Cellulase Production Under Solid-State Fermentation by Trichoderma reesei RUT C30: Statistical Optimization of Process Parameters

Sugar cane bagasse was used as substrate for cellulase production using Trichoderma reesei RUT C30, and the culture parameters were optimized for enhancing cellulase yield. The culture parameters, such as incubation temperature, duration of incubation, and inducer concentration, were optimized for enhancing cellulase yield using a Box-Behnken experimental design. The optimal level of each parameter for maximum cellulase production by the fungus was determined. Predicted results showed that cellulase production was highest (25.6 FPAase units per gram dry substrate) when the inducer concentration was 0.331 ml/gds, and the incubation temperature and time were 33 degrees C and 67 h, respectively. Crude inducer generated by cellulase action was found to be very effective in inducing cellulases. Validation of predicted results was done, and the experimental values correlated well with that of the predicted.     

Bioconversion of Kraft Paper Mill Sludges to Ethanol by SSF and SSCF

Paper mill sludge is a solid waste material composed of pulp residues and ash generated from pulping and paper making processes. The carbohydrate portion of the sludge has chemical and physical characteristics similar to pulp. Because of its high carbohydrate content and well-dispersed structure, the sludges can be biologically converted to value-added products without pretreatment. In this study, two different types of paper mill sludges, primary sludge and recycle sludge, were evaluated as a feedstock for bioconversion to ethanol. The sludges were first subjected to enzymatic conversion to sugars by commercial cellulase enzymes. The enzymatic conversion was inefficient because of interference by ash in the sludges with the enzymatic reaction. The main cause was that the pH level is dictated by CaCO₃ in ash, which is two units higher than the pH optimum of cellulase. To alleviate this problem, simultaneous saccharification and cofermentation (SSCF) using cellulase (Spezyme CP) and recombinant Escherichia coli (ATCC-55124), and simultaneous saccharification and fermentation (SSF) using cellulase and Saccharomyces cerevisiae (ATCC-200062) were applied to the sludges without any pretreatment. Ethanol yields of 75–81% of the theoretical maximum were obtained from the SSCF on the basis of total carbohydrates. The yield from the SSF was also found to be in the range of 74–80% on the basis of glucan. The SSCF and SSF proceeded under stable condition with the pH staying near 5.0, close to the optimum for cellulase. Decrease of pH occurred due to carbonic acid and other organic acids formed during fermentation. The ash was partially neutralized by the acids produced from the SSCF and SSF and acted as a buffer to stabilize the pH during fermentation. When the SSF and SSCF were operated in fed-batch mode, the ethanol concentration in the broth increased from 25.5 and 32.6 g/L (single feed) to 45 and 42 g/L, respectively. The ethanol concentration was limited by the tolerance of the microorganism in the case of SSCF. The ethanol yield in fed-batch operation decreased to 68% for SSCF and 70% for SSF. The high-solids condition in the bioreactor appears to create adverse effects on the cellulase reaction.     

Xylanase production by Trichoderma reesei rut C-30 on rice straw

Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.     

Additional Paper Waste in Pulping Sludge for Biohydrogen Production by Heat-Shocked Sludge

Dark anaerobic fermentation is an interesting alternative method for producing biohydrogen (H₂) as a renewable fuel because of its low cost and various usable organic substrates. Pulping sludge from wastewater treatment containing plentiful cellulosic substrate could be feasibly utilized for H₂ production by dark fermentation. The objective of this study was to investigate the optimal proportion of pulping sludge to paper waste, the optimal initial pH, and the optimal ratio of carbon and nitrogen (C/N) for H₂ production by anaerobic seed sludge pretreated with heat. The pulping sludge was pretreated with NaOH solution at high temperature and further hydrolyzed with crude cellulase. Pretreatment of the pulping sludge with 3% NaOH solution under autoclave at 121 °C for 2 h, hydrolysis with 5 FPU crude cellulase at 50 °C, and pH 4.8 for 24 h provided the highest reducing sugar production yield (229.68 ± 2.09 mg/g_TVS). An initial pH of 6 and a C/N ratio of 40 were optimal conditions for H₂ production. Moreover, the supplement of paper waste in the pulping sludge enhanced the cumulative H₂ production yield. The continuous hydrogen production was further conducted in a glass reactor with nylon pieces as supporting media and the maximum hydrogen production yield was 151.70 ml/g_TVS.     

Cellulase production of Trichoderma reesei rut C 30 using steam-pretreated spruce

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hem icellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and logen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C30. However, the conversion of cellulose using these enzymes was higher than that obtained with logen or Celluclast cellulases using steam-pretreated spruce as substrate.     

Enzyme production of Trichoderma reesei rut C-30 on various lignocellulosic substrates

Economical production of cellulase enzyme is key for feasible bioethanol production from ligh ocellulosics using an enzyme-based process. On-site cellulase production can be more feasible with the process of separate hydrolysis and fermentation (SHF) than with simultaneous saccharification and fermentation, since the cost of enzyme is more important and a variety of substrates are available for the SHF process. Cellulase production using various biomass substrates available for SHF, including paper sludge, pretreated wood (steam exploded), and their hydrolysis residues, was investigated in shake flasks and a fermenter for their productivities and titers. Among the newspaper sludge, office paper sludge, and steam-exploded woods treated in various ways, the steam-exploded wood showed the best properties for substrate in cellulase production. The besttiter of 4.29 IU/mL was obtained using exploded wood of 2% (w/v) slurry in the shake flask, and the titer with the same substrate was duplicated to about 4.30 IU/mL in a 3.7-L fermenter. Also, the yield of enzyme reached 215 1U/g of substrate or 363 IU/g of cellulose. Despite various pretreatment attempts, newspaper and office paper substrate was inferior to the exploded-wood substrate for cellulase production. However, hydrolysis residues of papers showed quite promising results. The hydrolysis residue of office paper produced 2.48 IU/mL of cellulase in 7 d. Hence, the utilization of hydrolysis residues for cellulase production will be further investigated in the future.     

Production of Crude Cellulase and Xylanase From Trichoderma harzianum PPDDN10 NFCCI-2925 and Its Application in Photocopier Waste Paper Recycling

This paper implies production of cellulase and xylanase enzyme using a potent strain of Trichoderma harzianum for the efficient deinking of photocopier waste papers. Different nutritional and environmental factors were optimized for higher production of cellulase along with xylanase. After fermentation, maximum enzyme extraction was achieved from fermented matter using a three-step extraction process with increased efficiency by 26.6–29.3 % over single-step extraction. Static solid state was found as the best fermentation type using wheat bran (WB) as carbon source and ammonium ferrous sulfate (0.02 M) as nitrogen source. Subsequently, inoculum size (8?×?10⁶ CFU/gds), incubation days (4 days), temperature (34 °C), initial pH (6.0), and moisture ratio (1:3) significantly affected the enzyme production. Cellulase and xylanase activities were found to be maximum at pH 5.5 and temperature 55–60 °C with good stability (even up to 6 h). Furthermore, this crude enzyme was evaluated for the deinking of photocopier waste papers without affecting the strength properties with improved drainage as an additional advantage. The crude enzyme-deinked pulp showed 23.6 % higher deinking efficiency and 3.2 % higher brightness than chemically deinked pulp. Strength properties like tensile, burst indices, and folding endurance were also observed to improve by 6.7, 13.4, and 10.3 %, respectively, for enzyme-deinked pulp. However, the tear index was decreased by 10.5 %. The freeness of the pulp was also increased by 21.6 % with reduced drainage time by 13.9 %.     

Characterisation of Specific Activities and Hydrolytic Properties of Cell-Wall-Degrading Enzymes Produced by Trichoderma reesei Rut C30 on Different Carbon Sources

Conversion of lignocellulosic substrates is limited by several factors, in terms of both the enzymes and the substrates. Better understanding of the hydrolysis mechanisms and the factors determining their performance is crucial for commercial lignocelluloses-based processes. Enzymes produced on various carbon sources (Solka Floc 200, lactose and steam-pre-treated corn stover) by Trichoderma reesei Rut C30 were characterised by their enzyme profile and hydrolytic performance. The results showed that there was a clear correlation between the secreted amount of xylanase and mannanase enzymes and that their production was induced by the presence of xylan in the carbon source. Co-secretion of α-arabinosidase and α-galactosidase was also observed. Secretion of β-glucosidase was found to be clearly dependent on the composition of the carbon source, and in the case of lactose, 2-fold higher specific activity was observed compared to Solka Floc and steam-pre-treated corn stover. Hydrolysis experiments showed a clear connection between glucan and xylan conversion and highlighted the importance of β-glucosidase and xylanase activities. When hydrolysis was performed using additional purified β-glucosidase and xylanase, the addition of β-glucosidase was found to significantly improve both the xylan and glucan conversion.     

Crude Cellulase from Oil Palm Empty Fruit Bunch by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for Fermentable Sugars Production

Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2 % NaOH with autoclave, which was composed of 59.7 % cellulose, 21.6 % hemicellulose, and 12.3 % lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1 % of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5 % of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33 % and 19.11 %, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.     

Improved Cellulase Production by <Emphasis Type="Italic">Trichoderma reesei </Emphasis>RUT C30 under SSF Through Process Optimization

The major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which includes the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). The current study investigated the production of cellulase by Trichoderma reesei RUT C30 on wheat bran under SSF. Process parameters important in cellulase production were identified by a Plackett and Burman design and the parameters with significant effects on enzyme production were optimized for maximal yield using a central composite rotary design (CCD). Higher initial moisture content of the medium had a negative effect on production whereas incubation temperature influenced cellulase production positively in the tested range. Optimization of the levels of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold increase in production from 0.605 to 3.8 U/gds of cellulase. The optimal combination of moisture and temperature was found to be 37.56% and 30 °C, respectively, for maximal cellulase production by the fungus on wheat bran.     

Removal of Phenolic and Lignin Compounds from Bleached Kraft Mill Effluent by Fly Ash and Sepiolite

This study aimed to remove phenolic and lignin compounds from paper mill industry (4500 m³/h) wastewaters, which is discharged to sea from a plant located in the western Turkey. As adsorbent, fly ash, raw sepiolite and heat-activated sepiolite were used. The effect of factors such as, particle size, temperature and pH on adsorption process was investigated. From kinetic studies, equilibrium time was found as 1 h for both. The kinetic data supports pseudo-second order model but shows very poor fit for pseudo-first order model. Intraparticle model also shows that there are two separate stages in sorption process, namely, external diffusion and pore diffusion. Adsorption isotherms for fly ash and activated sepiolite were obtained at two different temperatures. From experiments carried out at different pHs, it was observed that pH plays an important role in the adsorption process in removing of both lignin and phenolic compounds, providing both ionizating the compounds and modifying sorbent surfaces. It was also observed that heat-activated sepiolite is more effective than raw sepiolite and fly ash to remove these compounds. Adsorption of lignin and phenolic compounds increases with decreasing particle size. In addition, the efficiency of adsorption decreases with increasing adsorption temperature for both fly ash and untreated sepiolite.     

Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production,Characterization, and Thermal Stability

A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g^?1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g^?1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K _M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.     

Analysis of Dehydroabietic Acid in Kraft Mill Effluents by High-Performance Liquid Chromatography

Abstract

A rapid high-performance liquid chromatographic method for the determination of dehydroabietic acid in kraft mill effluent is described. Selective solvent extraction of the samples with dichloromethane was performed. The samples were isocratically analysed on a Rad-Pak C18 column using 75% acetonitrile in water (0.1% acetic acid added). Detection was carried out at 220 nm and 267 nm on a variable wavelength detector with a detection limit of 0.01 mg/L.     

Valorization of Kraft Pulp and Paper Mill Slaker Grits and Biomass Fly Ash as Fillers in a Commercial Screed Mortar Formulation

Slaker grits (SG) and biomass fly ash (BFA), two waste streams generated in the pulp and paper industry, are commonly disposed of in landfills, a practice with a high economic and environmental burden. In this work, their individual valorization as fillers in a commercial screed mortar formulation was evaluated in order to achieve a more sustainable management practice. The waste streams were characterized in terms of true density, particle size and morphology, and chemical and mineralogical composition. The influence of their incorporation amount (5.0, 7.5, and 10.0 wt.% of the total solids) and pre-treatment (sieving and grinding) on the fresh (workability) and hardened state (density, water absorption by capillarity, and flexural and compressive strength) properties of the mortars were assessed. The results show that the addition of 10.0 wt.% of the SG after milling and sieving (<75 µm) and 7.5 wt.% of BFA in the as-received condition, or up to 10.0 wt.% after grinding and sieving (<63 µm), allowed for the production of mortar samples with properties within the recommended specifications and that were resistant to 25 consecutive freeze-thaw cycles. This waste valorization route could represent an economic benefit of up to 8.85 €/t_mortar and 2.87 €/t_mortar for mortar, and pulp and paper companies, respectively.     

Microwave Pretreatment of Substrates for Cellulase Production by Solid-State Fermentation

The agricultural residues, wheat bran and rice hulls, were used as substrates for cellulase production with Trichoderma sp 3.2942 by solid-state fermentation. Microwave irradiation was employed to pretreat the substrates in order to increase the susceptibility. Although the highest cellulase yield was obtained by the substrates pretreated by 450 W microwave for 3 min, pretreatment time and microwave power had no significant effect on cellulase production. The initial reducing sugar content (RSC) of substrates was decreased by microwave irradiation, but more reducing sugars were produced in later fermentation. Alkali pretreatment combined with microwave pretreatment (APCMP) of rice hulls could significantly increase cellulase yields and reducing sugar. The maximum filter paper activity, carboximethylcellulase (CMC)ase, and RSC were increased by 35.2%, 21.4%, and 13%, respectively, compared with those of untreated rice hulls. The fermented residues could produce more cellulase and reducing sugars than fresh rice hulls after they were treated by APCMP. The increased accessibility of the substrates by microwave pretreatment was mainly achieved by rupture of the rigid structure of rice hulls. However, for alkali pretreatment and APCMP, delignification and removal of ash played very important roles for increasing the acceptability of substrates.     

Structural Characterisation of the Coloured Organic Matter from an Eucalyptus Bleached Kraft Pulp Mill Effluent

Abstract

XAD-8 and XAD-4 resins in tandem were used to isolate and fractionate the coloured organic matter from an eucalyptus bleached Kraft pulp mill effluent which is discharged into river Vouga (Portugal) near the lagoon that exists in its terminal part. The composition of the isolated organic matter was investigated by FTIR and CPMAS-¹³C-NMR and compared to that of humic matter isolated before from the same river at a non-polluted site and a site very contaminated with the effluent of a sulphite pulp mill. The results put into evidence that the organic matter from both kinds of effluent is more aromatic and contains more lignin derived structural units than the humic matter from the unpolluted site. However, the organic matter from the sulphite pulp mill effluent is more soluble due to its content of sulphonic functional groups.     

Water Hyacinth as Carbon Source for the Production of Cellulase by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation, and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend.     

Elimination of Estrogenic Activity of Thermal Paper Using Laccase from Trichoderma sp NFCCI-2745

In thermal printing, bisphenol A (BPA) functions chemically as a developer and reacts with white or colorless dyes in the presence of heat, converting them to a dark color. BPA can transfer readily to skin in small amounts from these papers. Its damage to environment and organisms has caused an extensive concern. In the present study, thermal paper used at the local automated teller machine counters of India were analyzed for the presence of BPA, and the capability of the paper to produce estrogenicity were assessed using a yeast two-hybrid assay experimental system. The study also focused on eliminating the endocrine-disrupting properties with partially purified laccase from newly isolated ascomycete fungi. The results indicate that these papers can produce estrogen hormone-like effect on experimental systems. It should be noted that on a daily basis, tons of such receipts are being dumped in the environment. Estrogenic properties of thermal paper were effectively removed from the reaction mixture within 3 h of incubation with the partially purified enzyme. We propose the utilization of waste thermal paper as a cheap substrate for laccase production for a safer and cleaner environment.     

Characterization of Composts and Humic Acids from Pulp and Paper Mill Biosludges by DSC in Association with FT-IR Spectroscopy

Samples of pulp and paper mill biosludges with different maturation ages (fresh biosludge and composts collected after 3 weeks, 1 year and 2 years of composting) and their corresponding humic acids were analyzed by thermal analysis with DSC in association with FT-IR spectroscopy. The DSC curves obtained exhibited endotherms in the low-temperature region, assigned to dehydration processes, exotherms at around 370°C, attributed to the loss of peptidic structures, and high-temperature exotherms, assigned to the loss of functional groups; the lignins were thermostable, regardless of the maturation age of the compost. The results obtained provided evidence that, during composting, the organic matter evolved in the direction of higher molecular complexity and stability, leading to a more homogeneous product closely resembling humic-like substances. This revised version was published online in July 2006 with corrections to the Cover Date.