Filter Paper Cellulase Activity Research Articles

Maximizing biofuel production from algal biomass: A study on biohydrogen and bioethanol production using Mg[sbnd]Zn ferrite nanoparticles

Algal biomass is a promising renewable feedstock for biofuel production that does not compete with food crops or require complex pretreatment like lignocellulosic biomass. This study examined biofuel production from two algae: Alkalinema pantanalense (cyanobacteria) and Chlorella vulgaris (green alga). Although there was no significant difference in their biomass, A. pantanalense showed a higher carbohydrate content (204.96 mg L−1) than C. vulgaris (156.07 mg L−1). To maximize reducing sugar release, three pretreatments were tested: thermotacidic, biological using the new fungal isolate Trichoderma longibrachiatum, and biological with nanoparticles. Biological pretreatment with MgZn ferrite nanoparticles (MZF-nps) at 60 mg L−1 concentration gave the best results, significantly enhancing cellulase, β-glucosidase and filter paper cellulase activities by 20.94 % (A. pantanalense) and 18.63 % (C. vulgaris). For biohydrogen production, the co-culture of Klebsiella pneumoniae and Enterobacter cloacae resulted in faster fermentation and improved hydrogen evolution compared to individual cultures. A. pantanalense and C. vulgaris yields were 35.1 mL g−1 and 26.6 mL g−1 dry weight, with maximal cumulative production of 2478 mL L−1 and 1845 mL L−1, respectively. Optimized Saccharomyces cerevisiae bioethanol fermentation conditions included 72 h incubation, 5 % inoculum, 30 °C, pH 5 under shaking condition, yielded 11.2 g L−1 (A. pantanalense) and 7.2 g L−1 (C. vulgaris). Furthermore, MZF-nps hydrolysate significantly increased bioethanol production, by 4.2-fold (A. pantanalense) to 32.45 g L−1 and 3.48-fold (C. vulgaris) to 28.6 g L−1, compared to thermoacidic pretreatment. In summary, biological pretreatment demonstrates the potential of algal biomass as a renewable feedstock for sustainable biofuel production.

Response surface optimization of cellulase production by Aspergillus stellatus NFCCI 5299 in shake flask submerged fermentation using wheat bran.

The expense of cellulase enzymes is the main barrier to the enzymatic saccharification of biomass. Numerous tactics, such as the utilizing inexpensive lignocellulosic substrates as well as economically feasible fermentation techniques for the production of the enzyme may reduce the cost of cellulases. The present investigation was aimed to improve cellulase production employing potential cellulolytic soil fungi, Aspergillus stellatus NFCCI 5299 using wheat bran as substrate. Employing response surface methodology (RSM) with central composite design (CCD), the most efficient process parameters were determined. The ideal conditions for the synthesis of carboxy methyl cellulase (CMCase) and filter paper cellulase activity (FPase) were 6days of incubation, inoculum size of 4 mycelial disc, 125rpm of agitation, and 3.5% of wheat bran. The significant mycelial development and enzymatic digestion of wheat bran were discovered by scanning electron microscopy (SEM) and fourier transform infrared (FTIR) analysis. The findings suggested that it can be practicable to use wheat bran as substrate under submerged fermentation utilizing Aspergillusstellatus NFCCI 5299 for efficient cellulase production.

Degradation potential of different lignocellulosic residues by Trichoderma longibrachiatum and Trichoderma afroharzianum under solid state fermentation

To achieve an economically viable substitutive strategy for lignocellulosic residues use, cellulose degradation by efficient Trichoderma strains become a key alternative. This study aimed to explore the effects of various cultural conditions on the degradation of different lignocellulosic substrates (corn straw and licorice residues) by Trichoderma longibrachiatum and Trichoderma afroharzianum in solid state fermentation. The strains were isolated from the rhizospheric soils of medicinal plants. In addition, the best fermentation combination was selected to evaluate the degradation results. This study confirmed that fermentation conditions and different components of corn straw and licorice residues were both crucial factors affecting the cellulase activity and spore production of two Trichoderma strains. A combination of T. afroharzianum degrading corn straw was selected for Response Surface Methodology optimization. The best fermentation conditions were fermentation time of 5.72 days, initial material liquid ratio of 1:4.58, initial pH of 6.32 and fermentation temperature of 28.8 ℃, with a filter paper cellulase activity of 0.91 U/mL. After continuous fermentation, the spore production reached 6.18 × 109 spores/g, the crude fiber degradation rate reached 23.5 % with the reducing sugar content maintained at 11 mg/g. These results highlight the great potential of T. afroharzianum degrading corn straw for organic amendment.

Enzyme production and oil palm empty fruit bunch bioconversion to ethanol using a hybrid yeast strain.

Oil palm empty fruit bunch (OPEFB) is a lignocellulosic biomass generated in palm oil mills. It is a sustainable resource for fuels and chemicals. In this study, OPEFB was converted to ethanol by an integrative OPEFB conversion process including dilute alkaline pretreatment, cellulolytic enzyme production, separate OPEFB hydrolysis, and cofermentation using a hybrid xylose-fermenting yeast. OPEFB was pretreated using 1% (w/v) NaOH solution followed by 1% (v/v) H2 O2 . Further, cellulolytic enzymes were produced by submerged fermentation using Trichoderma reesei Rut C30 and used for OPEFB hydrolysis. The filter paper cellulase activity of the crude cellulolytic enzymes was 15.1IU/mL, which was higher than those obtained by reported Trichoderma strains under laboratory conditions. Glucose and xylose yields reached 66.9% and 74.2%, respectively, at 30 filter paper unit (FPU)/g-biomass enzyme dosage and 10% (w/v) biomass loading. The hybrid yeast strain ScF2 was previously constructed through recursive genome shuffling of Pichia stipitis and Saccharomyces cerevisiae and was used in OPEFB hydrolysate fermentation. About 16.9g/L ethanol was produced with an ethanol yield of 0.34g/g sugars, which was 67% of theoretical ethanol yield.

The overexpression of one single cbh gene making Trichoderma asperellum T-1 a better cellulase producer

Trichoderma asperellum T-1, a traditional bio-control strain, is previously found to be potentially useful in the degradation of natural waste lignocellulose as it can ferment the natural materials without pretreatment. Many problems caused by substrate pretreatment can be therefore avoided. In this study, we intended to engineer a new strain to enhance its lignocellulose degradation ability by modifying the genome of T. asperellum T-1. A genetic transformation system mediated by Agrobacterium tumefaciens AGL-1 (ATMT) was constructed on T. asperellum T-1. On this basis, the overexpressed strain was produced by transforming a recombinant cellobiosidase gene (cbh) under the control of inducible promoter of endo-1, 4-β-xylanase gene, into wild-type T. asperellum T-1. After resistance screening, multiple transmission, growth comparison, and enzyme activity determination, four transformants (M1, M2, M5, and M6) were obtained. Filter paper cellulase activity of these transformants reached, respectively, 36.2%, 30.6%, 32.9%, and 42.7% higher than the wild-type strain. Most importantly, the CMCase, β-glucosidase and xylanase activity were also increased, although only one cbh gene was overexpressed. This work indicated that the enhancement of cellulase production ability of T. asperellum T-1 can be promisingly feasible by genetic modification. And the xylanase gene’s promoter can be effectively used in genetic modification to promote T. asperellum T-1 to be more effectively used in lignocellulose degradation.

Characterization of novel roles of a HMG-box protein PoxHmbB in biomass-degrading enzyme production by Penicillium oxalicum.

High-mobility group (HMG)-box proteins are involved in chromatin organization in eukaryotes, especially in sex determination and regulation of mitochondrial DNA compaction. Although a novel HMG-box protein, PoxHmbB, had been initially identified to be required for filter paper cellulase activity by Penicillium oxalicum, the biological roles of HMG-box proteins in biomass-degrading enzyme production have not been systematically explored. The P. oxalicum mutant ∆PoxHmbB lost 34.7-86.5% of cellulase (endoglucanase, p-nitrophenyl-β-cellobiosidase, and p-nitrophenyl-β-glucopyranosidase) activities and 60.3% of xylanase activity following Avicel induction, whereas it exhibited about onefold increase in amylase activity following soluble corn starch induction. Furthermore, ∆PoxHmbB presented delayed conidiation and hyphae growth. Transcriptomic profiling and real-time quantitative reverse transcription-PCR revealed that PoxHmbB regulated the expression of major genes encoding plant biomass-degrading enzymes such as PoxCel7A-2, PoxCel5B, PoxBgl3A, PoxXyn11B, and PoxGA15A, as well as those involved in conidiation such as PoxBrlA. In vitro binding experiments further confirmed that PoxHmbB directly binds to the promoter regions of these major genes. These results further indicate the diversity of the biological functions of HMG-box proteins and provide a novel and promising engineering target for improving plant biomass-degrading enzyme production in filamentous fungi.

Improving cellulase production in submerged fermentation by the expression of a Vitreoscilla hemoglobin in Trichoderma reesei

Trichoderma reesei is well known as an industrial workhorse fungus in cellulase production. The low dissolved oxygen supply in the highly viscous medium of T. reesei remains a major bottleneck that hampers growth and cellulase production in submerged fermentation. Vitreoscilla hemoglobin (VHb) has been demonstrated to improve metabolism and protein production in different heterologous hosts under hypoxic conditions, but the use of VHb in T. reesei remains uninvestigated. This study examines the effect of VHb in improving T. reesei performance in submerged fermentation. The VHb gene (vgb)-expressing cassette was successfully transformed into the TU-6 strain, integrated into the genome of T. reesei, and functionally expressed with biological activity, which was confirmed by carbon monoxide difference analysis. Compared to the parent strain, the expression of VHb increased the glucose consumption rate of the transformant. Moreover, in cellulase-inducing medium total protein secretion of the VHb expressing strain was 2.2-fold of the parental strain and the filter paper cellulase activity was increased by 58% under oxygen-limiting conditions. In summary, our results demonstrate that VHb has beneficial effects on improving total protein secretion and cellulase activity of T. reesei in submerged fermentation.

Cellulase production and saccharification of rice straw by the mutant strain Hypocrea koningii RSC1

The production of cellulase using solid-state fermentation of rice straw by the mutant strain Hypocrea koningii RSC1 was studied. Optimization of culture conditions, such as the nitrogen source, pH, and temperature, resulted in a maximum filter paper cellulase activity of 44.15 U g(-1) substrate, a carboxymethylcellulase activity of 324.6 U g(-1) substrate, and a β-glucosidase activity of 7.45 U g(-1) substrate. Saccharification of untreated, 1% H(2)SO(4)-treated, and 2.5% NaOH-treated rice straw using the RSC1 cellulase resulted in 19, 17, and 34 g L(-1) of reducing sugar, respectively. Further studies on the morphological and compositional changes of rice straw upon treatment with the cellulase by scanning electron microscopy analysis and Fourier transform infrared spectroscopy revealed the disruption of the arrangement of fibers and changes in the functional groups that occur in cellulose. X-ray diffraction analysis revealed a reduction in crystallinity of the rice straw upon treatment with the cellulase. Our study shows that H. koningii RSC1 could be a good choice for the production of cellulase and reducing sugars from rice straw.

Trichoderma atroviride mutants with enhanced production of cellulase and β-glucosidase on pretreated willow

Trichoderma atroviride mutants were developed by using N-methyl- N′-nitro- N-nitrosoguanidine (NTG) treatment and UV-light followed by a semiquantitative plate clearing assay on Walseth-cellulose/agar plates. The parent strain of the mutants was an isolate from the Amazonas basin (TUB F-1505), whose identity was established by ITS 1 and 2 and tef1 gene sequence analysis. Strain F-1505 proved to be the most promising extracellular cellulase producer among 150 wild-type Trichoderma in a screening program performed in shake flask fermentation on pretreated willow. Reducing sugar content, soluble protein, filter paper cellulase activity (FPA), β-glucosidase activity and endoglucanase activity of the fermentation broths of the mutant strains were measured in both shake flask and lab-scale fermenters and compared with Trichoderma reesei Rut C30. Also, hydrolytic capacities of fermentation supernatants of T. reesei Rut C30, the parent strain (F-1505) and the best mutants were compared on pretreated willow as carbon source and hydrolysis substrate. The T. atroviride mutants produced high levels of extracellular cellulases as well as β-glucosidase, rendering the need for β-glucosidase supplementation in hydrolysis of cellulose or pretreated willow unnecessary. On the contrary, β-glucosidase supplementations were essential in order to obtain good glucose yields for all other cellulase preparations tested.

Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties.

Xylanase production by the Antarctic psychrophilic yeast Cryptococcus adeliae was increased 4.3 fold by optimizing the culture medium composition using statistical designs. The optimized medium containing 24.2 g l(-1) xylan and 10.2 g l(-1) yeast extract and having an initial pH of 7.5 yielded xylanase activity at 400 nkat (nanokatal) ml(-1) after 168-h shake culture at 4 degrees C. In addition, very little endoglucanase, beta-mannanase, beta-xylosidase, beta-glucosidase, alpha-L-arabinofuranosidase, and no filter paper cellulase activities were detected. Among 12 carbon sources tested, maximum xylanase activity was induced by xylan, followed by lignocelluloses such as steamed wheat straw and alkali-treated bagasse. The level of enzyme activity produced on other carbon sources appeared to be constitutive. Among the complex organic nitrogen sources tested, the xylanase activity was most enhanced by yeast extract, followed by soymeal, Pharmamedia (cotton seed protein), and Alburex (potato protein). A batch culture at 10 degrees C in a 5-1 fermenter (3.5-1 working volume) using the optimized medium gave 385 nkat at 111 h of cultivation. The crude xylanase showed optimal activity at pH 5.0-5.5 and good stability at pH 4-9 (21 h at 4 degrees C). Although the enzyme was maximally active at 45 degrees - 50 degrees C, it appeared very thermolabile, showing a half-life of 78 min at 35 degrees C. At 40 degrees - 50 degrees C, it lost 71%-95% activity within 5 min. This is the first report on the production as well as on the properties of thermolabile xylanase produced by an Antarctic yeast.

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml−1 (248 nkat mg−1 protein). In addition, α-L-arabinofuranosidase, β-xylanase, β-xylosidase, β-glucosidase, β-mannosidase, α-galactosidase, β-galactosidase and endoglucanase activities were detected at 32 nkat ml−1, 30 nkat ml−1, 16 nkat ml−1, 15 nkat ml−1, 0.1 nkat ml−1, 1 nkat ml−1, 0.5 nkat ml−1 and 8 nkat ml−1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998

Microbial production of 2,3-butanediol from water hyacinth

A scheme for microbial production of 2,3-butanediol from water hyacinth (WH), a nuisance weed, is presented. It consists in sun-drying WH followed by crushing, alkaline treatment at high temperature, enzymatic hydrolysis, and fermentation. A caustic soda concentration of 1% and steam pressure of 1 kg/cm 2 for 1 h were optimal for delignification and yielded 35 g delignified water hyacinth (DLWH)/100 g crude water hyacinth. A slurry concentration of 5%, an enzyme concentration yielding filter-paper cellulase activity (FPCA) of 14 IU/g DLWH, a temperature of 50°C, a pH of 4·8, and an incubation period of 48 h were optimal for enzymatic hydrolysis yielding 19 g/litre reducing sugars. Prefermentation hydrolysis followed by combined hydrolysis and fermentation (PH-CHF) was most efficient and yielded 15 g 2,3-butanediol per 100 g DLWH. An HRT of 4 h corresponding to a productivity of 1·4 g/litre/h of 2,3-butanediol with anaerobic-fixed-film and upflowanaerobic-sludge-blanket (UASB) reactor systems was achieved on WH hydrolysate in a continuous mode.

Cellulolytic enzymes of a thermotolerantAspergillus terreus strain and their action on cellulosic substrates

A thermotolerant fungal strainAspergillus terreus produced high activities of cellulolytic enzymes when grown in shake flasks for 8 days at 40°C or 14 days at 28°C in medium containing 2.5% (w/v) cellulose powder and 1% (w/v) wheat bran. There was little difference between the final activities of endo-(1,4)-β-glucanase (ca. 14.4 U/ml); filter paper activity (ca. 1.3 U/ml) and β-glucosidase (ca. 10 U/ml). Endoglucanase had maximum activity at 60°C and pH 3.8; the other two enzymes were optimal at 60°C and pH 4.8. The maximum hydrolysis of different cellulosic substrates (about 50%) was obtained within 48 h when 1.1 U/ml of filter paper cellulase activity were employed to saccharify 100 mg alkali-treated cotton, filter paper, bagasse, and rice straw at 50°C and pH 4.8. The major end-product, glucose, was produced from all substrates, with traces of cellobiose and other larger oligosaccharides being present in rice straw hydrolysates.