Re-supplementing and re-casing mushroom (<Emphasis Type="Italic">Agaricus bisporus</Emphasis>) compost for a second crop

Experiments were performed to determine the effect of adding nutrient supplements to colonized mushroom compost (MC) for the production of a second crop of mushrooms. Mushrooms were harvested for 1, 2 or 3 flushes, the casing removed and the MC then was fragmented and re-supplemented with delayed release supplements treated or non-treated with fungicide (thiophanate-methyl; Topsin M 70WP) and re-cased. Overall double-crop yields were higher when MC was re-supplemented after 1st flush (1st flush MC) as compared to re-supplementation after the 2nd or 3rd flushes. Mean double-crop BEs were 128, 119 and 109% when 1st-, 2nd- and 3rd-flush MCs were used, respectively. Treatment of delayed release supplement with thiophanate-methyl fungicide did not affect mushroom yields. Soluble salts and potassium concentrations increased 350 and 900%, respectively, in the casing overlay through three flushes suggesting that removal of the casing would help to alleviate the build up of these potential growth-limiting materials. Re-supplementing and re-casing of MC represents a potential opportunity for growers to increase revenues and reduce costs associated with preparation and disposal of compost. The ability to double-crop mushroom compost would provide growers a chance to increase yields by 40% or more, depending on whether they re-supplement and re-case after 1st, 2nd or 3rd flush.     

Diversity and dynamics of the DNA and cDNA-derived bacterial compost communities throughout the <Emphasis Type="Italic">Agaricus bisporus</Emphasis> mushroom cropping process

The cultivation of Agaricus bisporus involves the conversion of agricultural materials via fermentation into utilisable simple sugars as a nutrient source for the fungal crop during mushroom cropping. However, little is currently known about the role of the bacterial community contributing to the fermentation process. In this investigation we characterised the composition and dynamics of the DNA and cDNA-derived bacterial populations throughout a commercial mushroom cropping process using MiSeq sequencing. Both methods indicated substantial changes in the bacterial community structure after the first flush of the mushroom crop. However, differences were observed between the composition of the bacterial community determined by each of the two methods. The cDNA-derived community indicated that thermotolerant genera with known sulphur-reducing characteristics were highly active up to the first flush. Activity of the phyla Actinobacteria and Firmicutes was observed to increase as fermentation progressed, indicating that the members of these phyla played prominent roles in the conversion of wheat straw into utilisable sugars. The cDNA-derived community comprised genera with roles in the nitrification process that became highly active at post flush 1. Subsequent chemical analysis of extractable nitrate indicated that substantial nitrification occurred up until the termination of the cropping process. This study has demonstrated that a highly dynamic bacterial community is present throughout the mushroom cropping process.     

Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization.

Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).     

Changes in Bacterial and Fungal Communities across Compost Recipes,Preparation Methods,and Composting Times

Compost production is a critical component of organic waste handling, and compost applications to soil are increasingly important to crop production. However, we know surprisingly little about the microbial communities involved in the composting process and the factors shaping compost microbial dynamics. Here, we used high-throughput sequencing approaches to assess the diversity and composition of both bacterial and fungal communities in compost produced at a commercial-scale. Bacterial and fungal communities responded to both compost recipe and composting method. Specifically, bacterial communities in manure and hay recipes contained greater relative abundances of Firmicutes than hardwood recipes with hay recipes containing relatively more Actinobacteria and Gemmatimonadetes. In contrast, hardwood recipes contained a large relative abundance of Acidobacteria and Chloroflexi. Fungal communities of compost from a mixture of dairy manure and silage-based bedding were distinguished by a greater relative abundance of Pezizomycetes and Microascales. Hay recipes uniquely contained abundant Epicoccum, Thermomyces, Eurotium, Arthrobotrys, and Myriococcum. Hardwood recipes contained relatively abundant Sordariomycetes. Holding recipe constant, there were significantly different bacterial and fungal communities when the composting process was managed by windrow, aerated static pile, or vermicompost. Temporal dynamics of the composting process followed known patterns of degradative succession in herbivore manure. The initial community was dominated by Phycomycetes, followed by Ascomycota and finally Basidiomycota. Zygomycota were associated more with manure-silage and hay than hardwood composts. Most commercial composters focus on the thermophilic phase as an economic means to insure sanitation of compost from pathogens. However, the community succeeding the thermophilic phase begs further investigation to determine how the microbial dynamics observed here can be best managed to generate compost with the desired properties.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

Comparison of the efficiency of bacterial and fungal laccases in delignification and detoxification of steam-pretreated lignocellulosic biomass for bioethanol production

This study evaluates the potential of a bacterial laccase from Streptomyces ipomoeae (SilA) for delignification and detoxification of steam-exploded wheat straw, in comparison with a commercial fungal laccase from Trametes villosa. When alkali extraction followed by SilA laccase treatment was applied to the water insoluble solids fraction, a slight reduction in lignin content was detected, and after a saccharification step, an increase in both glucose and xylose production (16 and 6%, respectively) was observed. These effects were not produced with T. villosa laccase. Concerning to the fermentation process, the treatment of the steam-exploded whole slurry with both laccases produced a decrease in the phenol content by up to 35 and 71% with bacterial and fungal laccases, respectively. The phenols reduction resulted in an improved performance of Saccharomyces cerevisiae during a simultaneous saccharification and fermentation (SSF) process, improving ethanol production rate. This enhancement was more marked with a presaccharification step prior to the SSF process.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.     

中亚热带地区两种森林植被类型土壤微生物群落结构

利用磷脂脂肪酸(PLFA)生物标记法分析了中亚热带地区罗浮栲天然林和相邻的杉木人工林土壤微生物群落结构特点.结果表明: 两种植被类型的磷脂脂肪酸总量、细菌特征脂肪酸、真菌特征脂肪酸、放线菌特征脂肪酸、革兰氏阳性菌和革兰氏阴性菌特征脂肪酸含量均为0～10 cm高于10～20 cm土层,罗浮栲天然林高于杉木人工林.在两种植被类型的两个土层中,细菌PLFAs含量均显著高于真菌PLFAs含量.两种植被类型中,细菌PLFAs含量约占PLFAs总量的44%～52%,而真菌仅占6%～8%,表明细菌在该地区两种植被类型土壤中处于优势地位.主成分分析表明,土壤微生物群落结构差异主要由植被类型差异引起,土层深度的影响相对较小.相关分析显示,革兰氏阴性菌、革兰氏阳性菌以及细菌的PLFAs含量与pH呈显著负相关,与含水量呈显著正相关;土壤微生物主要类群PLFAs含量与总氮、有机碳、C/N和铵态氮均呈显著正相关.     

刺芹侧耳栽培料及菌体中漆酶介体分子的挖掘

虽然真菌能够分泌漆酶从木质素中获取碳源,然而纯化后真菌漆酶降解木质素的能力却极低。本文利用萃取方法提取刺芹侧耳栽培料及菌体中可溶性化合物,通过快速漆酶介体体系（LMS）动力学实验确定其对漆酶催化氧化反应的促进或抑制能力,进一步利用HPLC-MS和代谢组学差异分析的方法从最高活性萃取液的柱层析组分中挖掘潜在的活性成分。研究结果表明栽培料中可能含有活性介体成分,生物信息学XCMS代谢产物组分析揭示了CTE05分离组分中富含的木质素代谢产物,涉及到软木脂单体合成、香豆素合成、丁香油酚或异丁香油酚合成、质体醌醇合成、由苯丙氨酸合成硫代葡萄糖苷和安息香酸合成等代谢途径。最后讨论了其中11种潜在的天然介体分子物理化学性质,其中4种为已有文献报道的天然介体小分子。本研究暗示木质素降解与合成代谢在分子机制上存在有某种联系,相关的代谢产物可能参与了真菌降解木质素的代谢过程。     

Increasing <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> laccase production by culture medium optimization and copper/lignin synergistic induction

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2^6–2 where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.     

Lignin-Degrading Enzymes of the Commercial Button Mushroom, Agaricus bisporus

Agaricus bisporus, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by A. bisporus. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in A. bisporus suggests significant roles for these two enzymes in lignin degradation by this fungus.     

中亚热带地区米槠天然林土壤微生物群落结构的多样性

为了解土壤微生物群落的结构,采用磷脂脂肪酸方法对武夷山和建瓯的米槠(Castanopsis carlesii)天然林土壤微生物群落的结构多样性进行了研究。结果表明,两地米槠天然林的土壤微生物群落组成十分丰富,多样性指数、丰富度指数和均匀度指数分别为2.92~3.01、25.84~28.23 和0.88~0.90。0~10 cm土层的磷脂脂肪酸总量、细菌特征脂肪酸、真菌特征脂肪酸、放线菌特征脂肪酸、革兰氏阳性菌和阴性菌特征脂肪酸含量均高于10~20 cm土层的,且建瓯万木林自然保护区的高于武夷山国家级自然保护区。10~20 cm土层的革兰氏阳性菌/革兰氏阴性菌高于0~10 cm土层的;细菌特征脂肪酸含量显著高于真菌,表明细菌在土壤微生物群落结构中处于优势地位。主成分分析表明,土壤微生物群落结构的差异主要是由采样地点的不同引起。     

Physiologic response of Agaricus subrufescens using different casing materials and practices applied in the cultivation of Agaricus bisporus

Casing materials and practices used in the cultivation of Agaricus bisporus were evaluated in the cultivation of Agaricus subrufescens, using the best techniques for optimization of production, including the possibility of re-casing of the compost for the production of a second crop of mushroom. Casing based on peat moss, loam soil or coir was compared to casing material mixed with or without spawn-run compost. Based on the results, we conclude that the casing layer used in the cultivation of A. subrufescens should not necessarily be the same as that used in the cultivation of A. bisporus. For the tested strain cultivated with loam soil as casing layer, the ruffling technique is highly superior to CACing and should be pursued in further research. The re-casing of compost in new cycles showed good results suggesting that the currently used compost could be improved.     

Recombinant Cellulase Accumulation in the Leaves of Mature,Vegetatively Propagated Transgenic Sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.     

Screening of a fungus capable of powerful and selective delignification on wheat straw

Aims: To screen and characterize a novel fungus with powerful and selective delignification capability on wheat straw. Methods and Results: A fungus capable of efficient delignification under solid‐state fermentation (SSF) conditions on wheat straw was screened. After 5 days of incubation, 13·07% of the lignin was removed by fungal degradation, and 7·62% of the holocellulose was lost. Furthermore, 46·53% of the alkali lignin was removed after 2 days of liquid fermentation. The fungus was identified as Fusarium concolor based on its morphology and an analysis of its 18S rDNA gene sequence. The molecular weight distribution of lignin was evaluated by gel permeation chromatography. Enzyme assay indicated that the fungus produced laccase, cellobiose dehydrogenase, xylanase and cellulase during the incubation period. Intracellular lignin peroxidase, manganese peroxidase and laccase were produced during liquid fermentation. Conclusions: We have successfully screened a fungus, F. concolor, which can efficiently degrade the lignin of wheat straw, with slight damage to the cellulose, after 5 days of SSF. Significance and Impact of the Study: The newly isolated strain could be used in pretreatment of lignocellulose materials prior to biopulping, bioconversion into fuel and substrates for the chemical industry.     

Laccase-Catalyzed Surface Modification of Thermo-Mechanical Pulp (TMP) for the Production of Wood Fiber Insulation Boards Using Industrial Process Water

Low-density wood fiber insulation boards are traditionally manufactured in a wet process using a closed water circuit (process water). The water of these industrial processes contains natural phenolic extractives, aside from small amounts of admixtures (e.g., binders and paraffin). The suitability of two fungal laccases and one bacterial laccase was determined by biochemical characterization considering stability and substrate spectra. In a series of laboratory scale experiments, the selected commercial laccase from Myceliophtora thermophila was used to catalyze the surface modification of thermo-mechanical pulp (TMP) using process water. The laccase catalyzed the covalent binding of the phenolic compounds of the process water onto the wood fiber surface and led to change of the surface chemistry directly via crosslinking of lignin moieties. Although a complete substitution of the binder was not accomplished by laccase, the combined use of laccase and latex significantly improved the mechanical strength properties of wood fiber boards. The enzymatically-treated TMP showed better interactions with the synthetic binder, as shown by FTIR-analysis. Moreover, the enzyme is extensively stable in the process water and the approach requires no fresh water as well as no cost-intensive mediator. By applying a second-order polynomial model in combination with the genetic algorithm (GA), the required amount of laccase and synthetic latex could be optimized enabling the reduction of the binder by 40%.     

Effects of bendiocarb and diflubenzuron on mushroom cropping

When mixed into the casing or compost layers of a mushroom bed in the absence of pests, bendiocarb decreased yield and number of mushrooms according to concentration. The most severe effects were on mushroom number at the two highest rates used (100 and 1000 μg/g), and there were large increases in mushroom size. Effects of bendiocarb incorporation in the compost diminished with time, and there was partial compensation in yield and numbers at the fourth flush. The action of bendiocarb persisted when it was mixed into the casing. Diflubenzuron showed some opposite effects at lower concentrations. When either mixed into, or drenched onto the casing at the commercial rate (30 μg/g), yield and size were both increased and the timing of the flushes was unaffected. At the two higher concentrations (180 and 1080 μg/g), reductions in yield and number and an increase in mushroom size were shown. However, these effects became more severe with time, especially those on mushroom number, possibly due to the accumulation of a toxic breakdown product.     

Effect of substrate on the production of antifungal volatiles from Bacillus subtilis

An antibiotic-producing strain of Bacillus subtilis has been shown to produce potent antifungal volatiles (AFV). These volatiles are active against a range of fungal species and are produced on a range of growth media and in loam-based compost. In vitro antifungal volatile activity on nutrient agar is enhanced with the addition of D-glucose, complex carbohydrates and peptones. The addition of L-glucose led to significantly less AFV activity than comparable levels of D-glucose. Growth studies in liquid culture revealed that B. subtilis failed to grow in response to L-glucose. Further growth studies on solid media showed no clear correlation between enhanced bacterial growth and increases in in vitro AFV activity in response to supply of substrates. Low level AFV activity was also detected from oilseed rape roots inoculated with B. subtilis . Gas chromatography mass spectrometry headspace analysis of B. subtilis cultures grown on various substrates revealed common similarities between substrates promoting AFV activity, although it was not possible to isolate individual antifungal compounds.     

Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose

The presence of lignin is known to reduce the efficiency of the enzymatic hydrolysis of lignocellulosic raw materials. On the other hand, solubilization of hemicellulose, especially of xylan, is known to enhance the hydrolysis of cellulose. The enzymatic hydrolysis of spruce, recognized among the most challenging lignocellulosic substrates, was studied by commercial and purified enzymes from Trichoderma reesei. Previously, the enzymatic hydrolysis of steam pretreated spruce has been studied mainly by using commercial enzymes and no efforts have been taken to clarify the bottlenecks by using purified enzyme components.Steam-pretreated spruce was hydrolyzed with a mixture of Celluclast and Novozym 188 to obtain a hydrolysis residue, expectedly containing the most resistant components. The pretreated raw material and the hydrolysis residue were analyzed for the enrichment of structural bottlenecks during the hydrolysis. Lignin was removed from these two materials with chlorite delignification method in order to eliminate the limitations caused by lignin. Avicel was used for comparison as a known model substrate. Mixtures of purified enzymes were used to investigate the hydrolysis of the individual carbohydrates: cellulose, glucomannan and xylan in the substrates. The results reveal that factors limiting the hydrolysis are mainly due to the lignin, and to a minor extent by the lack of accessory enzymes. Removal of lignin doubled the hydrolysis degree of the raw material and the residue, and reached close to 100% of the theoretical within 2 days. The presence of xylan seems to limit the hydrolysability, especially of the delignified substrates. The hydrolysis results also revealed significant hemicellulose impurities in the commonly used cellulose model substrate, making it questionable to use Avicel as a model cellulose substrate for hydrolysis experiments.