Available online at www.sciencedirect.com

ScienceDirect Fungal enzymes for environmental management Ursula Ku¨es Fungal ligninolytic enzymes have broad biotechnological applications. Particularly laccases and certain fungal class II peroxidases from white-rot basidiomycetes are considered in degradation of persistent organic pollutants. Promising processes with reusable immobilized laccases in special reactors have been developed up to pilot scale for degradation of pollutants in water. Bioremediation of chemically complex soils with their large indigenous microbial communities is more difficult. Living fungi and their enzymes are employed. Bioaugmentation, introduction of for example white-rots for enzyme production into a polluted soil, and biostimulation of suitable resident organisms by nutritional manipulations are strategies in degradation of pollutants in soil. Bioaugmentation has been successfully implemented on small scale for soils in biobeds and for specific materials such as olive mill wastes. Address Department of Molecular Wood Biotechnology and Technical Mycology, Bu¨sgen-Institute, University of Go¨ttingen, Bu¨sgenweg 2, Go¨tttingen D-37077, Germany Corresponding author: Ku¨es, Ursula ([email protected])

Current Opinion in Biotechnology 2015, 33:268–278 This review comes from a themed issue on Environmental biotechnology Edited by Spiros N Agathos and Nico Boon

http://dx.doi.org/10.1016/j.copbio.2015.03.006 0958-1669/# 2015 Elsevier Ltd. All rights reserved.

Introduction Saprotrophic fungi have crucial roles in ecosystem functioning. Primarily, they facilitate organic matter decomposition and nutrient recycling in favor of own and other organisms growth and can have additional indirect effects on above-ground and below-ground ecology and species composition [1,2]. Lignocellulose from plant cell walls with its three main components cellulose, hemicellulose and lignin represents the largest organic renewable resource on earth but it is also most recalcitrant to degradation. This is due to the structure of the cell wall microfibrils in which the elementary cellulose fibrils are coated and cross-linked by hemicellulose matrices and in which the lignin shelter is then covalently linked to the hemicellulose. It is thus the hydrophobic lignin that protects the cell walls from humidity and microbial Current Opinion in Biotechnology 2015, 33:268–278

degradation [3–5]. Specific basidiomycete fungi can enzymatically attack all the polymers in the complexstructured lignocellulose. The appearance of such whiterot fungi million years ago allowed for the first time massively the fast nutrient recycling from wood required for new plant growth, with evolutionary impact on plant diversification. Concomitantly with the innovation of fungal lignocellulolytic enzyme machineries, the Carboniferous period had found its end. Land plants were not anymore simply buried and chemically transformed to coal but instead could become effectually decomposed into their components [6]. Based on the ability to degrade lignin along or not with cellulose and hemicellulose, wood decay has traditionally been divided into white rot and brown rot mainly exerted by basidiomycetes and soft rot mainly performed by ascomycetes. As already indicated, the white-rots have the unique enzymatic abilities to selectively or simultaneously attack the persistent lignin to free the fermentable polycarbohydrates for enzymatic decomposition [7,8]. In brown rot, lignin is attacked by Fenton chemistry and chemically modified into a brown oxidized form which allows access of enzymes to the cellulose for oxidative depolymerisation [9,10]. Poorly understood soft rot with partial enzymatic degradation of cell wall polysaccharides and slight alterations of lignin can occur under high wood moisture content [11]. Typically, lignin degradation by white rots involves highly specialized class II peroxidases (PODs) with high-oxidation potential [7,8]. However, recent evaluation of the decay modes together with the genomes of the basidiomycetes Botryobasidium botryosum, Jaapia argillaceae, Cylindrobasidium torrendii and Schizophyllum commune suggests that forms of white rot exist independent of any PODs. Decay modes show features of in between white and brown rot and of soft rot [12,13]. In contrast, litter decomposing fungi might be best adopted to humic substances by expanding numbers of genes for specific types of enzymes, for example genes for heme-thiolate peroxidases, but these have also retained some enzymatic ability for white rot [14]. Loss or reductions of genes for similar groups of enzymes lead in the basidiomycetes on a number of occasions from white to brown rot and also to mycorrhizal lifestyles, respectively [6,10,15]. Increasing evidence supports that various mycorrhizal fungi have the abilities to act as occasional litter decomposers [17]. The mycorrhizal Paxillus involutus for instance has been shown to apply a trimmed brown rot mechanism with Fenton chemistry to plant litter [18,19], and Cortinarius species exhibit high peroxidase activity in soil for decomposition www.sciencedirect.com

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Box 1 Laccases (EC 1.10.3.2; p-diphenol oxygen oxidoreductases) are multi-copper-oxidases with their true biological functions and natural substrates little understood and known. Most fungal laccases are extracellular enzymes. In essence, these enzymes are biochemically characterized on artificial substrates. Laccases have a broad substrate range and act with low specificity on o-phenols and p-phenols and often also on aminophenols and phenylenediamines under transfer of four electrons from organic substrate to molecular oxygen. Importantly, the substrate range can become broaden and the kinetics of reactions enhanced by laccase-mediator-systems (LMSs) acting in a chain of electron transfers in which a compound is oxidized by the enzyme and the oxidized form then mediates the oxidation of a substrate that may not be a factual target of the enzyme (Figure 2). Peroxidases (EC 1.11.x; donor:hydrogen-peroxide oxidoreductases) comprise different superfamilies of phenoloxidases that use H2O2 or organic hydrogen peroxide as electron accepting cosubstrates. Main fungal high-redox class II peroxidases involved in biodegradation of lignocellulose with an exceptional broad organic and also inorganic substrate range are secreted heme-containing lignin peroxidases (LiPs; EC 1.11.1.14), manganese peroxidases (MnPs; EC 1.11.1.13), and versatile peroxidases (VPs; EC 1.11.1.16). Another family of largely unclarified biological functions but of high biotechnological interest for degradation of recalcitrant compounds presents dye-decolorizing peroxidases (DyPs; EC 1.11.1.19). DyPs are bifunctional enzymes with oxidative and hydrolytic activities on phenolic and non-phenolic organic compounds, some of which, for example some recalcitrant textile dyes and p-nitrophenol, are poorly accepted by other peroxidases. Halogenating chloroperoxidases (CPOs; EC 1.11.1.10) and unspecific or aromatic peroxygenases (UPOs/APOs; EC 1.11.2.1) belong to the heme-thiolate peroxidase (HTPs; haloperoxidases) superfamily. HTPs transfer peroxide-oxygen to substrate molecules. Among, UPOs have exceptionally broad reaction competences on a wide variety of substrates on which they perform various reactions including aromatic peroxygenation, double-bond epoxidation, hydroxylation of aliphatic compounds, ether cleavage, sulfoxidation, N-oxidation, bromide oxidation and more. Tyrosinases (EC 1.14.18.1; monophenol monooxygenases; phenolases; monophenol, o-diphenol:oxygen oxidoreductases; L-tyrosine,Ldopa:oxygen oxidoreductases) are type III copper proteins. Upon binding of molecular oxygen, tyrosinases catalyze o-hydroxylation of monophenols (monophenolase reaction cycle, reaction 1) to generate as intermediates o-diphenols that are subsequently oxidized into reactive oquinones (diphenolase reaction cycle, reaction 2). Tyrosinases are cytosolic enzymes that participate in pigment synthesis such as melanin. Best known for applications in biotechnology is Agaricus bisporus tyrosinase (mushroom tyrosinase) causing in its host mushroom browning. P450 cytochrome monooxidases (EC 1.14.14.1; unspecific monooxygenases; flavoprotein-linked monooxygenases; P450s; CYPs) are intracellular heme-thiolate-containing oxidoreductases acting on a wide range of substrates in stereo-selective and regio-selective manner under consumption of O2. Activated by a reduced heme iron, these enzymes add one atom of molecular oxygen to a substrate, usually by a hydroxylation reaction. However, various other reactions such as epoxidation, sulfoxidation, dealkylation and more can also occur. P450-catalyzed reactions require NAD(P)H as donors for electrons to be transferred via a flavoprotein or ferredoxin to the second oxygen atom from a cleaved O2 molecule. Members of the highly diverged and functionally very diverse P450 superfamily have essential roles in biosynthetic pathways of specific primary and secondary metabolites, others act in metabolization of xenobiotics. Glutathione transferases (EC 2.5.1.18; glutathione S-transferases; glutathione conjugating enzymes; GSTs) catalyze the nucleophilic attack by reduced glutathione (GSH) of an electrophilic carbon, nitrogen or sulfur atom in non-polar compounds. Conjugation of GSH to the electrophilic substrates makes the substrates more water-soluble. GSTs are intracellular enzymes present in different subcellular compartments. They have a broad substrate specificity and act in detoxification of various structurally different endogenous toxic metabolites, superoxide radicals and exogenous toxic chemicals. In fungi, there are at least eight distinct classes of GSTs (GTT1, GTT2, Ure2p, MAK16, EFb1, GSTFuA, GSTO, GHR).

of organic matter [16]. Mycorrhizal and typical saprotrophic species tend to be found distinctly in separate soil areas, along with specific functions in the rhizosphere and in the soil, respectively. Endophyte implies localization within plant tissues (endosphere) but such species also assemble in zones inhabited by typical saprotrophs [20,21]. Soil pH values as one parameter can determine whether soil-borne fungi colonize roots and tend toward an endophytic lifestyle of no harm to the host [22]. Under certain conditions, endophytes may change into pathogens [23], pathogens on one plant might be mycorrhizal on another [24,25], and litter and wood decay fungi may also have mycorrhizal properties [10,26]. There is apparently much continuum possible between the different lifestyles and situations of fungi in the soil. To verify such versatility, the soil-borne organisms will appoint and express to need different sets of enzymes. Enzymes that break down cellulose, hemicellulose and lignin are over-arching called cellulases, hemicellulases and lignin-modifying enzymes (LMEs), respectively. By sequence, catalytic mechanism and enzymatic specificity, these enzymes divide into multiple families and subfamilies, the constantly expanding information on which is compiled in the knowledge-based CAZy database together www.sciencedirect.com

with information on enzymes with auxiliary activities [27,28]. Enzymes in lignocellulose degradation are commonly extracellular, which is compulsory by the large molecule sizes of the envisaged substrates. Larger polymers are broken down into smaller fragments and finally into individual molecule units that might be taken up into the cells for eventual metabolic use [8] or for further detoxification by the xenome, that is the protein machineries for detection, transport and metabolism of xenobiotics [29]. Detoxification pathways of the xenome are constituted among others of multigenic families of intracellular cytochrome P450 monooxygenases and glutathione transferases, respectively (Box 1). These superfamilies of enzymes are particularly highly expanded in wood degraders (in white-rots and brown-rots) and in plant litter decay species but also to some extent in symbiotic species. Among other functional roles they have in primary and secondary metabolism, the enzymes likely diverged in different species to deal with the multiple harmful lignin metabolites and related compounds in humus generation and with the countless plant defense metabolites soil fungi are confronted with in nature [29,30,31]. There are multiple purposes in biotechnology as where ligninolytic enzymes [5,32–35] and enzymes for Current Opinion in Biotechnology 2015, 33:268–278

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Figure 1

Organic compounds

Extracellular enzymes Fixing to soil particles or organic matter

Nonenzymatic degradation

Persistent organic pollutants

Recycling of mediators

Oxygen radicals, photons, Fenton reactions

Enzymatic activation

Organic radicals

Enzymatic degradation (extracellular)

Indirect enzymatic degradation

Intermediates Polymers Degradation products

Fixing to soil particles or organic matter

Enzymatic degradation (intracellular)

Metabolites (intracellular) Excretion

Mineralization

CO2

Degradation (direct, indirect enzymatic; non-enzymatic)

Metabolites (extracellular)

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Simplified scheme of the very complex reactions possibly occurring with extracellular phenol oxidases and persistent organic pollutants (POPs) in environments. Organic pollutants might be free or (reversibly) fixed to soil particles or organic matter into a condition where they might be less harmful but also non-accessible for enzymes. For degradation, they may be attacked by non-enzymatic degradation mechanisms such as by oxygen radicals, photons and Fenton reactions or they may be transformed by direct or indirect enzymatic reactions. Functional extracellular enzymes might be free or immobilized such as on soil particles, organic matter or also on (producing) cells. Binding to such materials can lead to changes in enzyme properties, positively and negatively. Enzyme binding to soil particles or other matter could alternatively lead to full inactivation of the biocatalysts. In direct enzymatic action, the biocatalysts can use the pollutants as own substrates. In indirect enzymatic action, suitable organic compounds are enzymatically transformed into radicals which in turn attack as mediators the pollutants. By transfer of electrons, mediators can become regenerated for further cycles of reaction. Note that a direct enzyme substrate after enzymatic activation into a radical might also undergo further indirect transactions. If not binding to any soil matter, generated intermediates might undergo further rounds of nonenzymatic or direct or indirect enzymatic degradation (indicated in the scheme by double-sided arrows). One possible route can lead to polymerization of intermediates, another to smaller degradation products which might be taken up into cells if not binding to any soil matter. Within cells, these might be detoxified through the xenome employing cytochrome P450 monooxygenases and glutathione transferases ([29]; Box 1). Metabolites might be excreted from cells and undergo further extracellular reactions or might be fully mineralized by cells into CO2 [29]. Colors from red to yellow indicate arbitrary toxicity levels of compounds, green colors indicate less or non-toxic compounds.

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detoxification of xenobiotic compounds [36] bring benefits (Figure 1). Enzymes with high relevance to this report are shortly explained in Box 1. An emerging field in biotechnology lies in application of enzymes in environmental management. There are four main means of usage of ligninolytic enzymes in environmental management with partial overlaps: 1. Enzymes might be used to purify pollutions in contaminated water or solid materials prior to release into an environment. 2. Enzymes might be used in bioremediation within environments. 3. The environment might be manipulated in favor of organisms producing enzymes of environmental benefit. 4. Enzymes might be used in biosensors and as bioindicators to monitor pollution in the environment [29,37]. I will concentrate here on the first three points.

Enzymes in degradation of persistent organic pollutants in waste waters Comprehensibly, any contaminated liquid or solid material should not be released into the environment prior to purification. Wastes containing ordinary organic matter can be converted in common waste water, biogas and composting plants, respectively. Persistent organic pollutants (POPs) in contrast resist easy environmental degradation through biological, chemical and physical means. Such recalcitrant and frequently toxic pollutants often come in mixtures, also together with inorganic

contaminants, and may be still hazardous to health and environment when present in only minute amounts (micropollutants). POPs might be of natural or of artificial origin. Structures of synthetic origin can be identical to natural compounds or they may be newly contrived. Compounds of biological origin might accumulate by natural processes or by anthropogenic activities. Particularly distressing groups of POPs comprise natural and synthetic phenolic compounds and polycyclic aromatic hydrocarbons (PAHs) [37,38–40]. Natural or intentionally enforced chemical oxidation processes and photocatalysis can help in degradation of such compounds (Figure 1), in particular in wastewater treatments [38–40]. As biological means, numerous peroxidases and laccases and also tyrosinases (Box 1) of diverse fungal species are reported from multiple laboratory studies to be active in the degradation of POPs. By one-electron abstraction from organic substrates, the enzymes generate organic radicals that can undergo subsequent radical reactions [41–43]. Having themselves already pronounced substrate ranges, enzymatic activation of mediators, that is suitable enzyme substrates that can serve as electron shuttles between enzyme and other compounds, enhances kinetics of reactions and potentiates transformation activities exponentially also onto target molecules that are not direct substrates to an enzyme ([37]; Figures 1 and 2). Most often but not exclusively, sources

Figure 2

Phenolic substrate(red)

H2O

Laccase(ox) Phenolic product(ox)

O2

Mediator(red)

Phenolic or non-phenolic compound(ox)

Mediator(ox)

Phenolic or non-phenolic compound(red)

Laccase(red)

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Laccase reactions with phenolic enzyme substrates and laccase-mediator-system (LMS). Laccase in oxidized form (laccase(ox)) takes up electrons directly from phenolic substrates (phenolic substrate(red)) along with substrate oxidation (phenolic substrate(ox)) in order to transfer the electrons to molecular oxygen which in turn restores the laccase(ox) state. Specific laccase substrates called mediators can react in laccase-oxidized form (mediator(ox)) with other organic phenolic and non-phenolic compounds (phenolic compound(red) or non-phenolic compound(red)) including many non-laccase substrates to oxidize these (phenolic compound(ox) or non-phenolic compound(ox)) by uptake of electrons. This restores the mediator function (mediator(red)) for further cycles of laccase-mediator electron transfer chain reactions. Note that the oxidized phenolic and non-phenolic compounds can also be reactive and might also undergo further chemical reactions. www.sciencedirect.com

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of these oxidoreductases are white-rotting basidiomycetes [37,41–43]. Good mediators for applications should be highly effective in reaction, not inactivating to the enzyme, not be used up by action, recyclable, preferentially be small, also cheap, biodegradable and not themselves toxic. The in all aspects ideal mediator still needs to be found [44–47]. Attention has been given to exploit as natural co-oxidants small diffusible oxidative phenols originating from enzymatic degradation of lignin such as syringyl-type phenolics [48,49]. Cost of using free enzymes in purification of waste waters and solids on large scale can become high [50]. Recovery and reusability of free enzymes after use are restricted. Enzymes might be little stable in free solution and their catalytic properties can become inhibited by metal ions, salts, chelators, detergents and other compounds in the contaminated matter or they may be inactivated by binding to soil particles or organic matter (Figure 1). Application of enzymes free in solution is therefore little practicable, in particular not in large scale purification processes and under continuous conditions [51,52]. Various techniques of enzyme immobilization (carrier-surface-binding through ionic and covalent binding and hydrogen bonding; encapsulation in insoluble substances with pores; carrier-less cross-linking of enzymes to each other by bifunctional or multifunctional reagents) can improve all these drawbacks. Typically, immobilized biocatalysts are thermally and operationally stabilized and become recyclable for repeated and long-term use [51–53]. A focus general in immobilization and more specifically in application in waste water purification [51–53] is on laccases with broad substrate specificity that use molecular oxygen as co-substrate unlike PODs that depend on hazardous hydrogen peroxide ([41,42]; Box 1). Supports for enzyme immobilization should be biocompatible, in addition to being cheap and stable in activity also under various pH and temperature situations and under harsh chemical conditions of polluted effluents. Among others [51–53], mesoporous silica spheres, fumed silica nanoparticles, TiO2 nonoparticles, silane sol–gel matrix, chitosan, cellulose nanofibers, polyethersulfone and polyvinylidene membranes, macroporous polymeric cryogels, and crosslinked enzyme aggregates (CLEAs) are examples for carrier materials successfully been implemented and tested in largely empirical approaches in laccase immobilization for the target of waste water purification [54– 56,57,58–61,62,63]. Addition of suitable mediators can enhance actions of immobilized laccases as for the free enzymes [61,64]. Other than the enzyme laccase, mediators might also be immobilized [62]. Immobilization offers technically the possibility to combine enzymes for simultaneous or successive conversions in one-pot reactions. Laccases of different origin differ in characters (substrate ranges, pH optima). Combinations thereof Current Opinion in Biotechnology 2015, 33:268–278

can therefore be more versatile for converting mixtures of hazardous compounds under changing and poorly defined conditions [57], such as in municipal and industrial waste waters that inherently vary in compositions and amounts of their chemical burdens and can so also in pHs. Laccase and tyrosinase both need O2 for enzymatic action ([42,43]; Box 1) and have functionally been combined in so called combi-CLEAs [65]. Co-immobilization of laccase and horseradish peroxidase for the purpose of lignin bioprocessing has earlier been demonstrated [66]. A problem to be overcome with peroxidases is their need for H2O2 [41]. Co-immobilization of peroxidases and H2O2-generating enzymes for their support is a challenging route as a way out [67,68]. The feasibility of such approach has recently been exemplified in combiCLEAs with versatile peroxidase (VP) of Bjerkandera adjusta and H2O2-producing glucose oxidase of Aspergillus niger, to which moreover three distinct laccases of Trametes versicolor were added [69]. Toward application of immobilized enzymes in waste water purification several technical obstacles have to be faced such as the changing properties of waste waters, separation of enzymes for reiterative use and up-scaling of processes to sensible sizes [52]. Enzyme choices and type of immobilization are critical factors for stability under harsh environmental conditions as presented by properties of waste waters [52]. For enzyme separation, magnetic particles have been given attention to for iterative reuse since they can be easily recovered by electrical fields in magnetic bio-separation technology [55,63]. Multiple specific reactor designs preferentially for continuous mode (fluidized bed reactor; packed bed reactor; perfusion basket reactor; suspended nanoparticle reactor; nano-composite bio-catalytic membrane reactor; hybrid membrane-nanoparticle suspension system; hybrid bioreactor of hollow fiber microfilter membrane) are under test for practical use of immobilized laccases in waste water purification [51,54,58,60,62,63,68–76,77]. Most interesting for continuous application appear membrane reactor systems that keep the immobilized enzymes in place while passing the purified liquid through the membrane [54,58,68,75,77]. A first trial on pilot scale in longterm field tests was reported that used actual wastewater treatment plant effluents in tertiary treatment to eliminate any persistent xenobiotics (here bisphenol A) and a fixed bed reactor for settling any solids combined to a 460 L membrane reactor containing with on silica nanoparticles immobilized Thielavia laccase, with costs estimated to 0.130 s m3 purified water. Costs of the process as tested are comparable to chemical removal of xenobiotics through binding to powdered activated carbon and by ozonation. However, there is more potential in the process upon optimization of various running parameters (e.g. particle mixing, nanobiocatalyst load, hydraulic retention times) and by application of enzyme mixtures [77]. www.sciencedirect.com

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Enzymes in bioremediation of solid wastes and soils Solid organic wastes and contaminated soils present another challenge in POPs purification. Free enzymes mixed into the materials will only potentiate the problems on enzyme activities and stabilities observed in liquids. Enzymes applied to soil will interact with its occurring particles of specific natures and this may change enzyme properties — for the better and for the worse ([78]; Figure 1). A fungal laccase for example has been reported in experiments with individual materials to adsorb to soil iron and aluminum minerals with consequences of general reduction of enzyme activities through reduced enzyme-substrate affinities but at acidic pH the catalytic activities increased. Thermostability and temperature sensitivity were lower upon adsorption but resistance to proteolysis and enzymatic lifespan were enhanced [79]. Heterogeneous structures of organic wastes and even more of different types of soils make it however unpredictable of what will happen to the free enzymes under authentic conditions. Again, enzyme immobilization is considered in laboratory experiments for finding a solution to practical application, with clay or soil minerals as natural supports being tested as choice of carriers [79–81]. However, good dispersion and poor or no recovery of enzymes after use present problems for larger scale use. Fermentation with living enzyme-producing organisms is then a more practicable low cost alternative. Some practical experience exists with solid olive-mill wastes that contain many phytotoxic compounds of mainly phenolic character. A number of white-rot fungi in axenic laboratory cultures have been shown to degrade the toxic phenolic compounds [82,83]. Laccases, types of peroxidases and also aromatic peroxygenases (Box 1) appear to have roles in this [84–86]. Treated detoxified organic waste material might subsequently be used in fertilizing soil. Application of fungal fermented olivemill waste to loamy soil enhanced in greenhouse tests bacterial proliferation and it soon affected bacterial diversity, but to less degree as compared to amendments of the untreated material [87,88]. Changes in fungal community structure were also evident and some variation in diversity but only after longer run [89,90]. Enzymatic activities (b-glucosidase, urease) within soil were negatively affected with untreated material whereas phosphatase, b-glucosidase, and urease activities were enhanced with fungal transformed waste, possibly due to the input of extra nutrients helping microbial growth [90]. Further analysis revealed that functional diversity and microbial functional structures decreased by increase of some and loss of other groups of microbes with specialized metabolic functions [91]. More studies like these are needed to follow up what happens in terms of microbial communities, biodiversity and functionality in approaches with additions of fungal fermented waste materials to soils. www.sciencedirect.com

Enzymes and fungi still present and active in applied fermented material might react further with organic material within soil. This can be of particular interest when the soil contains any POPs. On laboratory scales, promising results were reported for degradation of for example creosote [92], PAHs [93], benzo(a)pyrene [94] and heptachlor and heptachlor epoxide [95] in soils upon addition of spent mushroom substrates (SMS) from Pleurotus and Agaricus cultivations. Transfer to real outside conditions might however be different by many factors, for instance due to soil structures and compositions, respective nutrient availabilities, moisture contents, aeration and climate conditions, actual pHs, and competition by already resident microbes [96,97]. The white-rot Phanerochaete velutina for example on small laboratory scale removed in three month 96% of 4-ring PAHs and 39% of 5-ring and 6-ring PAHs from contaminated sawmill soil. In larger field scale, P. velutina had then no recognizable effect since bacteria from added composted green waste were (also) active [98]. Choices of organisms for bioaugmentation (addition of actively growing specialized organisms into an existing microbial community to enhance degradation of pollutants) will be critical [96]. On the one side, an organism needs to be able to degrade the pollutants of concern and species differ in their reaction abilities toward individual compounds and ranges of pollutants [95,96]. On the other side, an effective organism needs to be competitive at place (for space and resources) and, moreover, needs be active in required enzyme production [98,99]. Preferentially, it should also not negatively shape the indigenous communities and trophic groups in a biotope [91,100]. Wood-degrading basidiomycetes with enzymes being most aggressive against POPs may have little potential to compete with the dominant inhabitant fungi in soil on sites since wood-rotting species will be adapted to the special nutritional conditions given by their lignocellulosic substrate wood and to possibly less competitive microbial communities of only small species numbers inhabiting together the wood. To establish an introduced wood degrader in soil, addition of much pre-grown fungal biomass and of extra nutrients might therefore be required. Preferentially, such extra nutrients will be provided for the wood degraders in form of some agricultural or forestry waste material rich in their favored substrate lignocellulose [101,102]. Other easily accessible nutrients might promote growth but lead to repression of required enzyme production [103]. With top soil, peat, straw and grass in low-cost biobeds as defined compartments for treatment of smaller amounts of pesticide contaminated matter, white-rot fungi can effectively be managed by nutritional manipulation through straw, and moisture and pH can be controlled through the peat in order to create an optimum environment for enzyme production required for the successful degradation of pesticides [104]. Current Opinion in Biotechnology 2015, 33:268–278

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Rather than adding an organism newly into a contaminated biotope, growth and enzyme production of indigenous microbes might alternatively be promoted in approaches of biostimulation through additions of suitable nutrients into environments [105]. Reactions upon nutrient additions within natural communities are however complex. Current knowledge is too restricted to foresee whether the actual types of organisms of interest will favorably grow and whether under good proliferation they will also produce the required enzymes for pollutant degradation. Advantages in biostimulation can however be that unknown and unexpected natural soil organisms might be able and stimulated to attack and detoxify POPs, and the possible presence of more than one suitable organism for potential synergistic actions. Detoxification abilities in nature might be broader distributed than expected on a first view and they are not restricted to wood-rots and litter-degrading fungi. For example, co-metabolic degradation of mono-fluorophenols in presence of glucose was recently observed by the ectomycorrhizal Pisolithus tinctorius [106]. Genome projects and observed changes in ecological behaviors of fungi support broad potentials and much versatility in extracellular degradation systems and in resident intracellular xenomic detoxification networks between species of different basic ecological functions, particularly in basidiomycetes (see Introduction). Little is so far known on what happens to individual enzymes secreted into the soil. Distribution of soil enzymes and linked decomposition rates are probably highly dynamic. Enzymes are naturally present from activities of organisms in soil. However, they are not evenly distributed throughout the soil and its distinctive horizons, not even within a horizon. Enzyme distributions can differ in small scales, down to millimeters [97]. Spatial restricted location of enzymes might contrast the distribution of POPs that might be much broader spread through the soil layers. Efficient in situ removal in soil by fungal enzymes would then ask for expensive soil mixing [96]. Presence and activities of enzymes tend to link to patches of nutrients and microbial biomass, thus the place of organisms that produced them [97]. Free diffusion of enzymes will more likely be low. Either they bind already to the producing cells as a form of natural immobilization [107,108] or, if freely secreted into the environment, they probably will bind to organic matter or soil particles ([78]; Figure 1). Soil type and properties (organic matter, minerals, pH, humic compounds) very much influence enzymatic activities. Between different types of soil, factors can be contrasting regarding effects on individual enzymes. It makes thus for example a big difference for activity of an enzyme whether being present in forest or in grassland soil [109]. Constantly changing abiotic parameters such as temperature and soil moisture content (by wetting and drying) in woodland Current Opinion in Biotechnology 2015, 33:268–278

soil very much effects soil enzyme activities in interactive but not necessarily in additive manner [110,111] and season plays a role [111]. There are thus multiple difficulties to be overcome when using and stimulating natural degradation systems.

Perspectives and future directions No doubt, basidiomycetous fungi with their large batteries of ligninolytic enzymes, among the different types of phenoloxidases with their different broad substrate ranges, and with their expanded but yet barely exploited xenomes provide us with an excellent ‘‘green’’ potential for handling of many problematical types of POPs. Application of fungal oxidative enzymes in waste water purification has been advanced to pilot studies and processes with immobilized enzymes are promising for applications at least in tertiary waste water treatments. Soil purification of POPs by white rot fungi and their enzymes has been implemented to applications on small scale such as in biobeds. However, for the ambitious task to purify polluted soils on site on larger scales by bioaugmentation or biostimulation, we lack much essential knowledge on general ecology, microbial communities and their behaviors, enzyme production and properties in complex chemical and physical soil conditions and changing climate conditions. Laborious systematic evaluations would be required not only for one site but for multiple sites considering for example just the multitude of existing different soil types. Soil purification of POPs by fungi and their enzymes is not simply a question of days but a longer-term task which would require long term manipulations. Nature offers through the fungi many solutions to us but we are far away from being masters of them.

Acknowledgements Andrzej Majcherzyk and Markus Euring are thanked for valuable critical discussions. Work on ligninolytic enzymes in our group received funding within the framework of a Common Lower-Saxony-Israel Project (Zn 2043) by the Ministry of Science and Culture in Hannover, Germany and by the German Federal Ministry of Education and Research (BMBF) as part of the BEST-Research Framework (PtJ0033L033A-IO-A5).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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A’Bear AD, Boddy L, Kandeler E, Ruess L, Jones TH: Effects of isopod population density on woodland decomposer microbial community function. Soil Biol Biochem 2014, 77: 112-120.

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Ruiz-Duen˜as FJ, Martı´nez A´T: Microbial degradation of lignin: how a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this. Microbial Biotech 2009, 2:164-177. www.sciencedirect.com

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Zeng Y, Zhao S, Yang S, Ding S-Y: Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels. Curr Opin Biotechnol 2014, 27:38-45.

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Floudas D, Binder M, Riley R, Barry K, Blanchette RA, Henrissat B, Martinez AT, Otillar R, Spatafora JW, Yadav ST et al.: The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 2012, 336:1715-1719. The authors compared the sets of lignocellulolytic enzymes as deduced from 31 different fungal genomes and brought gains and losses of respective genes onto an evolutionary time scale. Main conclusions of the study were that white-rot basidiomycetes appeared at the end of the Carboniferous period and that brown-rot and mycorrhizal species derived from these by specific loss of groups of genes in plant cell wall degradation. 7.

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10. Eastwood DC, Floudas D, Binder M, Majcherczyk A, Schneider P, Aerts A, Asiegbu FO, Baker SE, Barry K, Bendiksby M et al.: The plant cell wall-decomposing machinery underlies the functional diversity of forest fungi. Science 2011, 333:762-765. 11. Daniel G: Fungal and bacterial biodegradation: white rots, brown rots, soft rots, and bacteria. ACS Symp Ser 2014, 1158:23-58. 12. Riley R, Salamov AA, Brown DW, Nagy LG, Floudas D, Held BW,  Levasseur A, Lombard V, Morin E, Ottilar R et al.: Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi. Proc Natl Acad Sci U S A 2014, 111:9923-9928. Both, white-rot and brown-rot species showed a similar wide continuum in phylogenetically informed principal-components analysis (PCA) on the sets of carbohydrate-active and lignin-active enzymes deduced from their sequenced genomes (22 in total), indicating high degrees of heterogeneity in both modes of wood decay. The newly sequenced genomes of B. botryosum and J. argillacea lack genes for class II PODs but possess genes for diverse enzymes acting on crystalline cellulose. Both fungal species were shown to colonize aspen and pine wood and to locally erode all cell wall layers indicative of white rot decay. 13. Floudas D, Held BW, Riley R, Nagy LG, Koehler G, Ransdell AS,  Younus H, Chow J, Chiniquy J, Lipzen A et al.: Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequence of Fistulina hepatica and Cylindrobasidium torrendii. Fungal Genet Biol 2015 http://dx.doi.org/10.1016/ j.fgb.2015.02.002. Species have reduced content of genes for enzymes involved in lignin degradation which rendered wood decay modes from white rot to brown rot or to a type of soft rot in which middle lamellae of wood fibers are left undegraded. 14. Morin E, Kohler A, Baker AR, Foulogne-Oriol M, Lombard V,  Nagy LG, Ohm RA, Patyshakuliyeva A, Brun A, Aerts AL et al.: Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche. Proc Natl Acad Sci U S A 2012, 109:17501-17506. Humicolous fungi such as A. bisporus are adapted to growth in humic-rich environments of heterogeneous organic nature. This fungus is limited in enzymes for lignin degradation. It has however an expansion of secreted www.sciencedirect.com

heme-thiolate peroxidases (aromatic peroxygenases, chloroperoxidases) and also of b-esterases, likely for multiple catalytic activities on organic hydrocarbons and lignin-like aromatic compounds and substituted fatty acids. Agaricus is less well equipped with genes for cytochrome P450 oxygenases for intracellular metabolism of lignin metabolites and related compounds. 15. Kohler A, Kuo A, Nagy LG, Morin E, Barry KW, Buscot F, Canba¨ck B, Choi C, Cichocki N, Clum A et al.: Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists. Nature Genet 2015 http://dx.doi.org/ 10.1038/ng.3223. 16. Phillips LA, Ward V, Jones MD: Ectomycorrhizal fungi contribute to soil organic matter cycling in sub-boreal forests. ISME J 2014, 8:699-713. 17. Rineau F, Roth D, Shah F, Smits M, Johansson T, Canba¨ck B, Olsen BP, Persson P, Grell MN, Lindquist E et al.: The ectomycorrhizal fungus Paxillus evolutus converts matter in plant litter using a trimmed brown-rot mechanism involving Fenton chemistry. Environ Microbiol 2012, 14:1477-1487. 18. Rineau F, Shah F, Smits MM, Persson P, Johansson T, Carleer R, Troein C, Tunlid A: Carbon availability triggers the decomposition of plant litter and assimilation of nitrogen by an ectomycorrhizal fungus. ISME J 2013, 7:2010-2022. 19. Bo¨deker ITM, Clemmensen KE, de Boer W, Martin F, Olson A˚, Lindahl BD: Ectomycorrhizal Cortinarius species participate in enzymatic oxidation of humus in northern forest ecosystems. New Phytol 2014, 203:245-256. 20. Gottel NR, Castro HF, Kerley M, Yang ZM, Pelletier DA, Podar M, Karpinets T, Uberbacker E, Tuskan GA, Vilgalys R et al.: Distinct microbial communities within the endosphere and rhizosphere of Populus deltoides roots across contrasting soil types. Appl Environ Microbiol 2011, 77:5934-5944. 21. Danielsen L, Thu¨rmer A, Meinicke P, Bue´e M, Morin E, Martin F, Pilate G, Daniel R, Polle A, Reich M: Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities. Ecol Evol 2012, 2:1935-1948. 22. Postma JWM, Olsson PA, Falkengren-Grerup U: Root colonization by arbuscular mycorrhizal, fine endophytic and dark septate fungi across a pH gradient in acid beech forests. Soil Biol Biochem 2007, 39:400-408. 23. Eaton CJ, Cox MP, Scott B: What triggers grass endophytes to switch from mutualism to pathogenism? Plant Sci 2011, 180:1901-2195. 24. Hane JK, Anderson JP, Williams AH, Sperschneider J, Singh KB: Genome sequencing and comparative genomics of the broad host-range pathogen Rhizoctonia solani AG8. PLOS Genet 2014, 10:e1004281. 25. Rasmussen HN, Rasmussen FN: Seedling mycorrhiza: a discussion of origin and evolution in Orchidaceae. Bot J Linnean Soc 2014, 175:313-327. 26. Yagame T, Funabiki E, Nagasawa E, Fukiharu T, Iwase K: Identification and symbiotic ability of Psathyrellaceae fungi isolated from aphotosynthetic orchid, Cremastra appendiculata (Orchidaceae). Am J Bot 2013, 100:1823-1830. 27. Lombard V, Ramulu HG, Drula E, Coutinho PM, Henrissat B: The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 2014, 42:D490-D495. 28. Levasseur A, Drula E, Lombard V, Coutinho PM, Henrissat B:  Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotech Biofuels 2013, 6:41. The CAZy database collects and correlates sequence structures and molecular mechanisms for classification of enzymes involved in assembly and breakdown of complex carbohydrates (Carbohydrate-Active enZymes). Recently, the database has been extended to include also Auxiliary Activities (AA), formerly known as the FOLymes, that is enzymes involved in lignin degradation on which information was originally collected in a separate database (FOLy). 29. Morel M, Meux E, Mathieu Y, Thuillier A, Chibani K, Harvengt L,  Jacquot J-P, Gelhaye E: Xenomic networks variability and adaptation traits in wood decay fungi. Microbial Biotechnol 2013, 6:248-263. Current Opinion in Biotechnology 2015, 33:268–278

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Upon cellular uptake of a toxic compound, intracellular detoxification encompasses three main steps. In phase I, a molecule will be activated through oxidation, possibly performed by a respective cytochrome P450 monooxygenase. In phase II, the activated molecule will be conjugated to increase compound solubility and decrease its reactivity. Conjugative enzymes can be glutathione transferases, adding glutathione to substrates of very different structures. In phase III, the modified molecule will be either transported to storage places (vacuoles) or excreted or further catabolized (Figure 1). Wood-decay and plant litter-decay basidiomycetes and to a lesser degree mycorrhizal species possess extraordinary numbers of genes for cytochrome P450 monooxygenases and glutathione transferases, respectively. Many of these may act in the intracellular xenomic networks of compound detoxification. 30. Syed K, Shale K, Pagadala NS, Tuszynski J: Systematic identification and evolutionary analysis of catalytically versatile cytochrome P450 monooxygenase families enriched in model basidiomycete fungi. PLOS ONE 2014, 9:e86683. 31. Mathieu Y, Prosper P, Favier F, Harvengt L, Didierjean C, Jacquot J-P, Morel-Rouhier M, Gelhaye E: Diversification of a specific class A glutathione transferase in saprotrophic fungi. PLOS ONE 2013, 8:e80298. 32. Torres CE, Negro C, Fuente E, Blanco A: Enzymatic approaches in paper industry for pulp refining and biofilm control. Appl Microbiol Biotechnol 2012, 96:327-344. 33. Kuhad RC, Gupta R, Singh A: Microbial cellulases and their industrial applications. Enz Res 2011, 2011:280696. 34. Gangwar AK, Tejo PN, Prakadh R: Applicability of microbial xylanases in paper pulp bleaching: a review. Bioresources 2014, 9:3733-3754. 35. Kudanga T, Le Roes-Hill M: Laccase applications in biofuels production: current status and future prospects. Appl Microbiol Biotechnol 2014, 15:6525-6530. 36. Svobodova´ K, Mikeskova´ H, Petra´cˇkova´ D: Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions. Appl Microbiol Biotechnol 2013, 97:10263-10273. 37. Rao MA, Scleza R, Acevedo F, Diez MC, Gianfreda L: Enzymes as useful tools for environmental purposes. Chemosphere 2014,  107:145-162. The authors published the first comprehensive review on enzymes for use in environmental purposes, such as in decontamination of phenolic compounds, PAHs (polycyclic aromatic hydrocarbons) and organophosphates, and such as in monitoring of soil/water pollution and soil health. 38. Bolong N, Ismail AF, Salim MR, Matsuura T: A review of the effects of emerging contaminants in wastewater and options for their removal. Desalination 2009, 239:229-246. 39. Tijani JO, Fatoba OO, Petrik LF: A review of pharmaceuticals and endocrine-disrupting compounds: sources, effects, removal, and detections. Water Air Soil Pollut 2013, 224:1770. 40. Luo Y, Guo W, Ngo HH, Nghiem LD, Hai FI, Zhang J, Liang S, Wang XC: A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment. Sci Total Environ 2014, 473–474:619-641. 41. Hofrichter M, Ullrich R, Pecyna MJ, Liers C, Lundell T: New and classic families of secreted fungal heme peroxidases. Appl Microbiol Biotechnol 2010, 87:871-897. 42. Ku¨es U, Ru¨hl M: Multiple multi-copper oxidase gene families in basidiomycetes – what for? Curr Genom 2011, 12:72-94. 43. Halaouli S, Asther M, Sigoillot J-C, Hamdi M, Lomascolo A: Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications. J Appl Microbiol 2006, 100:219-232.

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44. Morozova OV, Shumakovich GP, Shleev SV, Yaropolov YI: Laccase-mediator systems and their applications: a review. Appl Biochem Microbiol 2007, 43:523-535.

60. Cabana H, Ahamed A, Leduc R: Conjugation of laccase from the white rot fungus Trametes versicolor to chitosan and its utilization for the elimination of triclosan. Bioresour Technol 2011, 102:1656-1662.

45. Euring M, Ru¨hl M, Ritter N, Ku¨es U, Kharazipour A: Laccase mediator systems for eco-friendly production of mediumdensity fiberboard (MDF) on pilot scale: physicochemical analysis of the reaction mechanisms. Biotech J 2011, 6:12531261.

61. Sathishkumar P, Kamala-Kannan S, Cho M, Kim JS, Hadibarata T, Salim MR, Oh BT: Laccase immobilization on cellulose nanofiber: the catalytic efficiency and recyclic application for stimulated dye effluent treatment. J Mol Catal B: Enzym 2014, 100:111-120.

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62. Jahangiri E, Reichelt S, Thomas I, Hausmann K, Schlosser D,  Schulze A: Electron beam-induced immobilization of laccase on porous supports for waste water treatment applications. Molecules 2014, 19:11860-11882. This is the first experimental test study that instead of immobilizing the catalyzing enzyme laccase applied mediators (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) = ABTS; syringaldehyde) immobilized in cryogels for degradation of bisphenol A (BPA) as model for harmful contaminants in waste water. Immobilization of mediators in combination with free enzyme resulted in considerable initial BPA removal rates as compared to the free enzyme alone. Future trials in one-pot reactions will be interesting with immobilized mediators along with also immobilized enzymes. 63. Kumar VV, Sivanesan S, Cabana H: Magnetic cross-linked laccase aggregates – bioremediation tool for decolorization of distinct classes of recalcitrant dyes. Sci Total Environ 2014, 487:830-839. 64. Macellaro G, Pezzella C, Cicatiello P, Sannia G, Piscitelli A: Fungal laccases degradation of endocrine disrupting compounds. Biomed Res Int 2014:614038. 65. Ba S, Haroune L, Cruz-Morato´ C, Jacquet C, Touahar IE, Bellenger J-P, Legault CY, Jones JP, Cabana H: Synthesis and  characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters. Sci Total Environ 2014, 487:748-755. Combined crosslinked enzymes aggregates (combi-CLEAs) of T. versicolor laccase TvL and mushroom (A. bisporus) tyrosinase were produced and tested in conversion of acetaminophen as model phenolic compound in municipal and hospital wastewaters. The enzymes transformed in parallel action between 80 and up to 100% of the acetaminophen into enzyme-specific metabolites. 66. Crestini C, Melone F, Saladino R: Novel multienzyme oxidative biocatalyst for lignin bioprocessing. Bioorg Med Chem 2011, 19:5071-5078. 67. Van Aken B, Ledent P, Naveau H, Agathos SN: Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus on porous silica beads. Biotechnol Lett 2000, 22:641-646. 68. Taboada-Puig R, Junghanns C, Demarche P, Moreira MT, Feijoo G, Lema JM, Agathos SN: Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: production, partial characterization and application for the elimination of endocrine disrupters. Bioresour Technol 2011, 102:6593-6599. 69. Touahar IE, Haroune L, Ba S, Bellenger JP, Cabana H:  Characterization of combined cross-linked enzyme aggregates from laccase, versatile peroxidase and glucose oxidase, and their utilization for the elimination of pharmaceuticals. Sci Total Environ 2014, 481:90-99. Combi-CLEAs of B. adjusta VP, A. niger glucose oxidase and three different T. versicolor laccases were tested in synthetic waste water supplemented with a cocktail of 14 different pharmaceutically active compounds. In combined action by VP and laccase and under supportive action of glucose oxidase, these multiple biocatalysts reacted at a pH closer to neutral as compared to the free enzymes and eliminated the five different compounds acetaminophen, naproxen, mefanamic acid, diclofenac and indomethacin from solution. 70. Galliker P, Hommes G, Schlosser D, Corvini PFX, Shahgaldian P: Laccase-modified silica nanoparticles efficiently catalyze the transformation of phenolic compounds. J Colloid Interface Sci 2010, 349:98-105. 71. Songulashvili G, Jimenez-Tobon GA, Jaspers C, Gratia JP, Debaste F, Penninckx MJ: Immobilized Coriolopsis sp. laccase for continuous elimination and transformation of phenolic micropollutants. Water Qual Res J Can 2014, 49:328-338. 72. Cabana H, Alexandre C, Agathos SN, Jones JP: Immobilization of laccase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals. Bioresour Technol 2009, 100:3447-3458. 73. Lloret L, Eibes G, Feijo G, Moreira MT, Lema JM: Continuous operation of a fluidized bed reactor for the removal of estrogens by immobilized laccase on Eupergit supports. J Biotechnol 2012, 182:404-406. www.sciencedirect.com

74. Lloret L, Hollmann F, Eibes G, Feijoo G, Moreira MT, Lema JM: Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor. Biodegradation 2012, 23:373-386. 75. Hou J, Dong G, Ye Y, Chen V: Bio-degradation of bisphenol-A with immobilized laccase on TiO2 sol–gel coated PVDF membrane. J Membr Sci 2014, 469:19-30. 76. Ba S, Jones JP, Cabana H: Hybrid bioreactor (HBR) of hollow fiber microfilter membrane and cross-linked laccase aggregates eliminate aromatic pharmaceuticals in wastewaters. J Hazard Mater 2014, 280:662-670. 77. Gasser C, Yu L, Svojitka J, Wintgens T, Ammann E, Shahgaldian P,  Corvini PFX, Hommes G: Advanced enzymatic elimination of phenolic contaminants in wastewater: a nano approach at field scale. Appl Microbiol Biotechnol 2014, 98:3305-3316. In a pilot scale experiment, a fixed bed reactor was used for removing first any suspended solids in the waste water treatment plant effluents to avoid any binding of phenolic compounds to the solids and thereby their escaping from the efficient targeting by the Thielavia laccase immobilized onto silica nanoparticles. The 1.27  0.18 nM bisphenol-A containing effluents were then fed into a membrane reactor with the laccase-nanoparticles. Bisphenol-A degradation was followed up for 43 days, partially in continuous mode, partially in batch mode. Once the system was stabilized, the bisphenol-A concentration dropped by about 66% in solution. At the end of the experiment, laccase activity was still 43% of the initially applied activity. The systems leaves room for several further improvements. 78. Burns RG, DeForest JL, Marxsen J, Sinsabaugh RL,  Stromberger ME, Wallenstein MD, Weintraub MN, Zoppini A: Soil enzymes in a changing environment: current knowledge and future directions. Soil Biol Biochem 2013, 58:216-234. This excellent and timely comprehensive review summarizes over a broad subject range the current knowledge on enzymes in soil. The reader finds newest information on enzyme production and regulation, spatial location and immobilization of enzymes, enzymatic activities in changing environmental conditions, and ecological functions of enzymes. Methods on detection of enzymes and enzyme activities are discussed, and thoughts on manipulation of extracellular enzymes for ecosystem services presented. 79. Wu Y, Jiang Y, Jiao JG, Liu MQ, Hu F, Griffiths BS, Li HX: Adsorption of Trametes versicolor laccase to soil iron and aluminium minerals: enzyme activity, kinetics and stability studies. Colloids Surf B: Biointerfaces 2014, 111:342-348. 80. Ahn MY, Zimmermann AR, Martinez CE, Archibald DD, Bollag JM, Dec J: Characteristics of Trametes villosa laccase adsorbed on aluminium hydroxide. Enzyme Microb Technol 2007, 41:141148. 81. Acevedo F, Pizzul L, Castillo MdP, Gonza´lez ME, Cea M, Gianfreda L, Diez MC: Degradation of polycyclic aromatic hydrocarbons by free and nanoclay-immobilized manganese peroxidase from Anthracophyllum discolor. Chemosphere 2010, 80:271-278. 82. Morillo JA, Abtizar-Ladislao B, Monteoliva-Sa´nchez N, RamosCormenzana A, Russell NJ: Bioremediation and biovalorisation of olive-mill wastes. Appl Microbiol Biotechnol 2009, 82:25-39. 83. Dermeche S, Nadour M, Larroche C, Moulti-Mati F, Michaud P: Olive mill wastes: biochemical characterization and valorization strategies. Process Biochem 2013, 10:1532-1552. 84. Aranda E, Sampedro I, Ocampo JA, Garcı´a Romera I: Phenolic removal of olive-mill dry residue by laccase activity of white rot fungi and its impact on tomato plant growth. Int Biodeterior Biodegrad 2006, 58:176-179. 85. Saparrat MCN, Jurado M, Diaz R, Garcı´a Romera I, Martı´nez MJ: Transformation of the water soluble fraction from ‘alpeorujo’ by Coriolopsis rigida: the role of laccase in the process and its impact on Azospirillum brasiliense survival. Chemosphere 2010, 78:72-76. 86. Reina R, Liers C, Ocampo JA, Garcı´a-Romera I, Aranda E: Solid state fermentation of olive mill residues by wood-dwelling and dung-dwelling Agaricomycetes: effects on peroxidase production, biomass development and phenol phytotoxicity. Chemosphere 2013, 93:1406-1412. Current Opinion in Biotechnology 2015, 33:268–278

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89. Siles JA, Gonza´lez-Mene´ndez V, Platas G, Sampedro I, Garcı´aRomera I, Bills GF: Effects of dry olive residue transformed by Coriolopsis floccosa (Polyporaceae) on the distribution and dynamic of a culturable fungal soil community. Microb Ecol 2014, 67:648-658.

100. Zhang J, Zeng G, Chen Y, Liang J, Zhang C, Huang B, Sun W, Chen M, Yu M, Huang H, Zhu Y: Phanerochaete chrysosporium inoculation shapes the indigenous fungal communities during agricultural waste composting. Biodegradation 2014, 25:669680.

90. Siles JA, Pe´rez-Mendoza D, Ibanez JA, Scervino JM, Ocampo JA,  Garcia-Romera I, Sampedro I: Assessing the impact of biotransformed dry olive residue application to soil: effects on enzyme activities and fungal community. Int Biodeterior Biodegrad 2014, 89:15-22. Soil enzyme activities such as of phosphatase, urease and b-glucosidase involved in P, N and C nutrient cycles are indicative for general microbial activities in soil. Together with species investigations, they can be used to monitor the impact of soil amendments such as fungal transformed olive waste on community structure and function.

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