黑曲霉与里氏木霉固态混合发酵产β-葡萄糖苷酶

对黑曲霉NL02与里氏木霉RUT-C30固态混合发酵产β-葡萄糖苷酶的发酵培养基进行优化,研究培养基含水率、C源、N源、接种量、温度和2种菌种不同延长接种时间与接种比例对β-葡萄糖苷酶活力的影响。研究表明：麸皮17.5 g、玉米芯7.5 g、（NH4）2SO4 0.40 g、尿素0.37 g、黑曲霉孢子接入量为107个接种到250 mL三角瓶中,温度30 ℃、摇床转速100 r/min时,里氏木霉以105个孢子与黑曲霉同时接入,每克干曲所得β-葡萄糖苷酶的活力为132.45 IU,较黑曲霉单独培养时的104.35 IU提高了26.94%。     

Cellulase production by Trichoderma reesei (RUT-C30) in fed-batch culture

Summary Fed-batch cultures of Trichoderma reesei RUT-C30 attained quasi-steady state conditions, in respect of biomass concentration and enzyme production rate, commensurate with a specific cell maintenance coefficient of 0.029 g cellulose.g biomass.^–1h^–1 and specific cellulase production rate of between 9.6 and 11.9 IU (filter paper activity).g biomass.^–1h^–1. A maximum enzyme yield of 57 IU.m1^–1 at an overall productivity of 201 IU.L.^–1h^–1 resulted from a cellulose feed rate of 1.0g.L.^–1h^–1.     

里氏木霉与黑曲霉混合发酵产纤维素酶及其水解特性

研究了利用里氏木霉和黑曲霉混合培养产纤维素酶,以黑曲霉孢子悬浮液的不同活化浓度及不同的活化时间来寻找2个菌种发挥最大协同作用的结合点以及所产纤维素酶的水解特性。以里氏木霉单一培养和黑曲霉单一培养为参照进行对比研究。底物为农林废弃物之一的玉米秸秆,经过蒸气爆破预处理后,用作产酶C源。结果表明:黑曲霉孢子悬浮液活化浓度为10个/mL,活化时间为12 h时,滤纸酶比酶活最高,达3.32 U/mL,高于里氏木霉单一培养的2.25 U/mL,β-葡萄糖苷酶比酶活达1.32 U/mL,高于里氏木霉单一培养的0.57 U/mL。为进一步验证混合菌产纤维素酶的水解效果,利用混合菌产纤维酶的酶液及里氏木霉产纤维素酶的酶液进行酶水解实验,当酶用量为20 U/g绝干纤维素,底物质量浓度为100 g/L条件下水解48 h,混合菌所产酶液酶解得率达70.00%,高于里氏木霉所产酶液的酶解得率63.05%。实验表明里氏木霉与黑曲霉混合培养产酶是可行的,并优于单一菌种培养。     

Reaction engineering analysis of cellulase production with Trichoderma reesei RUT-C30 with intermittent substrate supply

The effects of varying initial concentrations of microcrystalline cellulose on cellulase production with Trichoderma reesei RUT-C30 as well as the effects of varying lactose and ammonium sulfate concentrations in the feed medium were studied simultaneously in parallel-operated shake flasks and, alternatively, in parallel-operated stirred-tank bioreactors on a 10-mL scale. Fifteen experiments were performed as triplicates in shake flasks as well as in stirred-tank bioreactors in parallel to identify the parameters of second-order polynomials for the estimation of the final filter paper activity of T. reesei RUT-C30 after a process time of 96 h. Even though parameter estimation was not possible based on the results of the shake flasks due to final enzyme activities at or below the detection limit (with the exception of one shake flask), the identification of the second-order polynomial was successful with the results of the parallel-operated stirred-tank bioreactors on a 10-mL scale. Reaction conditions with 53.3 g L^?1 microcrystalline cellulose in the initial medium, no lactose feeding and 3.3 g L^?1 day^?1 intermittent ammonium sulfate addition were estimated to be optimal. The final experimental validation of the optimum substrate supply on a L-scale resulted in the production of 4.88 filter paper units (FPU) mL^?1 with T. reesei RUT-C30 after 96 h. This is an improvement by a factor of 3.6 compared to the reference batch process (1.35 FPU mL^?1).     

Application of image analysis in the fungal fermentation of Trichoderma reesei RUT-C30

Trichoderma reesei was grown in a stirred-tank bioreactor (STB) and a reciprocating plate bioreactor (RPB) at four different agitation speeds. A semiautomatic image analysis protocol that was developed to characterize the mycelium morphology during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms) was applied to study the effect of agitation on the morphology of T. reesei. It was shown via statistical validation that broth samples used for image analysis represented the whole population of the fungi in the bioreactor. High shear was found to be damaging to T. reesei grown in the STB. The gentler shear produced in the RPB was not detrimental to the microorganism even at higher agitation speed. Better productivity was obtained for T. reesei grown in the STB and the highest productivity, 0.121 IU/mL h, was obtained at 400 rpm. The morphological parameter, the hyphal growth unit, was found to be correlated to the productivity. Understanding the effect of agitation on the morphology and productivity of T. reesei could lead to the design of better bioreactors and the selection of operating conditions of bioreactors to optimize the production of cellulase.     

Effect of mechanical agitation on the production of cellulases by Trichoderma reesei RUT-C30 in a draft-tube airlift bioreactor

With the aim to produce cellulases and to study the effect of mechanical agitation, a 35 L draft-tube airlift bioreactor equipped with a mechanical impeller was developed and validated to grow Trichoderma reesei RUT-C30 in a cellulose culture medium with lactose and lactobionic acid as fed batch. Cultures carried out without mechanical agitation resulted in higher volumetric enzyme productivity (200 U L⁻¹ h⁻¹), filter paper activity (17 U mL⁻¹), carboxymethyl cellulase activity (11.8 U mL⁻¹) and soluble proteins (3.2 mg mL⁻¹) when compared to those with agitation. Stereo and polarized light microscopy analyses reveal that mechanical agitation resulted in shorter mycelial hyphae and larger numbers of tips.     

Lipid production by a cellulolytic strain of Aspergillus niger

Studies on lipid formation by a cellulolytic mould. Aspergillus niger AS-101, were carried out in shake flasks. The maximum amount of lipid accumulated comprised 17% and 14·9% of the mycelial dry weight on glucose and cellulose media, respectively. Unsaturated fatty acids comprised around 80% of the total fatty material, with linoleic acid predominating.     

Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30

The use of a fed-batch cultivation of the fungus Trichoderma reesei (C30) allows cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] production to occur under optimum conditions, and results in extremely high enzyme titres and productivities. Enzyme levels of 26 U ml^?1 at productivities >130 U l^?1 h^?1 have been achieved. These results are compared with the values obtained in two-stage continuous cultivation of the organism at optimum pH and temperature.     

Characterization of cellulolytic enzyme complexes obtained from mutants of Trichoderma reesei with enhanced cellulase production

Summary The cellulolytic enzyme complexes secreted by the fungus Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15, and MHC 22 were characterized by determining their specific filter-paper (FP)-, carboxymethylcellulase (C_x)-and -glucosidase (G)-activities. They were characterised further by measuring their C_x and G profiles after separation on an isoelectrofocusing column over the pH range 3–10. While the overall FP-activity was roughly equal in all preparations, the specific -glucosidase activity was highest in mutants MHC 15 and MHC 22 which are distingiushed morphologically from the parent strain, QM 9414, by a higher degree of branching of their hyphae. Two peaks of -glucosidase activity were detected by isoelectric focusing in preparations from QM 9414 and M 6, none in the enzyme from the mutant M 5 while 3 and 4 peaks respectively were found in preparations from morphological mutants MHC 15 and MHC 22. The higher -glucosidase activity in these last two preparations was also reflected in the higher glucose to cellobiose ratio in the initial stages of cellulose hydrolysis by the individual enzyme preparations.     

Hydrolysis of cellulose by a mixture of Trichoderma reesei cellobiohydrolase and Aspergillus niger endoglucanase

Two endoglucanase-containing fractions were separated from Aspergillus niger cellulase by gel filtration and fast protein liquid chromatofocusing (FPLC). They possessed no ability to bind to or hydrolyze insoluble microcrystalline cellulose (Avicel) but were active toward soluble carboxymethylcellulose. No synergism was observed between Trichoderma reesei cellobiohydrolase I and either endoglucanase from A. niger. These findings may indicate that the role of the endoglucanase component of cellulase in insoluble microcrystalline cellulose hydrolysis is dependent upon its ability to be adsorbed upon the substrate.     

Cellulase and xylanase production by Trichoderma reesei Rut C-30

Summary When grown on cellulose or xylan, Trichoderma reesei (strain Rut C-30) produced extra-cellular enzymes which could hydrolyze both cellulose and xylan to their respective monosaccharides. At low O₂ saturation, -glucosidase activity is greatly reduced for cellulose-grown but not xylan-grown cells.     

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse

Plant‐degrading enzymes can be produced by fungi on abundantly available low‐cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two “second generation” substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi‐)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non‐washed SCB. The sensitivity to non‐washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.     

曲霉与木霉纤维素酶系基因组的属间遗传表达相容性

选用三类典型重组子3a、3b、A7-1和双亲本菌株AspersillusnigerAMSH、TrichodermareeseiQM9414为材料,按照所设计的纤维素酶系基因的通用序列和同工酶分型方法,进行基因组DNA指纹和酶系同工酶多态性比较分析。旨在提供重组子中基因重组的分子证据,阐明远缘双亲本基因组间的遗传表达相容性,并讨论其杂种优势的分子基础。结果发现重组子中基因组DNA指纹的重组特征稳定遗传,并能够相容性增强表达重组后的羧甲基纤维素酶（CMCase）和β-葡萄糖苷酶（βGlase）同工酶组分。纤维素酶系杂种优势的分子基础多样性包括：（1）3b中来自于双亲本部分编码βGlase的基因的杂合迭加和增强表达;（2）3a和A7-1中对应继承双亲本部分编码CMCase和βGlase的基因间协调性增强表达,并导致相应酶组分蛋白合成量的显著增加。由此综合提出了一个由βGlase介导的纤维素酶系活性调节和诱导合成调控的“双重协同增效”模型。此外还建立了考察重组子中杂种优势分子基础及其遗传稳定性的可行方法。     

Pectinase production by Aspergillus niger LB-02-SF is influenced by the culture medium composition and the addition of the enzyme inducer after biomass growth

The production of pectinase by Aspergillus niger LB-02-SF was focused on a submerged cultivation, before it was evaluated in a solid-state process. This study involved the creation of a defined culture medium and an evaluation of the effects of the addition of the enzyme inducer, citrus pectin, to the medium after the intense biomass growth phase. A culture medium formulated without glucose allowed a reduction of biomass growth and greater pectinase production, facilitated by the control of process parameters such as mixing, pH and oxygen supply. The addition of pectin when a minimum pH of 2.7 was reached at 22 h of cultivation did not affect fungal growth. The maximum biomass concentration was 11.0 g/L at 48 h, a value similar to that observed for the control, in which pectin was included in the medium at the beginning of the process (11.5 g/L, at 41 h). However, this condition favored the production of 14 U/mL pectinase, which was approximately 40% higher than the value observed for the control. These results show that pectinase production by A. niger in a submerged cultivation is strongly affected by the medium composition as well as the delayed addition of pectin to the fermentation broth.     

Promoting pellet growth of Trichoderma reesei Rut C30 by surfactants for easy separation and enhanced cellulase production

It is desirable to modify the normally filamentous Trichoderma reesei Rut C-30 to a pellet form, for easy biomass separation from the fermentation medium containing soluble products (e.g., cellulase). It was found in this study that this morphological modification could be successfully achieved by addition of the biosurfactant rhamnolipid (at ≥ 0.3g/L) and the synthetic Triton X-100 (at ≥ 0.1g/L) to the fermentation broth before the cells started to grow actively. Thirteen other surfactants tested were not as effective. Furthermore, the added rhamnolipid and Triton X-100 increased the maximum cellulase activity (Filter Paper Units) produced in the fungal fermentation; the increase was 68 ± 7.8% for rhamnolipid and 73 ± 12% for Triton X-100. At the concentrations required for pellet formation, rhamnolipid had negative effect on the cell growth: with increasing rhamnolipid concentrations, the growth rate decreased and the lag-phase duration increased linearly. Triton X-100 caused no significant differences in growth rate or lag phase.     

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.     

Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during Growth on Sugarcane Biomass

Background

Our dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world''s second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.

Results

Analyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and β-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, β-glucosidases, β-xylosidases, endoxylanases, xyloglucanases, and α-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.

Conclusion

Our work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.