Fc	n.a.
	T 4	0.21 ± 0.01Aa	0.12 ± 0.01BCa	0.15 ± 0.00Bb	0.13 ± 0.03BCa	0.15 ± 0.01Ba	0.11 ± 0.00Cb	0.14 ± 0.02Ba	0.11 ± 0.00Cb	n.a.
	T 5	0.21 ± 0.01Aa	0.13 ± 0.01Ca	0.20 ± 0.01Aa	0.15 ± 0.02BCa	0.17 ± 0.01Ba	0.16 ± 0.01Ba	0.20 ± 0.02ABa	0.23 ± 0.01Aa	n.a.
Decolorization (%)	T 1	−25.82 ± 4.37Fe	2.41 ± 0.32Be	−0.99 ± 0.02De	14.23 ± 1.93Ab	−6.59 ± 0.41Ed	−2.35 ± 0.39Ce	−28.24 ± 1.88Gd	−23.22 ± 1.89Fd	0.44 ± 0.04b
	T 2	48.08 ± 3.71Ad	4.57 ± 0.54Ed	4.55 ± 1.20Ed	18.36 ± 0.84Bb	8.52 ± 1.30Dc	1.78 ± 0.25Fd	13.76 ± 1.52Cc	−21.44 ± 3.34Gd	0.68 ± 0.11ab
	T 3	56.84 ± 0.71Ac	7.31 ± 0.87Fc	17.99 ± 0.45Dc	25.21 ± 2.86Ca	35.16 ± 3.24Bb	11.72 ± 0.46Ec	21.09 ± 2.80Cb	17.68 ± 1.45Dc	0.89 ± 0.20a
	T 4	60.99 ± 0.78Ab	10.48 ± 1.53Eb	25.73 ± 1.32Db	27.09 ± 1.31Da	51.51 ± 2.57Ba	21.65 ± 1.80Db	40.64 ± 1.93Ca	23.43 ± 2.26Db	1.21 ± 0.14a
	T 5	64.01 ± 0.32Aa	13.86 ± 1.90Fa	33.63 ± 2.40Da	29.13 ± 0.57Ea	55.91 ± 1.64Ba	33.16 ± 2.19Da	48.12 ± 2.32Ca	38.76 ± 3.14Da	1.31 ± 0.13a
Total phenols reduction (%)	T 1	54.45 ± 3.98Ad	2.80 ± 0.41Dc	0.50 ± 0.09Ee	13.54 ± 3.73BCc	15.28 ± 0.79BCb	5.06 ± 0.69CDe	17.33 ± 1.23Bd	11.19 ± 3.55BCd	0.38 ± 0.04d
	T 2	72.13 ± 3.91Ac	48.57 ± 5.48Bb	9.57 ± 0.93Ed	18.42 ± 1.60Db	16.02 ± 1.60Db	8.85 ± 0.93Ed	32.14 ± 1.62Cc	16.61 ± 1.23Dd	0.58 ± 0.15c
	T 3	81.80 ± 2.59Ab	53.80 ± 3.20Bab	22.03 ± 1.23Dc	19.32 ± 2.28Db	54.45 ± 0.64BCa	18.60 ± 1.72Dc	43.30 ± 3.21CCb	46.94 ± 5.96BCc	1.10 ± 0.09b
	T 4	86.73 ± 2.11Ab	60.66 ± 2.16Ca	30.69 ± 0.91Db	23.83 ± 1.99Eb	56.67 ± 0.61Ca	29.43 ± 2.12Db	69.15 ± 2.50Ba	56.15 ± 3.86Cb	1.45 ± 0.37ab
	T 5	92.39 ± 1.39Aa	61.03 ± 3.21Ca	47.48 ± 1.78Da	45.63 ± 3.60DEa	58.89 ± 0.58Ca	41.74 ± 2.57Ea	76.01 ± 2.39Ba	70.59 ± 3.60Ba	2.14 ± 0.42a
G.I. (%)	T 1	7.58 ± 1.17Ad	0.00	0.78 ± 0.09Cd	0.00	6.51 ± 0.68Ad	1.42 ± 0.06Bd	0.71 ± 008Cd	0.73 ± 0.14 Cc	0.00
	T 2	9.23 ± 0.74Ad	6.62 ± 1.31Bb	2.77 ± 0.41Dcd	5.26 ± 0.94Bb	8.78 ± 1.20Ad	3.27 ± 0.16Cc	1.49 ± 0.10Ec	1.85 ± 0.36Ec	0.00
	T 3	20.47 ± 0.57cA	8.91 ± 0.87Cb	4.05 ± 0.71Dbc	6.41 ± 1.16CDb	14.49 ± 0.47Bc	3.91 ± 0.40Dc	2.13 ± 0.41Ec	4.27 ± 0.85Db	0.00
	T 4	24.89 ± 1.57Ab	13.28 ± 0.47Ba	5.92 ± 1.02CDab	7.33 ± 0.54Cb	15.78 ± 0.12Bb	6.76 ± 0.16Cb	4.75 ± 0.51Db	6.76 ± 1.52Ca	0.00
	T 5	30.01 ± 0.97Aa	17.57 ± 1.47Ba	8.11 ± 2.03Ca	15.01 ± 2.12Ba	17.04 ± 0.78Ba	8.89 ± 0.26Ca	8.11 ± 0.71Ca	8.89 ± 0.94Ca	0.00

All selected strains produced abundant mycelium in static batch cultures; yet four out of the eight strains (A. biennis, H. croceus, P. djamor, and P. pulmonarius) provided significantly higher yields (200–230 mg) than the rest (Table 4). Nevertheless, biomass production at initial growth stages was rather poor since substrate's content in readily assimilated nutrients was low [2]. In general, fungal growth was accompanied with varying degrees of enzymes production among the various strains examined. A. biennis was the only fungus to demonstrate a particularly high laccase activity within the initial cultivation period; that is, a peak value of 205 U L−1 was obtained after 10 days only, and then it subsided and remained rather constant at values of 115–122 U L−1 until the end of the cultivation (Figure 2(a)). The rest of the strains demonstrated either significantly lower laccase activities or no activities at all. Within the former group, laccase production for both Pleurotus spp. and G. carnosum increased in parallel with biomass growth and reached the highest value by the end of cultivation (63–79 U L−1), whereas H. croceus and Ph. chrysosporium presented much lower values (3.6–5.1 U L−1). In contrast, in the cases of H. erinaceus and I. lacteus no laccase activity was detected.

Figure 2

Enzyme activities (U L−1) for laccase (a), manganese peroxidase, (b) and manganese-independent peroxidase (c) exhibited on OMW-based media (25% v/v in water) by eight selected macrofungi during a growth period of 20 to 30 days and in five different time points (T 1 to T 5) as follows: 4, 8, 12, 16, and 20 days for H. croceus HCC522 and Ph. chrysosporium PHL322; 5, 10, 15, 20, and 25 days for A. biennis ABL436, G. carnosum GCL448, H. erinaceus HEL801, and I. lacteus ILC238; 6, 12, 18, 24, and 30 days for P. djamor PDC855 and P. pulmonarius PPL111.

As regards Mn peroxidase (MnP), a large variability was detected among strains in terms of both the peak activity values and the time periods where these were produced (Figure 2(b)). Hence, the highest MnP activities were observed for H. erinaceus and G. carnosum (18 and 11 U L−1, resp.) at the end of their growth. A. biennis, H. croceus, and I. lacteus presented their peak MnP values at or near the middle of the cultivation period (1.6–5.9 U L−1). On the other hand, Ph. chrysosporium and the two Pleurotus spp. were early MnP producers since their maximum production (0.4–3.0 U L−1) was noted within the first 12 days of growth, and then it gradually subsided to levels of no detection at the end of cultivation. Production of MnP was previously associated with secondary metabolism activated by lack of adequate nitrogen and/or carbon [41]. This was apparently not the case during the early stages of incubation of Ph. chrysosporium and Pleurotus spp. used in this study, but it might well be the case for the other five strains which presented their peak activities with a time delay. On the other hand, no Mn independent peroxidase (MnIP) activities were detected by the two Pleurotus spp. tested (Figure 2(c)). All the other strains produced their peak MnIP values towards the second half of their growth period starting from day 12 (H. croceus, 2.3 U L−1) and ending at day 25 (Irpex lacteus and A. biennis, 32 U L−1 and 7.1 U L−1, resp.).

In general, monitoring of enzymes production with respect to fungal growth indicated that P. djamor and P. pulmonarius shared identical patterns for all three activities, and this was also the case for H. croceus and Ph. chrysosporium. On the other hand, A. biennis presented common production patterns with I. lacteus as regards MnP and MnIP and so did G. carnosum with H. erinaceus.

Although in some cases the findings of the present study are in line with previous literature reports on enzyme activities in relation to fungal growth, for example, MnP activity for Pleurotus and Ganoderma spp. [32, 39], different types of data were also obtained. For example, MnIP activities in Ganoderma spp. showed their peak production earlier during the growth period [32] in comparison to the findings of the present study. Furthermore, previous pertinent results on P. eryngii and P. ostreatus revealed late production of MnP [32], which is in contrast with the results of this work (albeit with different Pleurotus spp.). While differences in enzyme activity values among strains used in various investigations could be attributed to both experimental conditions (including initial substrate composition and dilution rate) and properties of the particular fungus used, variations in enzyme production with respect to time could be primarily explained by the latter factor (i.e., variability of the biological material employed).

Total phenolic content was markedly reduced by all strains; however, particularly impressive was the reduction achieved by A. biennis, which after only six days of incubation decreased the initial phenols by more than 50%, while by the end of the experiment the respective figure reached 92% (Table 4). Very good performers (although significantly inferior than the first) were the two Pleurotus species (71%–76% of decrease), followed by I. lacteus and G. carnosum. The latter was the second fastest (after A. biennis) in reducing total phenols since a 49% decrease was measured after just 12 days of incubation. It is noteworthy that other Ganoderma species/strains were reported to present similar behavior as regards the time needed to decrease to a similar extent OMW's phenolics, that is, by 41–44% within a 10-day incubation period and by reaching a total reduction exceeding 64% [26, 32]. On the other hand, selected P. ostreatus and P. eryngii strains demonstrated a rather delayed phenols degradation which exceeded the 50% level only after the completion of the first half of growth [32] much like as it was observed for P. djamor and P. pulmonarius in the present study. In certain cases (e.g., for A. biennis, G. carnosum, and I. lacteus), a notable deceleration in the reduction rate of total phenols reduction was observed during the second half of the growth period, which could be attributed to the recalcitrance of certain phenolics remaining in culture (e.g., trans-cinnamic acids which are not degraded by laccase) [42].

Of special interest was that total phenol reduction was significantly correlated with laccase and MnP activities (r 2 = 0.43 and r 2 = 0.71, resp.) when data from all eight fungi were collectively evaluated (Table 5), while these figures were found to be particularly high in the individual calculations made for H. croceus, Ph. chrysosporium, and Pleurotus spp. for laccase (r 2 = 0.94–1.00), A. biennis, G. carnosum, H. erinaceus, and I. lacteus for MnP (r 2 = 0.71–0.94), and A. biennis, I. lacteus, and Ph. chrysosporium for MnIP (r 2 = 0.81–1.00). Such correlations were detected not only during the biodegradation of OMW by P. ostreatus [32, 39] but also when effluents from the debittering of green olives were treated by several white-rot fungi [43]. In this category of microorganisms, production of laccase is often induced by the presence of the appropriate substrate in the growth environment [44]. Especially as regards OMW media, such induction for Pleurotus spp. was associated with the concentration of the effluent in the culture substrate; high laccase activities were detected when phenolics exceeded 1.5 g L−1 [39, 45, 46], which is also the case with the initial phenolics content in the samples of this study (>2.0 g L−1). Most of the fungi examined here (with the only exception of A. biennis) demonstrated a lag phase for laccase production indicative of the time they needed to adapt their growth requirements to OMW [24, 47].

Table 5

Coefficient of determination (r 2) values in comparison between all degradation parameters (biomass, total phenolics reduction, decolorization, and germination index) and enzyme activities on the basis of the entire dataset obtained from all eight macrofungi examined. *Statistically significant at 5%; **statistically significant at 1%.

OMW degradation parameters and enzymes	Fungal biomass	Laccase activity	Mn independent peroxidase activity	Mn peroxidase activity	Decolorization	Total phenolics reduction	Germination index
Fungal biomass	—	0.50**	0.56**	0.56**	0.86**	0.76**	0.88**
Laccase activity		—	0.00	0.28	0.34*	0.63**	0.51**
Mn independent peroxidase activity			—	0.55**	0.46**	0.32*	0.60**
Mn peroxidase activity				—	0.33*	0.47**	0.62**
Decolorization					—	0.71**	0.71**
Total phenolics reduction						—	0.84**
Germination index							—

As previously noted with phenols content, A. biennis was the most efficient fungus in decolorizing OMW by reaching a 64% value by the end of the cultivation period (Table 4). During the process, color increased (for this and some other strains) during the first measurement(s) as a consequence of the oxidation and/or polycondensation of phenolic compounds in other darker-colored forms [44, 48]. Still, A. biennis achieved fast a very high decolorization rate (48% after 12 days) which is significantly higher than the respective values obtained from the other fungi during that same period (2%–18%). I. lacteus and Pleurotus spp. provided the next best total decolorization (56% to 39%), whereas G. carnosum performed rather poorly (14%). On the other hand, H. erinaceus was the only fungus that performed significantly better during the first half of the growth period, and then its decolorization subsided considerably.

OMW decolorization efficacy by A. biennis is among the best reported in literature for basidiomycetes. In other pertinent studies, Pleurotus spp., Ganoderma applanatum, Lentinula edodes, Pycnoporus coccineus, Coriolopsis polyzona, and Lentinus tigrinus demonstrated similar magnitude of decolorization; however, in several of these cases initial medium and cultivation conditions varied considerably [26, 32, 49, 50]. In addition, the outcome of this study demonstrates that decolorization is significantly correlated with total phenol reduction (r 2 = 0.71), which is in line with previous results associating OMW decolorization with the degradation of high molecular-mass polyphenols [47, 51]. Furthermore, high correlations were detected for decolorization not only versus laccase activity for majority of the fungi examined (r 2 = 0.83–0.99, in the cases of G. carnosum, H. croceus, Ph. chrysosporium, and Pleurotus spp.), but also versus MnP (r 2 = 0.66–0.99, for A. biennis, G. carnosum, H. erinaceus, and I. lacteus) and versus MnIP (r 2 = 0.83–0.99, in the cases of G. carnosum, I. lacteus, and Ph. chrysosporium). In the past, OMW decolorization was associated with laccase production for other white-rot fungi as well [24, 32, 52].

As regards the phytotoxicity evaluation, A. biennis was again the best performer by demonstrating 30% increase of the germination index with respect to untreated OMW, while G. carnosum, I. lacteus, and H. erinaceus provided significantly lower values (15%–18%). In these cases, increase of plant seed germination was particularly slow during the first 1 to 2 weeks of incubation, which is in line with previous reports on P. ostreatus and P. eryngii achieving 19%–27% increase of germination index towards the end of the growth period on OMW [32]. On the other hand, the other four strains produced a very low increase in seed germinability (<10%) that could be possibly attributed to the formation of phenoxy radicals and/or quinonoids that are more toxic than the initial phenolic compounds [53, 54]. In general, toxicity of OMW was associated with the presence of aromatic compounds of low molecular weight and with synergistic inhibition caused by phenolics [55-57]. Increase in seed germination was significantly correlated with reduction in phenolics and decolorization (r 2 = 0.84 and 0.71, resp., in cumulative calculations made for all strains tested); this high correlation with the former factor is in accordance with past reports on the germination of Triticum and Lepidium plant seeds in OMW treated by L. edodes and Pleurotus spp., respectively [24, 32].

When the results presented above were reassessed by taking into consideration the time needed for obtaining the final values for total phenols reduction, decolorization, and increase of plant seed germination, classification of selected strains did not change considerably. For example, A. biennis was again significantly more efficient for all parameters evaluated; that is, it demonstrated a daily average of 3.70 percentage units of total phenols reduction, 2.56 percentage units of decolorization, and 1.20 percentage units of seed germination increase; Pleurotus spp., G. carnosum, H. croceus, and I. lacteus followed as regards total phenols reduction (with values of 2.35–2.53), I. lacteus for decolorization (2.24), and G. carnosum, I. lacteus, and H. erinaceus for seed germination increase (0.60–0.70).

It is particularly noteworthy that when the experimental data from all eight fungi were assembled and evaluated vis-à-vis their total phenol reduction, decolorization, plant seed germination increase, and enzymes activity (Table 5), then biomass and germination index showed statistically significant correlations in all comparisons (r 2 = 0.55–0.88 and r 2 = 0.51–0.88, resp.). Laccase production was significantly correlated with total phenolics reduction and decolorization (Figure 3), and so was decolorization with MnIP (r 2 = 0.46). Last, MnP, and MnIP activities were also correlated (r 2 = 0.55).

Figure 3

Coefficient of determination (r 2) values for OMW decolorization and total phenolics reduction versus the laccase activities presented by the eight selected macrofungi as calculated from the respective data obtained at five time points (T 1 to T 5) during their entire growth period.

4. Conclusions

All wood-rot macrofungi selected for the purposes of this study were particularly effective in colonizing and degrading OMW through an efficient enzyme-producing mechanism. Especially A. biennis and secondly I. lacteus, G. carnosum, and Pleurotus spp. achieved total phenol reduction and decolorization values, which were significantly correlated with laccase and/or peroxidases activities. Enhancement of plant seed germination was less pronounced but it demonstrated high correlation with the other two biodegradation parameters. This approach seems to be suitable (alone or in combination with other techniques) in the development of a sustainable methodology leading to the efficient treatment of OMW.