黄孢原毛平革菌对松木、稻草和芦苇去木质化作用的研究

为确定黄孢原毛平革菌对不同植物材料的去木质化作用,以pH、干物质重、半纤维素、纤维素和木质素为主要技术指标,比较黄孢原毛平革菌对松木、稻草和芦苇降解能力的差异。松木、芦苇在发酵过程中pH呈下降趋势,稻草呈上升趋势。在干物质重、半纤维素、纤维素降解率三个指标上皆为松木〈芦苇〈稻草,在木质素降解率上则为松木〈稻草〈芦苇,且差异显著。表明黄孢原毛平革菌对不同植物材料去木质化能力有较大差异,其中芦苇的木质素降解率为13％,是三种材料中最易于被去木质化的。     

中国食（药）用真菌发酵工程研究进展

通过回顾食(药)用真菌生产工艺的研究历史,阐述了现代食(药)用真菌发酵工程包括液体发酵和固体发酵的研究状况,着重指出新型与多样性固体发酵工程的创建和发展可为人类提供取之不尽、用之不竭的传统生产工艺难以获得的珍贵目的产物。     

药用植物内生真菌及活性物质多样性研究进展

药用植物具有丰富的物种多样性,是人类生存与发展的重要自然资源。内生真菌广泛存在于健康植物组织内部,是植物微生态系统的重要组成部分,各种药用植物中蕴藏着非常丰富的内生真菌。通过与药用植物的“协同进化”,某些内生真菌具有了产生与宿主植物相同或相似的生物活性物质的能力。内生真菌产生的各种活性物质,在生物制药、农业生产、工业发酵等方面都表现出美好的应用前景,受到世界各国专家的广泛关注。利用内生真菌发酵实现生物活性物质的工业化生产,可以提高产量、降低产品成本,满足人们日益增长的需求;同时有利于珍稀、濒危药用植物资源的保护,对减少野生药用植物多样性的破坏具有重要意义。本文从药用植物内生真菌物种多样性与产生生物活性物质多样性等方面总结近年最新的研究进展,提出了内生真菌及活性物质研究的未来发展方向。     

Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius

Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [(sup14)C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn(sup2+) to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.     

Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics

The biological delignification of lignocellulosic feedstocks, Prosopis juliflora and Lantana camara was carried out with Pycnoporus cinnabarinus, a white rot fungus, at different scales under solid-state fermentation (SSF) and the fungal treated substrates were evaluated for their acid and enzymatic saccharification. The fungal fermentation at 10.0 g substrate level optimally delignified the P. juliflora by 11.89% and L. camara by 8.36%, and enriched their holocellulose content by 3.32 and 4.87%, respectively, after 15 days. The fungal delignification when scaled up from 10.0 g to 75.0, 200.0 and 500.0 g substrate level, the fungus degraded about 7.69–10.08% lignin in P. juliflora and 6.89–7.31% in L. camara, and eventually enhanced the holocellulose content by 2.90–3.97 and 4.25–4.61%, respectively. Furthermore, when the fungal fermented L. camara and P. juliflora was hydrolysed with dilute sulphuric acid, the sugar release was increased by 21.4-42.4% and the phenolics content in hydrolysate was decreased by 18.46 and 19.88%, as compared to the unfermented substrate acid hydrolysis, respectively. The reduction of phenolics in acid hydrolysates of fungal treated substrates decreased the amount of detoxifying material (activated charcoal) by 25.0–33.0% as compared to the amount required to reduce almost the same level of phenolics from unfermented substrate hydrolysates. Moreover, an increment of 21.1–25.1% sugar release was obtained when fungal treated substrates were enzymatically hydrolysed as compared to the hydrolysis of unfermented substrates. This study clearly shows that fungal delignification holds potential in utilizing plant residues for the production of sugars and biofuels.     

荧光定量PCR方法检测白酒发酵过程中Aspergillus tubingensis生物量

【目的】为了更好地分析霉菌在白酒发酵过程中的作用,需要快速准确地测定发酵过程中霉菌生物量的变化,本实验以白酒酿造中常用的塔宾曲霉(Aspergillus tubingensis)为例,建立一套快速准确定量塔宾曲霉生物量的方法。【方法】优化从酒醅中提取基因组的方法,设计和验证专一性引物,建立实时荧光定量PCR(Real-time quantitative PCR)方法,验证方法的有效性并应用于白酒发酵过程中塔宾曲霉生物量的检测。【结果】用原位机械破碎法提取酒醅中总基因组,其DNA的浓度能够达到1.060×105 ng/g酒醅;同时建立了一套快速准确测定固态基质中霉菌生物量的方法,并应用于白酒生产(制曲、堆积发酵和窖池发酵过程)中塔宾曲霉生物量的定量。【结论】实时荧光定量PCR方法能够快速准确地测定固态基质中霉菌的生物量,且检测限较低,对今后的相关研究具有借鉴意义。     

Production of D-xylanases by thermophilic fungi using different methods of culture

Summary Seven thermophilic strains of fungi were examined for their ability to produce D-xylanase in Liquid and solid-state fermentations. It was confirmed that the best producers of xylanase, among microorganisms used, wereH. Lanuginosa andS. thermophile in liquid fermentation, andT. aurantiacus andH. lanuginosa in solid-state fermentations. The higher productivity of xylanase, namely 18,72 IU/ml, was obtained in liquid culture ofH. lanuginosa. The pH and temperature optima of enzymes from liquid and solid-state cultures of fungi used were also presented.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

Optimisation of the biological pretreatment of wheat straw with white-rot fungi for ethanol production

The biological pretreatment of lignocellulosic biomass for the production of bioethanol is an environmentally friendly alternative to the most frequently used process, steam explosion (SE). However, this pretreatment can still not be industrially implemented due to long incubation times. The main objective of this work was to test the viability of and optimise the biological pretreatment of lignocellulosic biomass, which uses ligninolytic fungi (Pleurotus eryngii and Irpex lacteus) in a solid-state fermentation of sterilised wheat straw complemented with a mild alkali treatment. In this study, the most important parameters of the mechanical and thermal substrate conditioning processes and the most important parameters of the fungal fermentation process were optimised to improve sugar recovery. The largest digestibilities were achieved with fermentation with I. lacteus under optimised conditions, under which cellulose and hemicellulose digestibility increased after 21 days of pretreatment from 16 to 100 % and 12 to 87 %, respectively. The maximum glucose yield (84 %) of cellulose available in raw material was obtained after only 14 days of pretreatment with an overall ethanol yield of 74 % of the theoretical value, which is similar to that reached with SE.     

Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues

A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.     

Stoned olive pomace fermentation with Pleurotus species and its evaluation as a possible animal feed

The use of stoned olive pomace (SOP) as an unconventional feedstuff for livestock is limited by its inherently low crude protein (CP) content and by the presence of anti-nutritional compounds such as phenols. Aim of this study was to assess whether solid-state fermentation of SOP with selective lignin-degrading fungi might ameliorate nutritional properties of the waste. Incubation of SOP, mixed (25%, w/w) with various conventional feedstuffs (i.e., wheat bran, wheat middlings, barley grains, crimson clover, wheat flour shorts and field beans), with Pleurotus ostreatus and Pleurotus pulmonarius led to significant CP increases, ranging from 7 to 29%, and marked removal (from ca. 50–90%) of phenols after 6 weeks. Both species, however, led to moderate delignification associated with significant consumption of hemicelluloses. Consequently, no improvements of both organic matter digestibility (OMD) and net energy of SOP–feedstuff mixtures occurred after the fungal colonization.     

Kinetics of wheat straw solid-state fermentation with Trametes versicolor and Pleurotus ostreatus — lignin and polysaccharide alteration and production of related enzymatic activities

Summary The kinetics of straw solid-state fermentation (SSF) with Trametes versicolor and Pleurotus ostreatus was investigated to characterize the delignification processes by these white-rot fungi. Two successive phases could be defined during straw transformation, characterized by changes in respiratory activity, changes in lignin and polysaccharide content and composition, increase in in-vitro digestibility, and enzymatic activities produced by the fungi. Lignin composition was analysed after CuO alkaline degradation, and decreases in syringyl/guaiacyl and syringyl/p-hydroxyphenyl ratios and cinnamic acid content were observed during the fungal treatment. An increase in the phenolic acid yield, revealing fungal degradation of side-chains in lignin, was produced by P. ostreatus. The highest xylanase level was produced by P. ostreatus, and exocellulase activity was nearly absent from straw treated with this fungus. Lactase activity was found in straw treated with both fungi, but lignin peroxidase was only detected during the initial phase of straw transformation with T. versicolor. High levels of H₂O₂-producing aryl-alcohol oxidase occurred throughout the straw SSF with P. ostreatus. Offprint requests to: A. T. Martínez     

Production of polyhydroxyalkanoates (PHAs) from waste materials and by-products by submerged and solid-state fermentation

Polyhydroxyalkanoates are biodegradable polymers produced by prokaryotic organisms from renewable resources. The production of PHAs by submerged fermentation processes has been intensively studied over the last 30 years. In recent years, alternative strategies have been proposed, such as the use of solid-state fermentation or the production of PHAs in transgenic plants. This paper gives an overview of submerged and solid-state fermentation processes used to produce PHAs from waste materials and by-products. The use of these low-cost raw materials has the potential to reduce PHA production costs, because the raw material costs contribute a significant part of production costs in traditional PHA production processes.     

Development of a low-cost sterilization biological indicator using <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis> by solid-state fermentation

The production of biological indicators involving bacterial sporulation and multi-step downstream processes has been described. The goal of the present work was to use fermented material as the final product in a biological indicator, thereby reducing processing steps and costs. The performance of three different inexpensive supports (vermiculite, sand, and sugarcane bagasse) was assessed by determining Bacillus atrophaeus sporulation during solid-state fermentation and by assessing the direct use of the fermentation products in the subsequent steps of the process. All three supports allowed spore production of between 10⁷ and 10⁹ CFU g⁻¹. Sand proved to be the best inert support enabling the direct use of the fermented product due to its easy homogenization, filling properties, and compatibility with recovery medium. Bacterial adhesion to the sand surface was supported by biofilm formation. The resistance to sterilization of the dried fermentation product was evaluated. For dry-heat resistance (160°C), the D value was 6.6 min, and for ethylene oxide resistance (650 mg/L), the D value was 6.5 min. The cost reduction of this process was at least 48%. No previous studies have been published on the application of sand as a support in solid-state fermentation for the production of biological indicators.     

Production of Xylanase by Thermophilic Melanocarpus albomyces IIS-68

Melanocarpus albomyces IIS-68, a thermophilic fungus was used for the production of extracellular xylanase on various agroresidues in solid-state fermentation (SSF). Growth on untreated wheat straw and sugar cane bagasse supported xylanase production, while rice straw and rice husk did not. Alkali treatment and acid chlorite treatment of these latter substrates, which lead to extensive delignification, enhanced xylanase production. In contrast, these treatments caused a decline in xylanase activity on wheat straw and bagasse. Acetyl esterase was produced concurrently with xylanase, maximal activity being produced on bagasse. Enzyme production was higher in SSF than in submerged fermentation (SmF). Studies with electron micrographs indicated that culture filtrate proteins were able to degrade wall polymers.     

Rapid estimation of single cell oil content of solid-state fermented mass using near-infrared spectroscopy

Calibration model using near-infrared reflectance spectroscopy (NIRS) for estimation of SCO content in solid-state fermented mass was established. The NIRS calibration model was derived by partial least-squares (PLS) regression and prediction of SCO contents of independent solid-state fermented mass samples fermented by different oleaginous fungi showed the model to be rapid and accurate, giving R(2)-value higher than 0.9552 and root mean standard error of prediction (RMSEP) value lower than 0.5772%. The established NIRS calibration model could be used to estimate the SCO contents of the solid-state fermented masses and will provide much convenience to the research of SCO production in solid-state fermentation.     

Solid-state and membrane-surface liquid cultures of micromycetes: Specific features of their development and enzyme production (a Review)

Specific features in the development of micromycetes, typical mechanisms of their enzyme production, and conditions providing for an increase in enzyme secretion by the microscopic fungi in solid-state (on natural substrates and inert carriers) and membrane-surface liquid cultures are considered. The prospects and advantages of these fermentation methods for the production of extracellular enzymes are discussed and compared with submerged cultures.     

Improvement of dry matter digestibility of water hyacinth by solid state fermentation using white rot fungi

Feeding value of water hyacinth biomass colonized by three species of white rot fungi during solid-state fermentation was investigated. All three organisms proved to be efficient degraders and enhanced dry matter digestibility. Loss of organic matter was maximum (23.6+/-0.1% dry wt) after 48 days by P. ostreatus. C. indica showed maximum cellulose degradation (18.5+/-0.1% dry wt) than other two fungi after 48 days of incubation. In all cases, an extensive removal of hemicellulose at the initial growth period and a delayed degradation of lignin were observed. Hemicellulolysis was maximum (46.3+/-0.1% dry wt) by C. indica, but delignification (14.2+/-0.2% dry wt) by P. sajor-caju after 48 days. The amount of reducing sugar in the degraded biomass decreased at early stages, but increased as degradation progressed in all three cases (maximum 1.1+/-0.05% dry wt after 48 days by C. indica). Soluble nitrogen content increased only during 16-32 days of incubation (highest 1.1+/-0.1% dry wt after 32 days by P. sajor-caju). Crude protein of the bioconverted biomass increased gradually up to 32 days but decreased thereafter (maximum 10.3+/-0.1% dry wt after 32 days by P. sajor - caju). Per cent change in in vitro dry matter digestibility of degraded substrates enhanced gradually after 8 days and reached maximum after 32 days but thereafter decreased (highest + 20.4+/-0.3% dry wt by P. sajor-caju). The results demonstrated the efficient degrading capacity of the test fungi and their potential use in conversion of water hyacinth biomass into mycoprotein-rich ruminant feed, more so by P. sajor-caju.     

Water and glucose gradients in the substrate measured with NMR imaging during solid-state fermentation with Aspergillus oryzae

Gradients inside substrate particles cannot be prevented in solid-state fermentation. These gradients can have a strong effect on the physiology of the microorganisms but have hitherto received little attention in experimental studies. We report gradients in moisture and glucose content during cultivation of Aspergillus oryzae on membrane-covered wheat-dough slices that were calculated from (1)H-NMR images. We found that moisture gradients in the solid substrate remain small when evaporation is minimized. This is corroborated by predictions of a diffusion model. In contrast, strong glucose gradients developed. Glucose concentrations just below the fungal mat remained low due to high glucose uptake rates, but deeper in the matrix glucose accumulated to very high levels. Integration of the glucose profile gave an average concentration close to the measured average content. On the basis of published data, we expect that the glucose levels in the matrix cause a strong decrease in water activity. The results demonstrate that NMR can play an important role in quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation, which is needed to improve our understanding of the response of fungi to this nonconventional fermentation environment.