Banana Stalk Research Articles

Lignocellulose degradation and production of lignin modifying enzymes by Schizophyllum commune IBL-06 in solid-state fermentation

Abstract The modification of lignin is recognized as an important aspect of the successful refining of lignocellulosic biomass. Schizophyllum commune , a white rot basidiomycete was studied for ligninolytic enzymes (manganese peroxidase, lignin peroxidase and laccase) production in solid-state fermentation (SSF) of rice straw. Various physiological factors such as incubation time, culture pH, incubation temperature, C:N ratio and addition of mediators were optimized to enhance enzymes productivity. Maximum enzyme recoveries were obtained at pH, 5.0; temperature, 35 °C; C:N ratio, 20:1; mediator, MnSO 4 ; inoculum size, 4 mL after incubation time of 144 h. The crude ligninolytic extract thus produced was used for delignification of various agro-industrial residues. The enzyme extract caused 61.7%, 47.5%, 72.3% and 67.2% lignin removal from banana stalk, corn cobs, sugarcane bagasse, and wheat straw, respectively. The optimally delignified substrate was enzymatically digested by crude cellulase extract from Trichoderma harzaianum that resulted 47.3% and 69.4% cellulose hydrolysis from the native and pre-treated bagasse, respectively. The results suggested that lignocellulosic waste could be utilized as low-cost substrate for the production of enzymes which play significant role in many industrial and biotechnological sectors.

Employing peroxidase from Thai indigenous plants for the application of hydrogen peroxide assay

Thai local plants known as banana stalk, banana blossom, banana, sugar-cane, oroxylum indicum fruit, sesbania grandiflora fruit, and pigeon pea fruit were utilized for screening peroxidase enzyme to replace costly horseradish peroxidase in the hydrogen peroxide assay. The highest peroxidase activity was found in banana stalk extracted solution. The kinetic parameters, i.e., Michaelis–Menten constant (Km) and maximum velocity (Vmax) of banana stalk peroxidase were carried out. The optimum pH and thermal stability of this enzyme were also studied. Furthermore, crude banana stalk peroxidase was applied for the determination of hydrogen peroxide in a disinfection solution without any purification. The influent parameters affecting the developed method were cautiously studied and optimized. The calibration curve of standard hydrogen peroxide was achieved between 2.0 and 10.0 μmol L−1 with correlation coefficient (r2) 0.995. The method validations of detection limit (LOD), limit of quantification (LOQ) and precision were investigated. The concentrations of hydrogen peroxide achieved by the developed method were correlated with the enzymatic method using commercial available horseradish peroxidase.

Lignocellulose-degrading enzyme production by Pleurotus sapidus WC 529 and its application in lignin degradation / Lignoselüloz-çözücü enzim üretiminde Pleurotus sapidus WC 529 ve lignin parçalanmasındaki uygulamaları

Abstract Objective: Cost-effective production of industrially important enzymes is a key for their successful utilization on large scale. Present study was conducted to appraise Pleurotus sapidus WC 529 potential to produce oxidative (lignin peroxidase, manganese peroxidase, laccase) and hydrolytic (endoglucanase, exoglucanase and β-glucosidase) enzymes, grown on wheat straw, corn cobs, corn stover, rice straw, banana stalk and sugarcane bagasse in solid state fermentation (SSF) process. The crude extract thus produced was used for the delignification of various agro-industrial residues. Methods: Different agricultural wastes were assessed for lignocellulolytic enzymes production. The process parameters were optimized through response surface methodology (RSM) with central composite design (CCD) for maximum enzyme production. Results: P. sapidus secreted lower levels of hydrolytic enzymes but expressed high laccase, MnP and LiP activities in SSF (228.25, 153.45 and 59.20 UmL-1 respectively) of banana stalk. The optimal conditions for yielding highest enzyme activities were: initial pH, 4; temperature, 35°C; moisture level, 60%; inoculum size, 4 mL, and incubation time, 120 h. The enzyme extract removed 56.54, 51.08, 65.81 and 57.4% lignin from rice straw, sugarcane bagasse, corn cobs and wheat straw, respectively, after 48 h at 35°C. Conclusion: High ligninolytic activities were obtained in SSF of banana stalk by P. sapidus. The enzyme production was considerably improved by optimization through RSM. The results suggest that lignocellulolytic enzymes could be produced using cost-effective carbon sources and implicated to digest lignocellulosic biomass.

Sustainable waste mitigation: biotemplated nanostructured ZnO for photocatalytic water treatment via extraction of biofuels from hydrothermal carbonization of banana stalk

Working at the food-energy-water nexus, we identified an abundant agricultural waste with little intrinsic value – banana stalk – that can be converted to biofuels and a template for water treatment materials via hydrothermal carbonization.

Maturity and Safety of Compost Processed in HV and TW Composting Systems

Two composting systems: passive aerated system, horizontal-vertical system; active aerated system, turned windrow system was designed, constructed and studied for thirteen weeks to compare the maturity and safety of compost processed in them. Waste materials with the following percentage composition: river reed (75%), clay (10%), banana stalk / stem (5%), cow manure / dung (4%), rice chaff (4%), cocoa seed husk (1%), and poultry manure (1%) was processed in the two different systems. Compost materials were weekly sampled from top, bottom and mid portions of the compost masses in each of the systems and bulked to constitute a representative sample for the respective systems. From the weekly representative samples for the two systems, 5g subsamples were used for the determination of the germination index and microbial load for the compost masses. However temperature readings of the compost masses in both systems were recorded insitu daily at three different points using a long stem thermometer and the their respective averages used as the weekly readings for the systems. Temperature and the germination indices of composts processed in the two systems were used as parameters to assess the maturity; and the microbial load and its survival to assess the safety of the compost churned out. There were significant differences in the temperatures recorded in the two different systems during the composting period (p-value = 4.75 x 10-7, at α = 0.05). The total viable counts recorded in HV and TW ranged between 6.90 - 7.75logCFU/g of compost and 7.11–7.79logCFU/g of compost respectively which were significantly different (p-value = 0.027, at α = 0.05). The total fungi counts recorded in HV and T-W ranged between 1.11 – 2.32logCFU/g of compost and 1.68 - 2.40logCFU/g of compost respectively. Compost masses in all the systems had germination indices more than 150%.T-W had a higher rate of decomposition and maturity comparatively, hence a better composting system based on compost maturity. Penicillium spp. survived the process and it is known to produces mycotoxins which cause illnesses in humans. It is recommended that compost end-users, farmers, and producers use protectives and observe good hygiene in order to safeguard their health.

Screening of indigenously isolated fungi for lovastatin production and its in vivo evaluation.

Seven indigenously isolated fungal strains (Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Penicillium citrinum, Penicillium notatum, Pleurotus ostreatus and Trichoderma viradae) were tested for their potential to produce cholesterol lowering drug lovastatin by using different agro-industrial wastes (Corn cobs, corn stover, banana stalk, wheat straw, wheat bran, bagasse) in submerged as well as solid state fermentation. Aspergillus terreus showed maximum production of 18.74 mg/100 mL by wheat bran in solid state fermentation. The fermentation parameters (pH, temperature, Inoculum size, moisture contents and fermentation time) were also optimized for optimum production of lovastatin. It was found that Aspergillus terreus could produce 27.14 mg/100 mL lovastatin under optimum condition of pH (6), temperature (30°C), Inoculum size (2 mL), moisture contents (60%) and fermentation time (120 hrs) in solid state fermentation. The optimized lovastatin was extracted from fermented broth and orally administered to rats. The hypocholesterolemic effect of fermented lovastatin was evaluated on serum ALT, AST, HDL-C, LDL-C, TG and TC level of rats. It was concluded from the study, fermented lovastatin effectively lower the cholesterol level of rats.

Evaluation of Biosorptive Capacity of Banana (<i>Musa paradisiaca</i>) Stalk for Lead(II) Removal from Aqueous Solution

Raw Banana Stalk (RBS), Acid Activated Banana Stalk (AABS) and Base Activated Banana Stalk (BABS) prepared from banana stalk were used as biosorbents to remove Lead(II) from aqueous solution. The biosorbents were characterised using proximate analysis and Fourier Transform Infrared (FTIR) spectroscopy. Pb(II) of 1000 mg/L concentration was prepared from Pb(NO3)2 salt and other concentrations were obtained from this stock through serial dilution. Effects of adsorbent dose, temperature, initial metal concentration, contact time and pH on the percentage Pb(II) removal were evaluated. The Pb(II) concentrations in the solutions were analysed using Atomic Absorption Spectrophotometer. Kinetic, isotherm and thermodynamic parameters were determined. FTIR spectroscopy showed that RBS, AABS and BABS are rich in carboxyl, hydroxyl and phenolic functional groups. At an equilibrium time of 180 minutes, the percentage Pb(II) removal was 63.97%, 96.13% and 66.90% for RBS, AABS and BABS, respectively. Pseudo-second order kinetics best described the process with R2 (0.95, 0.98, 0.97) for RBS, AABS and BABS, respectively. Langmuir isotherm (AABS) has the maximum adsorption capacity (qmax) of 13.53 mg/g and R2 (0.99). Thermodynamic parameters obtained were △G0 (?18.75 kJ/mol), △H0 (12.63 kJ/mol), △S0 (0.05 kJ/mol·K) and Ea (4.37 kJ/mol). Banana stalk has viable characteristics for preparing biosorbents. Acid activated banana biosorbent is more efficient for removal of lead ions from its aqueous solution.

An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as K m = 27.5 ± 4.33 mg/mL, V max = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol.

Biogas Yield Potential Research of the Wastes from Banana Manufacturing Process under Mesophilic Anaerobic Fermentation

In this study is to verify the feasibility of the biogas yield potential of the wastes from banana manufacturing process and the feasibility of practical application through testing the fermentation performance of banana stalk and peel respectively at mesophilic temperature (35°C) under different concentration of fermentation. The research results indicated that the banana stalk and peel could be used as raw materials of anaerobic fermentation. The effect of gas production of low concentration of banana peel fermentation was the best and the biogas yield potential was 758 mL/g TS and 856 mL/g VS. But the fermentative broth acidified rapidly after the fermentation starting in high concentration of fermentation with banana peel. Therefore, the fermentation technology should be optimized during the practical application.

Optimization of Ligninolytic Enzymes Production through Response Surface Methodology

There is an increasing demand for green chemistry technologies that can cope with environmental waste management challenges. Agro-industrial residues are primarily composed of complex polysaccharides that support microbial growth for the production of industrially important enzymes such as ligninolytic enzymes. Schyzophyllum commune and Ganoderma lucidum were used alone, as well as mixed/co-culture, to produce crude ligninolytic enzymes extracts using corn stover and banana stalk as a substrate during solid state fermentation (SSF). In the initial screening, the extracted ligninolytic enzymes from S. commune produced using corn stover as the substrate showed higher activities of lignin peroxidase (1007.39 U/mL), manganese peroxidase (614.23 U/mL), and laccase (97.47 U/mL) as compared to G. lucidum and the mixed culture. To improve the production of ligninolytic enzymes by S. commune with solid state fermentation (SSF), physical factors such as pH, temperature, moisture, inoculum size, and incubation time were optimized by varying them simultaneously using response surface methodology (RSM) with a central composite design (CCD). The optimum SSF conditions were (for a 5 g corn stover substrate size): pH = 4.5; temperature = 35°C; inoculum size = 4 mL; and moisture content = 60%. Under optimum conditions, the activities of lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase were 1270.40, 715.08, and 130.80 IU/mL, respectively.

Hyper-production of Alkaline Protease by Mutagenic Treatment of Bacillus subtilis M-9 using Agroindustrial Wastes in Submerged Fermentation

Green chemistry technologies are the powerful tool for the management of environmental wastes challenges. Agro industrial residues are composed of complex polysaccharides that support the microbial growth for the production of useful products (enzymes, organic acids, drugs, etc.). Disposal and environment friendly management of these wastes has become a global priority. The aim of present investigation was to improve the alkaline protease yield by treating the parent Bacillus subtilis M-9 strain with different mutagens UV-irradiations, N-methyl-N-nitro- N-nitrosoguinidine (NTG), Ethidium bromide (EB), using agroindustrial wastes (banana stalk and corn stover) in submerged fermentation. Fifteen positive mutants were selected on skim milk agar plates for shake flask experiments. BSU-5 mutant strain showed 81.21± 3.24 PU/mL alkaline protease activity higher than parent strain (23.57 ± 1.19 PU/mL) in optimized fermentation medium. The fermentation profile like pH (9), temperature (45°C), inoculum size (2 mL), incubation time (24 hrs, and kinetic parameters such as u (h-1), Yp/s, Yp/x, Yx/s, qs, Qs, qp also confirmed the hyper proteolytic activity of alkaline protease produced from BSU-5 mutant strain over parent strain and other mutants. Finally, the BSU-5 mutant strain was immobilized by entrapping it in calcium alginate beads and agar. Alkaline protease production and stability of biocatalyst were investigated in both free and immobilized cells. It was concluded from the study, immobilized cells were more efficient for enzyme production then free cells when used repeatedly.

Bioethanol production from rice straw residues.

A rice straw - cellulose utilizing mold was isolated from rotted rice straw residues. The efficient rice straw degrading microorganism was identified as Trichoderma reesei. The results showed that different carbon sources in liquid culture such as rice straw, carboxymethyl cellulose, filter paper, sugar cane bagasse, cotton stalk and banana stalk induced T. reesei cellulase production whereas glucose or Potato Dextrose repressed the synthesis of cellulase. T. reesei cellulase was produced by the solid state culture on rice straw medium. The optimal pH and temperature for T. reesei cellulase production were 6 and 25 °C, respectively. Rice straw exhibited different susceptibilities towards cellulase to their conversion to reducing sugars. The present study showed also that, the general trend of rice straw bioconversion with cellulase was more than the general trend by T. reesei. This enzyme effectively led to enzymatic conversion of acid, alkali and ultrasonic pretreated cellulose from rice straw into glucose, followed by fermentation into ethanol. The combined method of acid pretreatment with ultrasound and subsequent enzyme treatment resulted the highest conversion of lignocellulose in rice straw to sugar and consequently, highest ethanol concentration after 7 days fermentation with S. cerevisae yeast. The ethanol yield in this study was about 10 and 11 g.L−1.

Purification and characterization of LiP produced by Schyzophyllum commune IBL-06 using banana stalk in solid state cultures

Lignin peroxidase was produced from Schyzophyllum commune IBL-06 through solid state fermentation of an abundantly available agro-industrial waste, banana stalk, under pre-optimized conditions. LiP was fractionated by 65% saturation with NH4SO4 and dialysis to 1.5-fold purification. The enzyme was further purified by Sephadex G-100 gel filtration chromatography to 2.34 fold with specific activity of 468 U/mg. A single band of 80 kDa was obtained on native gel while on sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), and two bands having molecular weight of 33 & 47 kDa were obtained, suggesting that LiP was a two polypeptide oligomeric protein. The present LiP from S. commune IBL-06 was optimally active at pH 5 and 35oC. The stability assay showed that LiP retained activity in an acidic pH range of 4 to 6 and a temperature of 25 to 45°C after 24 h of incubation. Lignin peroxidase oxidized the vertry alcohol and showed kinetic constants KM and Vmax values of 0.46 mM and 388 mM/min, respectively. All organic and inorganic compounds inhibited S. commune LiP, but EDTA, β-Marcaptoethanol, and Pb(NO3)2 were the most inhibitory.

Adsorptive features of banana (Musa paradisiaca) stalk-based activated carbon for malachite green dye removal

Chemically prepared activated carbon derived from banana stalk (BSAC) was used as an adsorbent to remove malachite green (MG) dye from aqueous solution. BSAC was characterised using thermogravimetric analyser, Brunauer Emmett Teller, Fourier transform infrared spectrometry, scanning electron microscopy, pHpzc, elemental analysis and Boehm titration. The effectiveness of BSAC in adsorbing MG dye was studied as a function of pH, contact time, temperature, initial dye concentration and repeated desorption–adsorption processes. pHpzc of BSAC was 4.5 and maximum dye adsorption occurred at pH 8.0. The rate of dye adsorption by BSAC was very fast initially, attaining equilibrium within 120 min following a pseudo-second-order kinetic model. Experimental data were analysed by Langmuir, Freundlich and Dubinin–Raduschevich isotherms. Equilibrium data fitted best into the Langmuir model, with a maximum adsorption capacity of 141.76 mg·g−1. Δ G 0 values were negative, indicating that the process of MG dye adsorption onto BSAC was spontaneous. The positive values of Δ H 0 and Δ S 0 suggests that the process of dye adsorption was endothermic. The regeneration efficiency of spent BSAC was studied using 0.5 M HCl, and was found to be in the range of 90.22–95.16% after four cycles. This adsorbent was found to be both effective and viable for the removal of MG dye from aqueous solution.

Production and optimization of ligninolytic enzymes by white rot fungus Schizophyllum commune IBL-06 in solid state medium banana stalks

The present study deals with production of ligninolytic enzymes from an indigenous white rot fungus Schizophyllum commune IBL-06 by using banana stalk as substrate through the process of solid state fermentation. The production process was further improved by optimizing a number of physical parameters such as incubation time, moisture level, pH, temperature, inoculums size and nutritional factors (carbon and nitrogen ratio, mediators and metal ions). By optimization of different parameters, the maximum activities of enzymes synthesized by S. commune IBL-06 were observed as 3745 IU/ml for manganese peroxidase (MnP), 2700 IU/ml for lignins peroxidase (LiP) and 345 IU/ml for laccase after 3 days incubation at рH 4.5 and 35°C temperature with 3 ml inoculum size, 60% moisture content, 20:1 C:N ratio (glucose and ammonium nitrate as carbon and nitrogen supplements), 1ml of 1mM MnSO 4 as mediator, and 1ml of 1mM MgSO 4.7H 2O2. This High activities of ligninolytic enzymes produced by the fungus suggest its potential for commercial scale production of these enzymes for diverse industrial applications.

Enhanced citric acid production by Aspergillus niger EB-3 mutant using an inert solid support in molasses medium

Aspergillus niger EB-3 was used to enhance citric acid production through optimization of nutritional parameters using banana stalk as carrier/inert support in solid state fermentation (SSF) of sugarcane molasses. The effects of moisture level, nitrogen sources, incubation time, metabolic inhibitors and metal complexing chemicals were studied on citric acid production in SSF. A. niger EB-3 mutant could produce 97.52 mg/ml citric acid with sugar utilization of 104.07 ± 1.98 mg/ml in 96 h by optimizing moisture level (60%) and nitrogen source (0.2% w/w ammonium nitrate). Addition of different surfactants to the medium increased fungal biomass, sugar utilization and citric acid production and Tween-80 was the best surfactant that caused maximum enhancement in citric acid yield at 0.2% concentration. The metabolic inhibitors like naphthol, resorcinol and cresol also enhanced citric acid production when used in lower concentrations, and cresol was the most effective inhibitor at 0.4% (w/w) level. Addition of metal complexing compounds also stimulated citric acid production by A. niger EB-3 and EDTA was the most effective metal chelator at 0.4% (w/w) level. Under optimum conditions, 112.42 mg/ml citric acid was produced with sugar utilization of 121.84 mg/ml. Key words: Citric acid, hyperproducting mutant, Aspergillus niger EB-3, optimization, metabolic inhibitors, metal complexing compounds.

Hyperproductivity of extracellular enzymes from indigenous white rot fungi (P. chrysosporium IBL-03) by utilizing agro-wastes

An indigenous locally isolated white rot fungal strain Phanerochaete chrysosporium IBL-03was investigated for the hyper-production of ligninolytic enzymes from different agro-industrial wastes including wheat straw, rice straw, banana stalks, corncobs, corn stover, and sugarcane bagasse as substrate material in still culture fermentation technique. Screening experiments were performed at 30oC from 1 to 10 days and maximum enzyme activities were recorded after the 5th day of incubation on banana stalk. P. chrysosporium IBL-03 produced highestactivities of lignin peroxidase (LiP) and manganese peroxidase (MnP) but no laccase activity was detected in any fermented culture media. Production of ligninolytic enzymes was substantially enhanced through the optimization process. When banana stalk at 66.6 % moisture level and pH 4.5 was inoculated with 5mL spore suspension of P. chrysosporium IBL-03 at 35oC in the presence of molasses (1%) as carbon source, ammonium sulfate (0.2%) as nitrogen supplement, (1%) Tween-80 (0.3 mL) as surfactant and mediators (MnSO4 and veratryl alcohol) enhanced the LiP and MnP production up to 1040 and 965 (U/mL), respectively.

Adsorption of pesticides from aqueous solution onto banana stalk activated carbon

Activated carbon was prepared from banana stalk by potassium hydroxide (KOH) and carbon dioxide (CO 2) activation and its ability to remove the pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and bentazon was explored. The banana stalk activated carbon (BSAC) was characterized by Fourier transform infrared spectroscopy (FT-IR) analysis. The adsorption kinetic data were analyzed using two kinetic models: the pseudo-first-order and pseudo-second-order models. The adsorption kinetics was better represented by the pseudo-second-order model. The equilibrium adsorption data obtained at 30, 40 and 50 °C were analyzed by Langmuir and Freundlich isotherm models and results showed that it was better described by the Freundlich model. Higher adsorption capacity observed for 2,4-D than bentazon were attributed to the presence of electron-withdrawing –Cl groups on the aromatic ring and smaller molecular size of 2,4-D. The thermodynamic parameters, Δ G°, Δ H° and Δ S° determined, showed that the adsorption of 2,4-D and bentazon onto BSAC was feasible, spontaneous and exothermic. The results showed that BSAC is an efficient adsorbent for the adsorptive removal of 2,4-D and bentazon from aqueous solutions.

Purification and characterization of laccase produced by Schyzophylum commune IBL-06 in solid state culture of banana stalks

Shyzophyllum commune IBL-06 produced 367 IU/mL of laccase in solid state bioprocess of banana stalk under optimum physical and nutritional parameters. The optimum SSF conditions were рH 4.5; temperature, 35°C; inoculum size, 3mL (106-108 mL-1); moisture content, 60%; C:N ratio in the medium, 15:1 (glucose and ammonium nitrate as additional carbon and nitrogen sources, respectively), ABTS (1mM), 1 mL, and CuSO4 (1mM), 1mL. Laccase was purified 3.95-fold with specific activity of 158 U/mg by ammonium sulfate precipitation, followed by gel filtration chromatography using Sephadex G-100 column. Molecular weight of the laccase was 63 KDa on SDS-PAGE. Purified laccase had an optimum рH of 6.0 and was stable in the рH range from 6 to 7. The optimum temperature was 40 °C, and it displayed considerable stability within the range 30 to 35 °C with 24 h incubation. Km and Vmax were 0.025 mM and 80 mM/min, respectively, using ABTS as a substrate. Copper sulfate increased the activity of purified laccase when used at low concentration, but silver nitrate exerted the strongest inhibition.

Cultivation of Pleurotus sajor-caju on banana stalk and Bahia grass based substrates

Banana stalks and Bahia grass were utilized as basic starting materials for the production of the mushroom Pleurotus sajor-caju. Banana stalks were combined with other waste or supplement products (wheat bran, coast-cross hay, bean straw and cotton textile mill) to obtain different nitrogen concentrations. Since Bahia grass is relatively rich in protein, it was combined with other substrates (banana stalk, coast-cross hay and bean straw) to maintain a substrate nitrogen concentration of about 1.5%. Banana stalks and Bahia grass were both more efficient in the production of the mushroom P. sajor-caju when utilized without the addition of other substrates, with biological efficiencies of 74.4% and 74.12%, respectively. When combined with other substrates or grasses, there was a drop in biological efficiency, independent of the concentration of nitrogen. Furthermore, the addition of protein-rich waste to banana stalks resulted in a decrease or absence of fructification, which indicates that high concentrations of nitrogen in the cultivation substrate may hinder the cultivation of this mushroom. On the other hand, results reveal that the ideal concentration of nitrogen may depend on other physicochemical factors and these factors may determine the success in cultivating P. sajor-caju. Therefore, we conclude that P. sajor-caju may be cultivated on banana stalk and Bahia grass as pure substrates, not being necessary their supplementation or combine them with another substrates.