Or Organi nic wa wastes fo for bios biostim timula lation tion - - PowerPoint PPT Presentation

Or Organi nic wa wastes fo for bios biostim timula lation tion of

• f Agaricus

bisporus and and Pleurotus

• streatus

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

1Department of Agricultural Chemistry and Food Sciences, University Autónoma of Madrid, 28049 Madrid, Spain 2Department 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
• Versatil Peroxidase

Bioremediation

• Oxidation by free radicals
• Degradation of organic pollutans

Edible mushroom

• Residues generation
• Solid waste and mycelia

Degradation improvement Media conditions Genetical modification

Mushroom cultivation

straw Compost Deinking sludge from paper mill

Enzyme production is influence by:

• Moisture
• C/N ratio
• pH (4.5 optimum for fungi growth)
• Microbial competition

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.

• Compost from goat manure (GC)
• Wheat Straw (WS)
• Deinking sludge from paper mil

(PW)

• Spent mushroom substrate of

P.ostreatus and A.bisporus (SM)

Chemical characterization

• Fungi: A.bisporus and

P.ostreatus

• Amendments: GC, SM, WS, PW
• Treatments: Sterilized (E) and no

sterilized (X)

Fungi Inoculation

• Time: 28 days
• Dark
• Room Temperature
• Aired every 48 hours for 30

minutes

• Sampled at 14 and 28 days

Incubation

• Ergosterol
• Ligninolitic enzymes activities

Analysis

Chemical Characterization of amendments

• C and N determination
• LECO CHNS‐932 Elemental Chemical Analyzer

Elemental analysis

• pH and EC determined in the aqueous extract 1:10 (w/v)
• For WSOC, H2SO4 and K2Cr2O7 were added to the aqueous extract to determine the Cr6+ by valoration with Möhr salt.

Electrical Conductivity (EC), pH and water‐soluble

• rganic carbon (WSOC)
• Microwave digestion
• Determination by atomic absorption spectroscopy (λ: 324. nm, and 279.5 nm for Cu and Mn respectively)

Cu and Mn

• Bruker AV‐400‐WB unit at 300 K.
• 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.

Solid‐State nuclear magnetic resonance,

13C‐CP‐MAS NMR

Sample analysis

‐ Ergosterol

Sonication

• 0,5 of

sample + 3 mL of KOH 10% at 70 oC for 90 min

washing

• Filtered (42

nm) and washed with 3 mL of metahnol

Extraction

• 3 times with

3 mL of hexane

Drying

• N2 sparging

Analysis

• Redisolved

with 3 mL of metanol

• HPLC‐PDA

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

Laccase Mn Peroxidase (MnP) Versatil Peroxidase (VP) Analysis Colorimetry Oxidation ABTS (2,2’‐azino‐bis (3‐ ethylbenzothiazoline‐6‐sulphonic acid) to ABTS+* Mn2+ to Mn3+ ABTS in presence of H2O2 λ (nm) 420 270 310 Reactives 880 µL of potassium acetate buffer pH 4.5, 110 µL of ABTS 1.8 M and 10 µL of sample 950 µL of malonic sodium malonate buffer pH 4.5, 10 µL of MnSO4 1 mM, 10 µL H2O2 10 mM and 30 µL of sample 870 µL of potassium acetate buffer pH 4.5, 110 µL of ABTS 0.2 mM, 10 µL

• f H2O2 0.1 mM and 10 µL of sample

Enzymatic extraction 30 mL of Tris‐HCl 0.1 M pH 7.5 buffer + 1.5 g of sample Washed in water‐ ice (4oC) bath for 60 min under agitation (160 rpm) Centrifugation for 10 min at 5000 rpm Analysis of supernatant

RESULTS

C bond (δ) ppm % Area GC (X) SM (X) WS (X) PW (X) Alkyl (0‐50) 25.1 16.8 3.8 23.3 N‐alkyl (50‐60) 10.9 5.0 3.7 4.0 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 (190‐220) 1.0 0.1 1.1 0.8

Characterization of solid wastes: Solid state nuclear magnetic resonance (13C CP‐MAS NMR)

Mayor region in the O-alkyl fraction  belongs to cellulose and hemicellulose  WS Peak at 173 ppm is associated to de carboxylic C  raises with the compost maturation rate  GC more maturation rate than the SM

e3

Slide 10 e3 Poner characterization of solid wastes

enrique.eymar@uam.es; 14/6/2018

Wastes characteristics

GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 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

‐ C/N ratio higher in WS ‐ Cu higher in PW while Mn is higher in GC

e1 MBMR2

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

Sterilization effect in the wastes

GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 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 pH E 7.58 7.17 5.55 b 8.01 X 7.65 7.20 5.82 a 7.76 EC (dS/m) E 3.24 7.47 a 3.53 a 0.40 X 3.38 6.99 b 2.84 b 0.44 WSOC (mg/kg) E 584.6 a 580.5 a 2347.3 a 304.4 X 236.7 b 350.4 b 1831.4 b 282.2

‐ C/N ratio higher in WS ‐ Cu higher in PW while Mn is higher in GC ‐ pH: WS more acidic conditions

e2 MBMR1

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

GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 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 pH E 7.58 7.17 5.55 b 8.01 X 7.65 7.20 5.82 a 7.76 EC (dS/m) E 3.24 7.47 a 3.53 a 0.40 X 3.38 6.99 b 2.84 b 0.44 WSOC (mg/kg) E 584.6 a 580.5 a 2347.3 a 304.4 X 236.7 b 350.4 b 1831.4 b 282.2

‐ C/N ratio higher in WS ‐ Cu higher in PW while Mn is higherin GC ‐ pH: WS more acid conditions. ‐ Sterilization affects: ‐ EC: increasing in SM and WS a 20 % and a 6.5 % respectively.

Sterilization effect in the substrates

GC SM WS PW C (%) X 14.2 23.7 43.8 23.0 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 pH E 7.58 7.17 5.55 b 8.01 X 7.65 7.20 5.82 a 7.76 EC (dS/m) E 3.24 7.47 a 3.53 a 0.40 X 3.38 6.99 b 2.84 b 0.44 WSOC (mg/kg) E 584.6 a 580.5 a 2347.3 a 304.4 X 236.7 b 350.4 b 1831.4 b 282.2

‐ C/N ratio higher in WS ‐ Cu higher in PW while Mn is higherin GC ‐ pH: WS more acid conditions. ‐ Sterilization affects: ‐ EC: increasing in SM and WS a 20 % and a 6.5 % respectively. ‐ WSOC increases in GC, SM and WS.

• A. bisporus
• P. ostreatus

GC

(E)

21.0 ± 0.4c 61 ± 8 c

(X)

21 ± 1 c 60 ± 5c SM

(E)

11.5 ± 0.9b 34 ± 4ab

(X)

19 ± 1 b 38 ± 11ab WS

(E)

18 ± 2b 41 ± 3b

(X)

13.7 ± 0.5b 35 ± 5b PW

(E)

6.5 ± 0.8 a 19 ± 2a

(X)

9.8 ± 0.8 a 24 ± 3a

Er Ergos goster erol

• l co

conte ntent

‐ Sterilization: No significant differences ‐ Fungi: P. ostreatus > A. bisporus ‐ Waste: In both fungi GC > WS > SM > PW

MBMR3 MBMR4

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

5 10 15 20 25

Laccase Activity U/g

5 10 15 20 25

Laccase Activity U/g

t14 t28

a) b)

Ligninolytic activities

Laccase

Sterilized No sterilized

‐ Under non‐sterile conditions laccase activities were slightly lower than in sterilized conditions. ‐ WS and PW were the most effective wastes to preserve the laccase activity of both fungi. ‐ Laccase activity was not related to ergosterol. ‐ Cu concentration could have some incidence in the expression of laccase in PW

Mn Peroxidase ‐ Behaviour in the 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

a) Sterilized b) No sterilized

Selection Selection of the the mos

• st effectiv

ective tr trea eatm tmen ent

Substrates GC and SM Low enzymatic activity Even their high Mn2+ concentracion pH (7.10 ‐7.65) Favours bacterial growth Low C/N rate PW High enzymatic activities pH (7.7 – 8.0) Medium C/N rate WS High enzymatic activities Laccase and MnP mantain their activity over time Peroxidases activities triplicate

• ther treatments

acid pH High C/N rate

Selection Selection of the the mos

• st effectiv

ective tr trea eatm tmen ent

The values of no sterile treatments (X) were lower than the sterile treatment (E). This fact suggested that the amendments should be incubated for at least 14 days in this condition before their application. P.ostreatus A.bisporus Enzymatic activity ‐ Persisted over time ‐ ↑ Peroxidase ↑ Laccase and MnP Ergosterol ↑ More competive and vigorous

Conclusions

WS was the best waste for bioremediation purposes because of its capability of enzymatic stimulation which was also the most persistent over time. Regardless the specie, PW was the waste which caused more enhanced stimulation of laccase activity due to its high Cu content, its presence could affect more than the specie factor in laccase activity. The sterilization of amendments did not have influence in fungi growth of P.ostreatus specie but it had a little impact on A.bisporus. P.ostreatus was competitive and adequate to bioremediation the waste factor had also influence in the enzymatic activity.

Apl Aplicati tions

• ns

Biofilters

Removal of antibiotics (Poster 52)

Straw inoculated with Pleurotus Biofilters Development Further research has been done in different media conditions and with more fungi species. (Poster 51 and 69)