兼性厌氧纤维素降解菌的筛选和产酶研究

从我国天津地区堆肥中分离筛选出一株兼性厌氧的纤维素分解细菌D1,初步鉴定为纤维单孢菌(Cellulomonas sp.)。产酶最适碳源为葡萄糖,氮源为复合蛋白胨,酶的最适作用温度和pH值是50℃和6．4,在70℃以下和pH在5．2～8．4里稳定。     

Cultivation of filamentous fungi in the disc fermenter

Summary A method of growing filamentous fungi in a novel disc fermenter is described. Growth occurs as a layer on the discs and the fermenter design permits aseptic sampling of the attached biomass. Observations concerning oxygen and nutrient exchange between medium and biomass are reported.     

Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.     

Novel process for the production of cellulolytic enzymes

A novel process for the production of extracellular carboxymethylcellulase (CMCase) and xylanase by fermentation under nonaseptic or nonsterile conditions is described. The fermentation process is carried out under very acidic conditions of pH 2.0 by using a acidophilic cellulolytic fungus. Microbial contamination is avoided or minimized to an insignificant level under this acid pH condition. The culture medium for this production consists of a carbon source from cellulosics or lignocellulosics, such as Na-CMC, xylan, Avicel cellulose, cellulose powder, alpha-cellulose, sawdust, etc., or a mixture of the forementioned together with simple ingredients such as (NH(4))(2)SO(4), K(2)HPO(4), MgSO(4) and NaNO(3). The fermentation is carried out at room temperature (28-30 degrees C), under aerobic conditions, and without controlling the pH. The CMCase and xylanase produced are stable under very simple storage conditions, such as in the fresh culture medium not containing the substrate for a period of 3 days, at any temperature from 0 to 30 degrees C. These extracellular enzymes have an optimum pH around 3, with the best range of pH from 2.0 to 3.6, for any temperature between 15 and 60 degrees C. The optimum temperatures are 55 degrees C for CMCase activity and 25-50 degrees C for xylanase activity, at any pH between 2.0 and 5.2. The apparent Michaelis constants Km are 2.6 and 1.5 mg/mL for CMCase and xylanase of the culture filtrate, respectively.     

A semi-continuous culture system for production of cellulolytic and xylanolytic enzymes by the anaerobic fungus Piromyces sp. strain E2

Summary A system was developed for the semi-continuous cultivation of an anaerobic fungus, Piromyces sp. strain E2 (isolated from an Indian elephant), on Avicel (microcrystalline cellulose). The fungus was grown in a semi-continuous culture system: solids and fungal biomass was retained by means of a simple filter construction whereas the culture fluid was removed continuously. The production of fermentation products (acetate, ethanol, formate, lactate, hydrogen or methane), cellulolytic and xylanolytic enzymes, and protein by the fungus in monoculture or co-culture with Methanobacterium formicicum during growth on Avicel was monitored up to 45 days. These productions stabilized after an adaptation period of 24 and 30 days in the semi-continuous co-culture and monoculture, respectively. After this period the average (±SD) avicelase, -glucosidase, endoglucanase, and xylanase production in the semi-continuous monoculture were 27±6, 140±16, 1057±120 and 5012±583 IU.l^–1.dya^–1, respectively. Co-culture with the methanogen caused a shift in fermentation products to more acetate, and less ethanol and lactate. Furthermore, the production of all cellulolytic enzymes increased (40%) and xylanolytic enzyme production decreased (35%).Correspondence to: H. J. M. Op den Camp     

Fermentation of cassava peels for the production of cellulolytic enzymes

Cassava peels were used as a substrate for the production of cellulolytic enzymes. Under solid substrate fermentation conditions and a Rhizopus sp., thermostable cellulolytic enzymes were produced. Optimal production temperature and pH were 45°C and 5.6 respectively. Kinetic studies of the enzymes showed that the cellulase C₁ activity was optimal at pH 5.0 and 50°C, whereas that of cellulase C_x was optimal at pH 7.0 and 60°C. The enzymes degraded ca 44% of sorghum grains in 6 h, thus suggesting a possible use in saccharification processes. The results also showed the possibility of re-cycling cassava peels as a cheap substrate for the enzyme industry. and accepted 6 June 1989     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H₂ production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H₂ production rate and yield of 57.7 ml/h/L and 2.03 mol H₂/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H₂ fermentation.     

Cultivation techniques for the production of ectomycorrhizal fungi

Ectomycorrhizal fungi have a valuable role to play in current agricultural practices. In order to produce inocula for use in laboratory, nursery and field trials it is first necessary to isolate the organism, grow it in axenic culture and produce sufficient quantities of the fungus. The methods currently employed to cultivate this group of fungi are considered.     

Effects of glycerol on the growth,adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species,Ruminococcus flavefaciens andFibrobacter succinogenes subsp.succinogenes, and of an anaerobic fungal species,Neocallimastix frontalis, was studied. At low concentrations (0.1–1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity ofN. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.     

Dilution rate increases production of plant cell-wall degrading enzymes by anaerobic fungi in continuous-flow culture

The gut anaerobic fungi,Neocallimastix hurleyensis and aOrpinomyces sp., were grown in 100 mL batch and continuous-flow cultures on wheat straw at a concentration of 80 g dry matter/L of culture liquid. In batch cultures,N. hurleyensis and Orpinomyces sp. degraded only ca. 9% and 5% of the wheat straw, respectively. In continuous-flow cultures, however, the two fungi degraded 52-56% of the apparent dry matter of wheat straw. Both fungi were able to produce greater quantities (up to x 30) of cell-wall degrading enzymes (CMCase, xylanase, beta-glucosidase and beta-xylosidase) in continuous-flow cultures than in the corresponding batch cultures. Increasing the dilution rate in continuous-flow culture resulted in the production of increased enzyme activity for all the measured cell-wall degrading enzymes, with proportional relationships between dilution rate and the cumulative activities of beta-glucosidase and beta-xylosidase. Dilution rates, however, had no consistent effect on the cumulative production of the fermentation end-products, acetate, formate, D- and L-lactate from both fungi. In addition to acetate and formate,N. hurleyens is produced D- and L-lactate in both batch and continuous-flow cultures, whereas only trace amounts of L-lactate were detected in the Orpinomyces sp. cultures.     

Development of a culture medium for large-scale production of cellulolytic enzymes by Trichoderma reesei

Culture filtrates of CL-847 strain of Trichoderma reesei grown on different carbon sources have been compared. The highest enzyme production is obtained with Whatman C 41 cellulose: 17.9 mg/mL of soluble proteins and 13.7 units of filter paper (FP) activity. Wood pulps gave lower production values and more viscous culture media. About one-third of maximal enzyme production is obtained on lactose as the sole carbon source. Addition of 0.5% cellulosic inducer to 6% lactose media enhances enzyme production up to the following levels: 14.1 mg/mL of soluble proteins and 8.4 units of FP activity.     

厌氧真菌纤维降解酶及其应用潜力的研究进展

反刍动物的瘤胃是已知的纤维降解能力最强的天然发酵罐,其发酵粗纤维的能力很大程度上依赖于其中栖息的各类微生物的功能。厌氧真菌作为瘤胃内的一类低丰度菌群,最先定殖到宿主动物摄入的纤维质饲料上,并通过分泌大量高效的碳水化合物活性酶降解粗纤维。然而,由于缺乏足够的基因组信息和有效的厌氧真菌遗传操作系统,目前国内外对厌氧真菌分泌的纤维降解酶及其降解机制的研究进展有限。本文对厌氧真菌的分类及已发表的基因组信息进行概述,介绍了各类纤维降解酶及纤维小体的组成结构和催化机制特点,并对纤维降解酶在生物质能、饲料处理、纺织造纸及食品加工等方面的应用进行总结。研究厌氧真菌纤维降解酶的作用特性,将有助于完善其在瘤胃环境中有效竞争资源并降解粗纤维的知识体系,也将进一步了解其生物技术应用潜力,为工业生产中应用酶制剂提供新思路。     

Experimental and theoretical analysis of a novel deep-bed solid-state bioreactor for cellulolytic enzymes production

A novel deep-bed solid-state bioreactor was designed and fabricated for cellulolytic enzymes production using mixed fungal cultures. Better temperature and moisture control was achieved through a unique bioreactor design comprising an outer wire-mesh frame with internal air distribution along with near-saturation conditions within the cabinet. Without airflow through the internal distributors, maximum temperatures of 48 °C and 52 °C were observed during half- and full-capacity operation. These were reduced to 44 °C and 43 °C on resumption of airflow. In terms of cellulolytic enzyme production, no significant differences occurred in filter paper activity with depth in half-capacity operation; however, in full-capacity operation, top-level filter paper activity (5.39 FPU/g-solids) was significantly different from middle- and bottom-level activity. Top level beta-glucosidase, endocellulase, and xylanase activities were significantly (P < 0.05) different from middle and bottom levels in both half- and full-capacity operation. A two-phase coupled heat and mass transfer model was developed that predicted the experimental trends reasonably well. Model predictions confirmed that cabinet temperature of 30 °C and distributor airflow rate of 3.42 kg h⁻¹ during operation enabled effective temperature control.     

Miniaturized enzyme production and development of micro-assays for cellulolytic and xylanolytic enzymes

Miniaturized fungal cultivation and enzyme assays were developed. Cultivation for enzyme production was performed in 50 mL conical tubes. In addition, the miniaturized enzyme assays reduced the amount of enzymes and reagents necessary. These procedures can be adopted in screening fungi to determine if they produce cellulolytic and xylanolytic enzymes.     

Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides

For the commercial production of CoQ₁₀, batch-type fermentations were attempted in a 150-l fermenter using a mutant strain of R. sphaeroides. Optimum temperature and initial aeration rate were found to be 30°C and 2 vvm, respectively. Under optimum fermentation conditions, the maximum value of specific CoQ₁₀ content was achieved reproducibly as 6.34 mg/g DCW after 24 h, with 3.02 g/l of DCW. During the fermentation, aeration shift (from the adequate aeration at the early growth phase to the limited aeration in active cellular metabolism) was a key factor in CoQ₁₀ production for scale-up. A higher value of the specific CoQ₁₀ content (8.12 mg/g DCW) was achieved in fed-batch fermentation and comparable to those produced by the pilot-scale fed-batch fermentations of A. tumefaciens, which indicated that the mutant strain of R. sphaeroides used in this study was a potential high CoQ₁₀ producer. This is the first detailed study to demonstrate a pilot-scale production of CoQ₁₀ using a mutant strain of R. sphaeroides.     

Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production

Nine cellulolytic bacterial strains were isolated from soil sample taken in southern Taiwan. Through 16S rRNA sequence matching; eight of those isolates belong to Cellulomonas sp., while the other one belongs to Cellulosimicrobium cellulans. The activity of cellulolytic enzymes (cellulases and xylanase) produced from those strains was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (xylan, rice husk and rice straw) used for growth. HPLC analysis confirmed the bacterial hydrolysis of these cellulosic substrates for soluble sugars production. The efficiency of fermentative H₂ production from the enzymatically hydrolyzed rice husk was examined with seven H₂-producing pure bacterial isolates. With an initial reducing sugar concentration of 0.36 g l⁻¹, only Clostridium butyricum CGS5 exhibited efficient H₂ production from the rice husk hydrolysates with a cumulative H₂ production and H₂ yield of 88.1 ml l⁻¹ and 19.15 mmol H₂ (g reducing sugar)⁻¹ (or 17.24 mmol H₂ (g cellulose)⁻¹), respectively.