Or Organi nic wa wastes fo for bios biostim timula lation tion of of Agaricus bisporus and and Pleurotus ostreatus R . C A M A C H O ‐ A R É VA LO 1 , J . G Ó M E Z ‐ H E R N Á N D E Z 1 , B . M AYA N S 1 2 , R . A N TÓ N 1 , C . E S C O L Á S T I C O 2 , C . G A R C I A ‐ D E LG A D O 3 E . E Y M A R 1 1 Department of Agricultural Chemistry and Food Sciences, University Autónoma of Madrid, 28049 Madrid, Spain 2 Department of Organic and Biorganic Chemistry. National Distance Education University (UNED) 3 Institute of Natural Resources and Agrobiology of Salamanca (IRNASA) Spain
Ligninolytic fungi Characteristics • Extracellular activities • Lacasse • Mn Peroxidase • Versatil Peroxidase Bioremediation • Oxidation by free radicals • Degradation of organic pollutans Edible mushroom • Residues generation • Solid waste and mycelia
Ligninolytic fungi Characteristics • Extracellular activities • Lacasse • Mn Peroxidase Media • Versatil Peroxidase conditions Bioremediation • Oxidation by free radicals Degradation improvement • Degradation of organic pollutans Edible mushroom Genetical modification • Residues generation • Solid waste and mycelia
Mushroom cultivation straw Enzyme production is influence by: Moisture Compost C/N ratio pH (4.5 optimum for fungi growth) Microbial competition Deinking sludge from paper mill
Objectives and experimental design To obtain an organic material from agricultural or industrial wastes to revitalize the mycelia remnant in spent mushroom substrates from A. bisporus and P. ostreatus. Fungi Analysis Inoculation • Compost from goat manure (GC) • Time: 28 days • Wheat Straw (WS) • Dark • Fungi: A.bisporus and • Deinking sludge from paper mil • Room Temperature P.ostreatus • Ergosterol (PW) • Aired every 48 hours for 30 • Amendments: GC, SM, WS, PW • Ligninolitic enzymes activities • Spent mushroom substrate of minutes • Treatments: Sterilized (E) and no P.ostreatus and A.bisporus (SM) • Sampled at 14 and 28 days sterilized (X) Chemical Incubation characterization
Chemical Characterization of amendments • C and N determination Elemental analysis • LECO CHNS ‐ 932 Elemental Chemical Analyzer Electrical Conductivity (EC), • pH and EC determined in the aqueous extract 1:10 (w/v) pH and water ‐ soluble • For WSOC, H 2 SO 4 and K 2 Cr 2 O 7 were added to the aqueous extract to determine the Cr 6+ by valoration with Möhr salt. organic carbon (WSOC) • Microwave digestion Cu and Mn • Determination by atomic absorption spectroscopy ( λ : 324. nm, and 279.5 nm for Cu and Mn respectively) Solid ‐ State nuclear • Bruker AV ‐ 400 ‐ WB unit at 300 K. magnetic resonance, • Samples were packed in a 4 mm diameter zirconium rotor with Kel ‐ F cap, with a rotor spin rate set at 14 kHz. For each sample of about 100 mg, 28,500 scans were accumulated with a relaxation delay of 2 s and 5 ms of contact time. 13 C ‐ CP ‐ MAS NMR
Sample analysis ‐ Ergosterol Sonication washing Extraction Drying Analysis • 0,5 of • Filtered (42 • 3 times with • N 2 sparging • Redisolved sample + 3 nm) and 3 mL of with 3 mL of mL of KOH washed with hexane metanol 10% at 70 o C 3 mL of • HPLC ‐ PDA for 90 min metahnol HPLC Conditions PDA detector (Waters ‐ PDA 996) Column Phenomenex Luna C18 (250 mm x 4.60 mm; particle size 5 µm; pore size 100 Å) Movil phase methanol:water (94:5, v:v) Flow rate 1.0 mL/min Injection volumen 20 µL Time of analysis 30 min UV spectrum 200 ‐ 400 nm
‐ Ligninolytic enzymes Washed in water ‐ 30 mL of Tris ‐ HCl Enzymatic ice (4 o C) bath for Centrifugation for Analysis of 0.1 M pH 7.5 buffer extraction 60 min under 10 min at 5000 rpm supernatant + 1.5 g of sample agitation (160 rpm) Laccase Mn Peroxidase (MnP) Versatil Peroxidase (VP) Analysis Colorimetry ABTS (2,2’ ‐ azino ‐ bis (3 ‐ Mn 2+ to Mn 3+ Oxidation ethylbenzothiazoline ‐ 6 ‐ sulphonic ABTS in presence of H 2 O 2 acid) to ABTS +* λ (nm) 420 270 310 950 µL of malonic sodium malonate 880 µL of potassium acetate buffer 870 µL of potassium acetate buffer buffer pH 4.5, 10 µL of MnSO 4 1 mM, Reactives pH 4.5, 110 µL of ABTS 1.8 M and 10 pH 4.5, 110 µL of ABTS 0.2 mM, 10 µL 10 µL H 2 O 2 10 mM and 30 µL of µL of sample of H 2 O 2 0.1 mM and 10 µL of sample sample
RESULTS
e3 Characterization of solid wastes: Solid state nuclear magnetic resonance ( 13 C CP ‐ MAS NMR) % Area C bond ( δ ) ppm GC (X) SM (X) WS (X) PW (X) Mayor region in the O-alkyl Alkyl (0 ‐ 50) 25.1 16.8 3.8 23.3 fraction belongs to cellulose N ‐ alkyl (50 ‐ 60) 10.9 5.0 3.7 4.0 and hemicellulose WS O ‐ alkyl (60 ‐ 110) 42.9 59.4 81.1 52.4 Aromatic (110 ‐ 140) 5.8 7.7 2.4 11.2 O ‐ aryl (140 ‐ 160) 4.9 6.1 2.9 5.3 Carboxyl (160 ‐ 190) 9.4 4.9 3.0 2.8 C ketone and amide 1.0 0.1 1.1 0.8 (190 ‐ 220) Peak at 173 ppm is associated to de carboxylic C raises with the compost maturation rate GC more maturation rate than the SM
Slide 10 e3 Poner characterization of solid wastes enrique.eymar@uam.es; 14/6/2018
e1 MBMR2 Wastes characteristics ‐ C/N ratio higher in WS GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 ‐ Cu higher in PW while Mn is higher in GC N (%) X 1.5 2.5 0.4 0.4 C/N X 9.5 9.5 109.5 57.5 Cu (mg/Kg) X 10.90 50.29 1.15 195.90 Mn (mg/Kg) X 428.9 400.3 47.86 63.78
Slide 11 e1 qué significa la columna X? Aquyí sin que cuentes nada no veo el efecto de la esterilización que pones en el título enrique.eymar@uam.es; 14/6/2018 MBMR2 Significa no esterilizado MARIA BEGOÑA MAYANS RIVILLA; 14/6/2018
e2 MBMR1 Sterilization effect in the wastes ‐ C/N ratio higher in WS GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 ‐ Cu higher in PW while Mn is higher in GC N (%) X 1.5 2.5 0.4 0.4 C/N X 9.5 9.5 109.5 57.5 ‐ pH: WS more acidic conditions Cu (mg/Kg) X 10.90 50.29 1.15 195.90 Mn (mg/Kg) X 428.9 400.3 47.86 63.78 E 7.58 7.17 5.55 b 8.01 pH X 7.65 7.20 5.82 a 7.76 E 3.24 7.47 a 3.53 a 0.40 EC (dS/m) X 3.38 6.99 b 2.84 b 0.44 E 584.6 a 580.5 a 2347.3 a 304.4 WSOC (mg/kg) X 236.7 b 350.4 b 1831.4 b 282.2
Slide 12 e2 columnas X y E que quiere decir? enrique.eymar@uam.es; 14/6/2018 MBMR1 Son los esterilizados (E) y no esterilizados (X) MARIA BEGOÑA MAYANS RIVILLA; 14/6/2018
Sterilization effect in the substrates ‐ C/N ratio higher in WS GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 ‐ Cu higher in PW while Mn is higherin GC N (%) X 1.5 2.5 0.4 0.4 C/N X 9.5 9.5 109.5 57.5 ‐ pH: WS more acid conditions. Cu (mg/Kg) X 10.90 50.29 1.15 195.90 ‐ Sterilization affects: Mn (mg/Kg) X 428.9 400.3 47.86 63.78 E 7.58 7.17 5.55 b 8.01 pH ‐ EC: increasing in SM and WS a 20 % X 7.65 7.20 5.82 a 7.76 E 3.24 7.47 a 3.53 a 0.40 and a 6.5 % respectively. EC (dS/m) X 3.38 6.99 b 2.84 b 0.44 E 584.6 a 580.5 a 2347.3 a 304.4 WSOC (mg/kg) X 236.7 b 350.4 b 1831.4 b 282.2
Sterilization effect in the substrates ‐ C/N ratio higher in WS GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 ‐ Cu higher in PW while Mn is higherin GC N (%) X 1.5 2.5 0.4 0.4 C/N X 9.5 9.5 109.5 57.5 ‐ pH: WS more acid conditions. Cu (mg/Kg) X 10.90 50.29 1.15 195.90 ‐ Sterilization affects: Mn (mg/Kg) X 428.9 400.3 47.86 63.78 E 7.58 7.17 5.55 b 8.01 pH ‐ EC: increasing in SM and WS a 20 % X 7.65 7.20 5.82 a 7.76 E 3.24 7.47 a 3.53 a 0.40 and a 6.5 % respectively. EC (dS/m) X 3.38 6.99 b 2.84 b 0.44 E 584.6 a 580.5 a 2347.3 a 304.4 ‐ WSOC increases in GC, SM and WS. WSOC (mg/kg) X 236.7 b 350.4 b 1831.4 b 282.2
MBMR3 MBMR4 Er Ergos goster erol ol co conte ntent A. bisporus P. ostreatus (E) 21.0 ± 0.4 c 61 ± 8 c GC (X) 21 ± 1 c 60 ± 5 c ‐ Sterilization: No significant differences (E) 11.5 ± 0.9 b 34 ± 4 ab SM ‐ Fungi: P. ostreatus > A. bisporus (X) 19 ± 1 b 38 ± 11 ab (E) 18 ± 2 b 41 ± 3 b WS ‐ Waste: In both fungi GC > WS > SM > PW (X) 13.7 ± 0.5 b 35 ± 5 b (E) 6.5 ± 0.8 a 19 ± 2 a PW (X) 9.8 ± 0.8 a 24 ± 3 a
Slide 15 MBMR3 MARIA BEGOÑA MAYANS RIVILLA; 14/6/2018 MBMR4 Con el título me refiero a que cada enmienda influye en el nivel de ergosterol, quito lo de esterilización al no haber diferencias? MARIA BEGOÑA MAYANS RIVILLA; 14/6/2018
Ligninolytic activities Laccase ‐ Under non ‐ sterile conditions laccase activities were slightly Sterilized No sterilized a) b) lower than in sterilized 25 25 conditions. t14 t28 20 20 ‐ WS and PW were the most Laccase Activity U/g Laccase Activity U/g effective wastes to preserve 15 15 the laccase activity of both fungi. 10 10 ‐ Laccase activity was not related to ergosterol. 5 5 ‐ Cu concentration could have 0 0 some incidence in the expression of laccase in PW
Mn Peroxidase ‐ Behaviour in the no sterilized a) Sterilized b) No sterilized amendments was similar to the sterilized treatments but with lower values. ‐ P.ostreatus with sterilized straw trends to maintain its enzymatic activity over time, both laccase and MnP. ‐ VP activity was lower than laccase and MnP
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