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蘑菇的成长过程英文作文The Fascinating Lifecycle of a Mushroom: A Journey from Spore to Fungus.Mushrooms, the enigmatic inhabitants of the fungal kingdom, embark on an extraordinary journey from microscopic spores to visible fruiting bodies. This complex lifecycle involves a series of remarkable transformations, each stage playing a crucial role in the mushroom'ssurvival and reproduction.1. Spore Formation:The lifecycle of a mushroom begins with the formation of spores. Inside the mature mushroom's gills or pores, millions of tiny spores are produced, carried by microscopic threads called basidia. These spores are incredibly lightweight and can travel through the air for long distances, propelled by even the slightest breeze.2. Germination and Mycelium Growth:When a spore finds a suitable environment with favorable conditions such as moisture and nutrients, it germinates and develops into a new organism called a mycelium. The mycelium is a network of branching hyphae, which are threadlike filaments that spread through the substrate, usually soil or organic matter.3. Substrate Colonization:As the mycelium grows and expands, it secretes enzymes that break down organic matter, releasing nutrients that the mushroom uses for growth. This process enables the mycelium to colonize the substrate and establish a symbiotic relationship with the surrounding environment.4. Primordia Formation:Under specific environmental cues, such as temperature and light, the mycelium begins to form tiny structures called primordia. These primordia are the early stages ofthe mushroom's fruiting body, and they represent the transition from the vegetative mycelial stage to the reproductive stage.5. Fruiting Body Development:As the primordia mature, they differentiate into the visible mushroom that we are familiar with. The stalk, or stipe, elongates and supports the cap, or pileus. The gills or pores on the underside of the cap produce more spores, completing the reproductive cycle.6. Spore Dispersal:Once the mushroom reaches maturity, it releases its spores back into the environment. This dispersal is aided by various mechanisms, including wind, insects, and animals that consume the mushroom. The spores then can germinate and start the lifecycle anew, perpetuating the species' survival.Exceptional Adaptations:Mushrooms have evolved remarkable adaptations that contribute to their success in diverse ecosystems. For example, some species, such as bioluminescent mushrooms, emit light to attract insects for spore dispersal. Others, such as parasitic mushrooms, derive nutrients from living plants or other organisms.Ecological Significance:Mushrooms play a vital ecological role in nutrient cycling and decomposition. They decompose organic matter, releasing essential nutrients back into the environment. Additionally, many mushrooms form symbiotic relationships with plants, known as mycorrhizae, providing them with water and nutrients while receiving carbohydrates from the plant in return.Conclusion:The lifecycle of a mushroom is a fascinating and complex process that showcases the remarkable adaptationsand ecological importance of fungi. From the humble spore to the iconic fruiting body, mushrooms' journey through the plant and animal kingdoms underscores their enduring contribution to the intricate web of life on Earth.。
上海市松江区2024届高三一模英语试题(含听力）(4)一、听力选择题1.A．The harm done by single-use plastics.B．The topic for the woman’s composition.C．Environmental issues.D．Some recent hot news.2. Where are the speakers?A．At home.B．At a restaurant.C．At a stadium.3. Where does the conversation take place?A．In an office.B．In a restaurant.C．In a store.4. What does the woman think of teaching?A．It’s boring.B．It’s interesting.C．It’s difficult.5. Where are the speakers?A．In a supermarket.B．In a hotel.C．In a police station.二、听力选择题6. 听下面一段较长对话，回答以下小题。
1. What courses will the girl attend?A．Business and African music.B．Finance and English composition.C．Basic Spanish and English composition.2. On which day does the girl have piano classes?A．Monday.B．Tuesday.C．Thursday.7. 听下面一段较长对话，回答以下小题。
1. What tea does good to your lungs?A．Green tea.B．Dark tea.C．Black tea.2. How much will the woman pay?A．100 yuan.B．150 yuan.C．200 yuan.8. 听下面一段较长对话，回答以下小题。
真核生物英语Eukaryotes: The Diverse and Complex Organisms that Dominate the Biological WorldEukaryotes, a term derived from the Greek words "eu" meaning "true" and "karyon" meaning "nucleus," are a group of organisms that are characterized by the presence of a true nucleus within their cells. This defining feature sets them apart from the other major domain of life, the prokaryotes, which lack a distinct nuclear membrane. Eukaryotes encompass a vast and diverse array of organisms, ranging from single-celled microbes to the towering trees and majestic mammals that populate our planet.At the heart of eukaryotic cells lies the nucleus, a membrane-bound organelle that houses the genetic material, or DNA, of the cell. This genetic information is organized into linear structures called chromosomes, which are further compacted and organized within the nucleus. The presence of a true nucleus allows for the compartmentalization of various cellular processes, enabling a higher level of complexity and specialization within eukaryotic cells.Beyond the nucleus, eukaryotic cells are characterized by thepresence of numerous other membrane-bound organelles, each with its own specialized function. These organelles include the endoplasmic reticulum, which is responsible for the synthesis and transport of proteins; the Golgi apparatus, which modifies and packages these proteins for distribution; and the mitochondria, which serve as the "powerhouses" of the cell, generating the energy-rich molecule ATP through the process of cellular respiration.One of the most striking features of eukaryotic cells is the presence of a cytoskeleton, a network of filamentous structures that provide structural support, facilitate intracellular transport, and enable cellular movement. This cytoskeleton is composed of three main types of filaments: microfilaments, intermediate filaments, and microtubules, each with their own unique properties and functions.Eukaryotes can be broadly divided into several major groups, including plants, animals, fungi, and protists. Each of these groups has its own distinct characteristics and evolutionary histories, but they all share the fundamental features that define the eukaryotic domain of life.The plant kingdom, for example, is characterized by the presence of chloroplasts, organelles that house the photosynthetic machinery responsible for converting sunlight, carbon dioxide, and water into glucose, the primary energy source for plant cells. Plants also possessa rigid cell wall made of cellulose, which provides structural support and protection.The animal kingdom, on the other hand, is defined by the presence of specialized cells that are capable of movement, such as muscle cells, and the ability to sense and respond to their environment, as exemplified by the complex nervous systems of many animals. Additionally, animal cells lack the rigid cell walls found in plants, instead possessing a more flexible cell membrane.Fungi, while often overlooked, play a crucial role in the global ecosystem. These organisms are characterized by the presence of chitin-based cell walls and the ability to obtain nutrients through the secretion of digestive enzymes and the absorption of the resulting molecules. Fungi can be found in a wide range of habitats, from the deep ocean to the highest mountain peaks, and they play vital roles in decomposition, nutrient cycling, and the formation of symbiotic relationships with other organisms.Protists, the most diverse and heterogeneous group of eukaryotes, encompass a wide range of single-celled organisms that do not fit neatly into the plant, animal, or fungal kingdoms. This group includes a variety of algae, protozoa, and slime molds, each with their own unique adaptations and ecological niches.The diversity of eukaryotic organisms is truly astounding, with estimates suggesting that there may be as many as 8.7 million species on Earth, the vast majority of which are yet to be discovered and described. This remarkable diversity is a testament to the evolutionary success of the eukaryotic domain, which has adapted to thrive in a wide range of environments and ecological niches.One of the key factors that has contributed to the success of eukaryotes is the development of sexual reproduction, a process that involves the fusion of two haploid cells to form a diploid zygote. This process allows for the shuffling of genetic material, creating genetic diversity and enabling the rapid adaptation of eukaryotic organisms to changing environmental conditions.Moreover, the complexity of eukaryotic cells, with their intricate organelles and specialized functions, has allowed for the evolution of multicellular organisms, which can coordinate the activities of individual cells to form complex tissues, organs, and systems. This level of organization has enabled the development of highly specialized and sophisticated life forms, such as the towering trees of the rainforest and the intricate neural networks of the human brain.Despite their diversity and complexity, eukaryotes face a range of challenges in the modern world. Environmental degradation, habitat loss, and the spread of disease have all contributed to the decline ofmany eukaryotic species, highlighting the fragility of the delicate balance that sustains life on our planet.In conclusion, the eukaryotic domain of life is a testament to the incredible diversity and complexity of the biological world. From the single-celled protists to the majestic mammals, eukaryotes have evolved to thrive in a wide range of environments, demonstrating the remarkable adaptability and resilience of life. As we continue to explore and understand the intricate workings of these remarkable organisms, we gain a deeper appreciation for the wonders of the natural world and the importance of preserving the delicate balance that sustains it.。
土壤多环芳烃污染根际修复研究进展许超，夏北成*中山大学环境科学与工程学院，广东广州510275摘要：多环芳烃(polycyclic aromatic hydrocarbons，PAHs)是环境中普遍存在的具有代表性的一类重要持久性有机污染物，具“三致性”、难降解性，在土壤环境中不断积累，严重危害着土壤的生产和生态功能、农产品质量和人类健康。
修复土壤多环芳烃污染已成为研究的焦点。
根际修复是利用植物-微生物和根际环境降解有机污染物的复合生物修复技术，是目前最具潜力的土壤生物修复技术之一。
对国内外学者近年来在土壤多环芳烃污染根际修复的效果、根际修复机理和根际修复的影响因素方面的研究进展作了较系统的综述，并分别分析了单作体系、混作体系、多进程根际修复系统和接种植物生长促进菌根际修复系统对土壤多环芳烃的修复效果。
指出根际环境对PAHs的修复主要有3种机制：根系直接吸收和代谢PAHs；植物根系释放酶和分泌物去除PAHs，增加根际微生物数量，提高其活性，强化微生物群体降解PAHs。
并讨论了影响根际修复PAHs 的环境因素如植物、土壤类型、PAHs理化性质、菌根真菌以及表面活性剂等。
植物-表面活性剂结合的根际修复技术、PAHs 胁迫下根际的动态调节过程、运用分子生物学技术并结合植物根分泌物的特异性筛选高效修复植物以及植物富集的PAHs代谢产物进行跟踪与风险评价将成为未来研究的主流。
关键词：根际；多环芳烃（PAHs）；根际修复；土壤中图分类号：X53 文献标识码：A 文章编号：1672-2175（2007）01-0216-07多环芳烃（polycyclic aromatic hydrocarbons，PAHs）是环境中普遍存在的具有代表性的一类重要持久性有机污染物（persistent organic pollutants，POPs）。
大量研究已经证明，多环芳烃具有慢性毒性和致癌、致畸、致突变的“三致”作用，是环境中一类危险而需重点研究的、也是各国优先控制的污染物。
对生物有害的英语作文英文回答：How Biological Agents Can Harm Humans。
Biological agents refer to pathogenic microorganisms or their products that can cause disease in humans and other organisms. These agents include bacteria, viruses, fungi, and parasites, and can pose a significant threat to public health. Understanding the mechanisms by which biological agents cause harm is crucial for developing effective strategies for prevention and control.Pathogenicity and Virulence。
Pathogenicity refers to the ability of a biological agent to cause disease, while virulence measures the severity of the disease it produces. Pathogenicity and virulence can vary widely among different agents, and are influenced by several factors, including:Toxins: Many biological agents produce toxins that target specific host tissues or cells, causing cell damage, tissue destruction, and organ failure.Enzymes: Some agents produce enzymes that facilitate their entry into host cells, promote their survival within the host, or damage host cells directly.Immune Evasion: Successful biological agents often have mechanisms to evade the host's immune system, allowing them to replicate and cause disease more effectively.Replication: The ability of a biological agent to replicate within the host plays a crucial role in its pathogenicity. Rapidly replicating agents can overwhelm the host's immune response and cause more severe disease.Mechanisms of Harm。
ISSN 0003 6838, Applied Biochemistry and Microbiology, 2012, Vol. 48, No. 1, pp. 17–20. © Pleiades Publishing, Inc., 2012.Original Russian Text © M.A. Kupryashina, N.Yu. Selivanov, V.E. Nikitina, 2012, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2012, Vol.48, No. 1, pp. 23–26.17INTRODUCTIONDiazotrophic Azospirillum bacteria known for syn thesizing biologically active substances, which posi tively affect plant growth and development, are used as model objects to study plant–bacterial associations. It has been recently shown that Azospirillum possesses phenoloxidase activity [1–3]. Screening of a variety of Azospirillum strains for the presence of phenoloxidases revealed Mn peroxidase activity [3].For the first time, Mn peroxidases (MnPs , EC 1.11.1.13) were isolated from Phanerochaete chrysos porium [4]. The enzyme demonstrated oxidative activ ity only in the presence of Mn 2+. Later, it was shown that the enzyme catalyzed peroxide dependent oxida tion of Mn 2+ to Mn 3+, which, in turn, appeared to be a strong oxidant. MnP is an enzyme with a molecular weight of 42–47 kD, which is usually located in extra cellular space [5].The enzyme is often used as a natural oxidant in contemporary biotechnologies, such as pulp and tex tile bleaching and wastewater and natural pool treat ment against biocides, dyes, and xenobiotics.Although MnPs of several species of basidiomycetes have been isolated and well studied, bacterial MnPs have been poorly investigated. To date, only one study ,which deals with the isolation and purification of MnPs from Bacillus pumilus and Paenibacillus , has been published [6]. The high oxidative capacity of MnPs, as well as their multifunctionality in different organisms, makes this enzyme interesting for both applied and basic research.The present study was aimed at the isolation and purification of extracellular MnPs from Azospirillum brasilense Sp245.EXPERIMENTALBacteria Cultivation Conditions. Strains of A.brasilense Sp245 from the microbial collection of the Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences,were used as an object of our study . The bacteria were cultivated in 250 ml Erlenmeyer flasks on a liquid medium with the following content: 0.1 g/l of KH 2PO 4, 0.4 g/l of K 2HPO 4, 0.1 g/l of NaCl, 0.002 g/l of Na 2MoO 4 ⋅ 7H 2O , 0.2 g/l of MgSO 4 ⋅ 7H 2O , 0.02 g/l of FeSO 4 ⋅ 7H 2O , 5 g/l of malic acid, 1.7 g/l of NaOH,1.0 g/l of NH 4Cl , 0.02 g/l of CaCl 2, MnSO 4 ⋅ 5H 2O at a concentration of 1 mM, and pH 6.8. A 12 h culture grown on the same medium was used as an inoculation material. The bacteria were cultivated at 37°С for 35 h.Induction of MnP Activity. To induce MnP activity ,the medium was supplemented with syringaldazine (Acros Organics, United States), 2,6 dimethoxyphe nol (Acros Organics, United States), 2,2' azino bis(3 ethylbenzothiazoline 6 sulfonate) (ABTS, Sigma,United States), and pyrocatechol (Acros Organics,United States) at concentrations of 0.1, 0.5, and 1mM. To measure the enzyme activity , samples were taken after 24 and 36 h, i.e., during the exponential and stationary growth phases of the culture, respec tively .Estimation of the MnP Activity. The activity of extracellular MnPs was measured spectrophotometra ically at each stage of the study , using a Specord M40spectrophotometer (Carl Zeiss, Germany), by the rate of oxidation of 2,6 dimethoxyphenol (ε = 30.5 mM –1cm –1) at 30°С [7]. The reaction mixture (2 ml) con tained 50 mM sodium tartrate buffer, pH 4.5, 1 mM 2,6 dimetoxiphenol, 1 mM MnSO 4 ⋅ 5H 2O , and an enzyme preparation. The reaction was initiated by the introduction of 100 μl of 1 mM H 2O 2. A unit change inIsolation and Purification of Mn Peroxidasefrom Azospirillum brasilense SP245M. A. Kupryashina, N. Yu. Selivanov, and V. E. NikitinaInstitute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Saratov, 410049 Russiae mail: Kupryashina_m@mail.ruReceived February 14, 2011Abstract —Homogenous Mn peroxidase of a 26 fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS PAAG. It was shown that the use of pyrocatechol and 2,2' azino bis(3 ethylbenzotiazoline 6 sulfonate) at concentrations of 0.1 and 1 mM as inductors increased the Mn peroxidase activity by a factor of 3.DOI: 10.1134/S000368381201009718APPLIED BIOCHEMISTRY AND MICROBIOLOGY V ol. 48 No. 1 2012KUPRYASHINA et al.the absorption at a 468 nm wavelength for 1 min was considered to correspond to 1 unit of enzymatic activ ity . The specific enzymatic activity was expressed as units per mg of protein. The protein concentration was estimated by the Bradford method.Enzyme Purification. A 36 h bacterial culture medium, containing 0.1 mM pyrocatechol as an inductor, was centrifuged at 10000 g for 20 min at 4°С.The obtained supernatant was desalinated on a G 25column against buffer A (0.025 M sodium acetate buffer, pH 5.0). A release of protein fractions was reg istered using a Uvicord S II detector (LKB, Sweden)at a λ = 280 nm wavelength. The collected active frac tions were purified by HPLC using a HPLC Smart Line 5000 device (Knauer, Germany). Separation was performed on an anion exchange TSK Bioassist Q column (Tosohaas, United States) preequilibrated with buffer A. Proteins were eluted with NaCl concen tration gradient in buffer A at a 1 ml/min flow rate using photometric detection at a λ = 280 nm wave length. Fractions, which possessed Mn peroxidase activity , were dialyzed against water and used for fur ther investigations.Electrophoresis. The homogeneity of the obtained enzyme preparation, as well as its molecular weight,were estimated by electrophoresis in denaturating gels (SDS PAAG) according to the method of Laemmli using protein molecular weight standards (Fermentas,Latvia) containing β galactosidase (116 kD), bovine serum albumin (66.2 kD), ovalbumin (45.0 kD), lac tate dehydrogenase (35.0), REase Bsp98I (25.0 kD),β lactoglobulin (18.4 kD), and lysozyme (14.4 kD).Electrophoresis in nondenaturating PAAG was per formed according to the same method, but the stage of boiling or adding SDS and mercaptoethanol wereomitted. The protein bands were stained with a solu tion containing silver.RESUL TS AND DISCUSSIONCultivation of A.brasilense Sp245 strains on a stan dard malate containing medium in the presence of 0.094 mg/l of Mn 2+ without inductors revealed low MnP activity , which did not exceed 1.2 units/mg by the oxidation of 2,6 dimethoxyphenol. The increase in the concentration of MnSO 4 ⋅ 5H 2O to 1 mM resulted in an increase in the MnP activity to 2.6units/mg (Fig. 1). The given data are typical of bacteria in the stationary phase of growth (36 h cul ture), while for 24 h cultures, lower enzymatic activity is commonly observed (data are not shown). It was previously shown that the increase in the manganese concentration in the medium led to an increase in the yield and activity of fungal MnPs [8].It is known that synthesis of extracellular enzymes significantly depends on the culture medium ually , high concentrations of complex organic sub stances in a medium lead to more intensive bacterial growth and an increase in production of extracellular enzymes. However, the opposite effect was observed for the studied extracellular MnPs. The introduction of a yeast extract to the medium resulted in a sharp decrease in the level of enzymatic activity . Therefore,it was not used any further.Based on the suggestion that bacterial MnPs, simi larly to fungal enzymes, are also inducible enzymes,the cultivation medium was supplemented with differ ent concentrations of aromatic substrates in order to increase MnP activity . It was shown that the introduc tion of phenol compounds, such as syringaldazine,2,6 dimethoxyphenol, ABTS, and pyrocatechol increased MnP activity . The data on the effect of phe nol compounds on MnP activity are shown in Fig. 1.An increase in the Mn peroxidase activity of A.brasilense Sp245, which were cultivated in the pres ence of inductors, was observed after 36 h similarly to the one was observed for bacteria grown in a regular malate containing medium. The highest level of MnP activity , which exceeded the control values by a factor of 3, was observed after the introduction of pyrocate chol and ABTS at concentrations of 0.1 mM and 1mM, respectively (Fig. 1).Based on the data obtained, A. brasilense Sp245were further cultivated in a medium supplemented with 0.1 mM pyrocatechol and 1.0 mM MnSO 4 ⋅5H 2O for 36 h.At the first stage of enzyme purification, the super natant was freed from low molecular weight sub stances by gel filtration on a G 25 Sephadex column preequilibrated with 0.025 M sodium acetate buffer,pH 5.0. The main obstacle was to separate the enzyme from the pigment, which was formed as a result of the enzymatic oxidation of the inductor. The pigment was only totally removed at the next stage of purification.765432100.1 mM0.5 mM 1 mM123units/mg IIIIIIIVIIIIIIIVIII IIIIVFig. 1. MnP activity in a culture medium of A. brasilense Sp245: (1) medium without inductors, (2) medium with 1.0 mM MnSO 4 ⋅ 5H 2O, (3) medium containing phenol inductors, (I)pyrocatechol, (II) syringaldazine, (III) 2,6 dimethoxyphenol, (IV) ABTS.APPLIED BIOCHEMISTRY AND MICROBIOLOGY V ol. 48 No. 1 2012ISOLATION AND PURIFICATION OF Mn PEROXIDASE19Further purification of the protein fraction was car ried out by HPLC on a TSK Bioassist Q anion exchange column. Chromatographic separation car ried out at low pH values and the use of a complex gra dient of NaCl concentration allowed us to remove the majority of bulk proteins at the stage of application of the sample on the column. Proteins, which possessed Mn peroxidase activity , were strongly bound to the matrix and were eluted with high concentrations of NaCl. The chromatographic curve obtained in the result of MnP purification on the TSK Bioassist Q col umn is shown in Fig. 2. An analysis of the enzymatic activity revealed that the enzyme was released from the column in two spikes (41 and 42 min), which were combined.An analysis of the homogeneity of the obtained enzyme preparation was performed by electrophoresis in nondenaturating (7.5% PAAG) (Fig. 3) and dena turating gels (12% SDS PAAG) (Fig. 4). A compara tive analysis of the obtained data revealed that extra cellular MnPs of A . brasilense Sp245 consisted of one subunit with a molecular weight of ~43 kD.The specific activity of the isolated bacterial MnPs was 125 units/mg protein, and the purity grade was 26 fold.The purification results of MnPs of A. brasilense Sp245are shown in the table.A comparative analysis of the data obtained in our study and data from the literature revealed that the activ ities of fungal MnPs were tenfold, or even 100 fold,higher than the activity of MnPs of A. brasilense Sp245. However, the specific activity of the enzyme isolated in our study appeared to be either similar to that of fungal enzymes or even higher [10].A comparison of MnPs of A. brasilense Sp245 with the MnPs isolated from Bacillus pumilus and Paeniba cillus sp. in [5] revealed one significant difference; i.e.,the enzymes obtained in [6] demonstrated a higher level of activity at alkaline pH values, whereas the MnP isolated in our study was active at low pH values (similarly to the known fungal MnPs). Doubtlessly ,there can be differences between fungal and bacterial enzymes. However, it is also possible that the enzymes previously isolated from the cultures of Bacillus pumi1015010050505010203040D 280mAU min12Fig. 2. Chromatographic curve of an A. brasilense Sp245preparation on a TSK Bioassist Q column: (1) protein, (2)NaCl concentration gradient.12116.066.245.035.018.414.4116.066.245.035.025.012Fig. 3. PAAG electrophoresis of homogenous MnPs from A. brasilense Sp245 after purification on a TSK Bioassist Q column under (a) nondenaturating conditions and (b) with SDS PAAG: (1) homogenous enzyme, (2) molecular weight markers.Results of purification of MnPs from A. brasilense Sp245Stage of purificationActivity, unitsProtein concen tration, mgSpecific activity,units/mgPurity gradeCulture medium4.00.84 4.81Gel filtration on Sephadex G 25 2.30.45.7 1.2on exchange chromatography on a TSK Bioassist Q column0.40.003212526(a)(b)20APPLIED BIOCHEMISTRY AND MICROBIOLOGY V ol. 48 No. 1 2012KUPRYASHINA et al.lus and Paenibacillus sp., which possessed such spe cific properties, belonged to another group of peroxi dases.The table and Figs. 2 and 3 demonstrate that anion exchange chromatography on the TSK Bioassist Q column allowed for the obtaining of bacterial MnPs of high purity and specific activity .Therefore, we have for the first time isolated homogenous bacterial MnPs from Azospirillum .The functional role of MnPs of Azospirillum is yet to be studied. However, it is common knowledge that aromatic metabolites play an important role in the vital functions and interactions of both microorgan isms and plants with the environment. In particular,phenol compounds are known to be involved in pro tective reactions of plant tissues [11]. It is known that specific phenol compounds, such as flavonoids, which are released by roots into the rhizosphere and provide the chemotaxis of rhizobacteria, work as primary sig nals during the formation of plant–bacterial associates [11]. Based on the data obtained, we suggested that the presence of a phenol oxidizing enzyme in A. brasilense Sp245, the activity of which correlated with the con centration of aromatic compounds in the medium,can be part of the adaptive mechanism aimed at increasing the survivability and competitiveness of Azospirillum in the rhizosphere owing to its ability to oxidize toxic phenol compounds.ACKNOWLEDGMENTSThis work was supported by the grant of the presi dent of the Russian Federation (project no. NSH 3171.2008.4).REFERENCES1.Diamantidis, G., Effosse, A., Potier, P ., and Bally ,R.,Soil Biol. Biochem., 2000, vol. 32, pp. 919–927.2.Faure, D., Bouillant, M. L., and Bally , R., Appl. Envi ron. Microbiol., 1994, vol. 60, no. 9, pp. 3413–3415.3.Nikitina, V.E., V etchinkina, E.P ., Ponomareva, E.G,and Gogoleva, Yu.V., Microbiology, 2010, vol.79, no. 3,pp. 327–333.4.Tien, M. and Kirk, T ., Science , 1983, vol. 221, no. 3,pp.661–662.5.Levit, M.N. and Shkrob, A.M., Bioorg. Khim., 1992,vol. 18, pp. 309–345.6.Lopes de Oliveira, P ., Duarte, M., Ponezi, A., and Dur rant, L., Brazilian J. Microbiol., 2009, vol. 40, pp. 818–826.7.Paszczynski, A., Crawford, R., and Huynh, V. B.,Methods Enzymol., 1988, vol. 161, pp. 264–270.8.Lisov , A.V., Leont’evskii, A.A., and Golovleva, L.A.,Biochemistry (Moscow), 2003, vol. 68, pp. 1256–1265.9.Elisashivili, V. and Kachlishivili, E., J. Biotechnol.,2009, vol. 144, pp. 37–42.10.Dzedzyulya, E.I. and Bekker, E.G., Biochemistry (Moscow), 2000, vol. 65, pp. 829–835.11.Zaprometov , M.N., Fiziol. Rast., 1992, vol. 39,pp.1197–1207.。
中国农业大学学报2021，26(6): 114-125 Journal of China Agricultural Universityhttp：//zgnydxxb. ijournals. cn D O I：10. 11841/j. issn. 1007-4333. 2021. 06. 12花生壳生物炭用量对猪粪堆肥温室气体和NH3排放的影响王义祥叶菁林怡刘岑薇李艳春(福建省农业科学院农业生态研究所/福建省红壤山地农业生态过程重点实验室，福州350013)摘要为研究不同花生壳生物炭添加比例对猪粪堆肥过程中温室气体和N H3排放的影响。
利用强制通风静态堆肥技术，研究0(对照）、3%、6%和9%花生壳生物炭添加比例（质量比）对猪粪堆肥过程C()2、C H4、N2()和N H:1排放和堆肥性质的影响。
结果表明：添加生物炭能够延长堆肥高温期持续天数，使p H提高0. 09〜0• 13个单位，E C提高11. 7%〜50. 6%;各堆肥处理C02、C H.,和N20排放速率均随发酵时间的延长呈先升高后降低的趋势.且C02、C H',和N2()排放速率均与p H具有显著的相关性；随生物炭用量的增加，猪粪堆肥过程中C()2排放速率表现为先升高后降低的变化趋势.其中以3%生物炭添加比例处理最高.其平均C02排放速率比对照增加12.9%;N20排放和N H,挥发均以9%生物炭添加比例处理最低，分别比对照降低12. 5%和29. 9%。
综上，在整个堆肥过程中，花生壳生物炭的添加降低了N2()和C H.,的累积排放量•且随花生壳生物炭添加比例的增加，温室气体减排效应增大。
关键词猪粪；堆肥；生物炭；温室气体；N H3中图分类号X713文章编号1007-4333(2021)06-0114-12 文献标志码AEffects of peanut shell biochar on greenhouse gas andNH3emissions during swine manure compostingWANG Yixiang，YE Jing, UN Y i, LIU Cenwei，LI Yanchun(A g r ic u ltu r a l E c o lo g y In s titu te, F u jia n A c a d e m y of A g ric u ltu ra l S c ie n c e s/F u jia n K e y L a b o ra to ry ofA g ric u ltu ra l E c o lo g ic a l P ro c e s s o f R ed S o il M o u n ta in, Fuzhou 350013, C h in a)Abstract T h e effects of different peanut shell biochar addition ratio on greenhouse g a s a nd N H3 emissions during swine m a n u r e composting w e r e investigated to provide scientific basis for nitrogen conservation and greenhouse gas emissions reduction. In this study，an experiment with peanut shell biochar addition rates of 0，3%，6%an d 9%w a s conducted to study greenhouse g a s (G H G) a nd N H3 emission and its correlation with environmental factors during swine m a n u r e composting using static forced-air composting boxes. T h e results s h o w e d that peanut shell biochar addition increased the high temperature duration of swine m a n u r e composting, the p H of c o m p o s t s increased by 0. 09 - 0. 13 units, a nd the E C increased by 11.7% - 50. 6%.T he C02,C H4,a nd N20emission rates increased f irst a nd then decreased with the increase of the composting time, a nd had a significant correlation with pH. With the increase of peanut shell biochar addition, the C02emission rate during swine m a n u r e composting first increased and then decreased. T h e average C02 emission rate of 3%peanut shell biochar treatment w a s the highest, a nd increased by12.9%than that of control. Both N20 emission a nd N H3 volatilization of 9%peanut shell biochar treatment w e r e thelowest, which decreased by 12.5%a nd 29.9%，respectively. In conclusion, during the entire composting process, the addition of peanut shell biochar reduced the cumulative emissions of N20 a nd C H4.T h e emission reduction effect increased with the increase of the peanut shell biochar.K e y w o r d s swine m a n u r e；co m p o s t i n g；biochar；greenhouse g a s；N H3收稿日期：2020-09-27基金项目：国家重点研发计划子课题（2016Y F D0501404-3);福建省科技厅公益项目（2020R1021003);福建省农科院科技创新团队建设项目第一作者：王义祥，研究员，主要从事农业废弃物资源化利用研究，E-ma i l:Sd_ w o l c m g@163.c o m第6期王义祥等：花生壳生物炭用量对猪粪堆肥温室气体和N H3排放的影响115猪粪含有较多的氮、磷、钾及蛋白质、脂肪、有机 酸、无机盐等，具有巨大的农业应用潜力。
Fungal Plant Pathology and DiseaseManagementFungal plant pathology is a critical field of study that focuses on the identification, prevention, and management of diseases caused by fungi in plants. Fungal pathogens can severely impact crop yields, leading to significant economic losses for farmers and threatening food security. Understanding the mechanisms through which fungi infect plant tissues and spread within a plant is essentialfor developing effective disease management strategies. One of the key challenges in fungal plant pathology is the identification of specific fungal species responsible for causing diseases in plants. Fungi have evolved a wide range of mechanisms to infect plants, including the production of enzymes that degradeplant cell walls, toxins that kill plant cells, and the ability to outcompete beneficial microorganisms in the plant's ecosystem. By studying the genetic and biochemical characteristics of fungal pathogens, researchers can developdiagnostic tools to quickly and accurately identify the causal agents of plant diseases. Once the fungal pathogens have been identified, the next step is to develop strategies to control and manage these diseases. Integrated disease management approaches that combine cultural practices, biological control agents, and chemical treatments are often utilized to minimize the impact of fungal diseases on plant health. For example, rotating crops, planting disease-resistant varieties, and managing irrigation practices can help reduce the spread of fungal pathogens in agricultural fields. Biological control agents, such as beneficial fungi and bacteria, can also be used to suppress the growth of pathogenic fungi in plants. These biocontrol agents compete with fungal pathogens for resources, produce antifungal compounds, and stimulate the plant's immune response to enhance its resistance to disease. Additionally, the use of fungicides and other chemical treatments can help prevent the spread of fungal diseases in crops, althoughcareful monitoring is required to prevent the development of resistance in fungal populations. Despite the advances in fungal plant pathology and disease management, challenges remain in effectively controlling fungal diseases in plants. Climate change, globalization, and the emergence of new fungal strains withincreased virulence pose ongoing threats to plant health. Furthermore, the use of chemical treatments can have negative impacts on the environment and human health, underscoring the need for sustainable and environmentally-friendly disease management practices. In conclusion, fungal plant pathology plays a crucial role in protecting plant health and ensuring food security around the world. By identifying fungal pathogens, developing integrated disease management strategies, and exploring sustainable solutions for disease control, researchers and farmers can work together to mitigate the impact of fungal diseases on crops. Continued research and collaboration in the field of fungal plant pathology are essential to safeguarding global food production and promoting sustainable agriculture for future generations.。
Fungal enzymes for environmentalmanagementUrsula Ku¨es Fungal ligninolytic enzymes have broad biotechnologicalapplications.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 specialreactors 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.AddressDepartment of Molecular Wood Biotechnology and Technical Mycology,Bu¨sgen-Institute,University of Go ¨ttingen,Bu ¨sgenweg 2,Go ¨tttingen D-37077,GermanyCorresponding author:Ku¨es,Ursula (ukuees@gwdg.de )Current Opinion in Biotechnology 2015,33:268–278This review comes from a themed issue on EnvironmentalbiotechnologyEdited by Spiros N Agathos and Nico BoonFor a complete overview see the Issue and the EditorialAvailable online 9th April 2015/10.1016/j.copbio.2015.03.0060958-1669/#2015Elsevier Ltd.All rights reserved.IntroductionSaprotrophic fungi have crucial roles in ecosystem func-tioning.Primarily,they facilitate organic matter decom-position 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 microbialdegradation [3–5].Specific basidiomycete fungi can enzymatically attack all the polymers in the complex-structured lignocellulose.The appearance of such white-rot 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 nd plants were not anymore simply buried and chemically trans-formed 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 simulta-neously attack the persistent lignin to free the ferment-able 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 poly-saccharides 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 evalua-tion of the decay modes together with the genomes of the basidiomycetes Botryobasidium botryosum ,Jaapia argilla-ceae ,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 decompositionAvailable online at ScienceDirectof organic matter[16].Mycorrhizal and typical sapro-trophic species tend to be found distinctly in separate soil areas,along with specific functions in the rhizosphere and in the soil,respectively.Endophyte implies localiza-tion 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 patho-gens[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 appar-ently 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 subfami-lies,the constantly expanding information on which is compiled in the knowledge-based CAZy database together with information on enzymes with auxiliary activities [27,28 ].Enzymes in lignocellulose degradation are com-monly extracellular,which is compulsory by the large molecule sizes of the envisaged rger poly-mers are broken down into smaller fragments andfinally 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 machin-eries for detection,transport and metabolism of xenobiotics [29 ].Detoxification pathways of the xenome are constituted among others of multigenic families of intra-cellular cytochrome P450monooxygenases and glutathione transferases,respectively(Box1).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 Fungal enzymes for environmental management Ku¨es269Box1Laccases(EC1.10.3.2;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 ccases 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(Figure2).Peroxidases(EC1.11.x;donor:hydrogen-peroxide oxidoreductases)comprise different superfamilies of phenoloxidases that use H2O2or 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;EC1.11.1.14), manganese peroxidases(MnPs;EC1.11.1.13),and versatile peroxidases(VPs;EC1.11.1.16).Another family of largely unclarified biological functions but of high biotechnological interest for degradation of recalcitrant compounds presents dye-decolorizing peroxidases(DyPs;EC1.11.1.19).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;EC1.11.1.10)and unspecific or aromatic peroxygenases(UPOs/APOs;EC1.11.2.1)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(EC1.14.18.1;monophenol monooxygenases;phenolases;monophenol,o-diphenol:oxygen oxidoreductases;L-tyrosine,L-dopa:oxygen oxidoreductases)are type III copper proteins.Upon binding of molecular oxygen,tyrosinases catalyze o-hydroxylation of monophenols(monophenolase reaction cycle,reaction1)to generate as intermediates o-diphenols that are subsequently oxidized into reactive o-quinones(diphenolase reaction cycle,reaction2).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.P450cytochrome monooxidases(EC1.14.14.1;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 O2molecule. Members of the highly diverged and functionally very diverse P450superfamily have essential roles in biosynthetic pathways of specific primary and secondary metabolites,others act in metabolization of xenobiotics.Glutathione transferases(EC2.5.1.18;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).270Environmental biotechnologyFigure 1Extracellular enzymesOrganic compoundsFixing to soil particles or organic matterEnzymatic activationOrganic radicalsOxygen radicals,photons,Fenton reactions Non-enzymatic degradation Fixing to soil particles or organic matterExcretionMineralizationDegradation (direct, indirect enzymatic;non-enzymatic)Enzymatic degradation (extracellular)Enzymatic degradation (intracellular)Indirect enzymatic degradationRecycling of mediatorsPersistent organic pollutantsIntermediatesDegradation productsMetabolites (intracellular)Metabolites (extracellular)CO 2PolymersCurrent Opinion in BiotechnologySimplified scheme of the very complex reactions possibly occurring with extracellular phenol oxidases and persistent organic pollutants (POPs)in anic 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 extracellularenzymes 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 non-enzymatic or direct or indirect enzymatic degradation (indicated in the scheme by double-sided arrows).One possible route can lead topolymerization 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 P450monooxygenases 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 CO 2[29 ].Colors from red to yellow indicate arbitrary toxicity levels of compounds,green colors indicate less or non-toxic compounds.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 biotech-nology 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 environ-ment might be manipulated in favor of organisms produc-ing 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 watersComprehensibly,any contaminated liquid or solid mate-rial 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 inorganiccontaminants,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 pounds of biological origin might accumulate by natural processes or by anthropogenic activities.Particu-larly distressing groups of POPs comprise natural and synthetic phenolic compounds and polycyclic aromatic hydrocarbons (PAHs)[37 ,38–40].Natural or inten-tionally enforced chemical oxidation processes and photocatalysis can help in degradation of such com-pounds (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 gener-ate organic radicals that can undergo subsequent radical reactions [41–43].Having themselves already pro-nounced substrate ranges,enzymatic activation of med-iators,that is suitable enzyme substrates that can serve as electron shuttles between enzyme and other compounds,enhances kinetics of reactions and potentiates transfor-mation activities exponentially also onto target mole-cules that are not direct substrates to an enzyme ([37 ];Figures 1and 2).Most often but not exclusively,sourcesFungal enzymes for environmental management Ku¨es 271Figure 2Phenolic or non-phenolic compound (red)Phenolic or non-phenolic compound (ox)Mediator (ox)Phenolic product (ox)Phenolic substrate (red)Mediator (red)Laccase (red)Laccase (ox)H 2OO 2Current Opinion in BiotechnologyLaccase 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.of these oxidoreductases are white-rotting basidiomy-cetes[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 oxida-tive 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(Figure1). Application of enzymes free in solution is therefore little practicable,in particular not in large scale purification processes and under continuous conditions[51,52].Vari-ous techniques of enzyme immobilization(carrier-sur-face-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 molecu-lar oxygen as co-substrate unlike PODs that depend on hazardous hydrogen peroxide([41,42];Box1).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 nanopar-ticles,TiO2nonoparticles,silane sol–gel matrix,chitosan, cellulose nanofibers,polyethersulfone and polyvinyli-dene 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 immobi-lization 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,med-iators might also be immobilized[62 ].Immobilization offers technically the possibility to combine enzymes for simultaneous or successive conversions in one-pot ccases of different origin differ in characters (substrate ranges,pH optima).Combinations thereof can therefore be more versatile for converting mixtures of hazardous compounds under changing and poorly defined conditions[57 ],such as in municipal and indus-trial waste waters that inherently vary in compositions and amounts of their chemical burdens and can so also in pHs. Laccase and tyrosinase both need O2for enzymatic action ([42,43];Box1)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 combi-CLEAs 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 proper-ties of waste waters[52].For enzyme separation, magnetic particles have been given attention to for itera-tive reuse since they can be easily recovered by electrical fields in magnetic bio-separation technology[55,63]. Multiple specific reactor designs preferentially for con-tinuous 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 bio-reactor of hollowfiber 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 mem-brane[54,58,68,75,77 ].Afirst trial on pilot scale in long-termfield tests was reported that used actual wastewater treatment plant effluents in tertiary treatment to elimi-nate any persistent xenobiotics(here bisphenol A)and a fixed bed reactor for settling any solids combined to a 460L membrane reactor containing with on silica nanoparticles immobilized Thielavia laccase,with costs estimated to0.130s mÀ3purified water.Costs of the process as tested are comparable to chemical removal of xenobiotics through binding to powdered activated car-bon 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 ].272Environmental biotechnologyEnzymes in bioremediation of solid wastes and soilsSolid organic wastes and contaminated soils present an-other challenge in POPs purification.Free enzymes mixed into the materials will only potentiate the pro-blems 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 ];Figure1).A fungal laccase for example has been reported in experiments with individual materials to adsorb to soil iron and aluminum minerals with conse-quences of general reduction of enzyme activities through reduced enzyme-substrate affinities but at acidic pH the catalytic activities increased.Thermostability and tem-perature sensitivity were lower upon adsorption but re-sistance 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 im-mobilization 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 main-ly 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(Box1) appear to have roles in this[84–86].Treated detoxified organic waste material might subsequently be used in fertilizing soil.Application of fungal fermented olive-mill 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 varia-tion in diversity but only after longer run[89,90 ].Enzy-matic 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.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 out-side conditions might however be different by many factors,for instance due to soil structures and composi-tions,respective nutrient availabilities,moisture con-tents,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 month96%of4-ring PAHs and39%of5-ring and6-ring PAHs from contami-nated sawmill soil.In largerfield 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 com-pete 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 bio-mass 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 produc-tion[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 environ-ment for enzyme production required for the successful degradation of pesticides[104].Fungal enzymes for environmental management Ku¨es273。
