Organic wastes for biostimulation of Agaricusbisporus and Pleurotus

ostreatusR .   C AMACHO ‐A R É VA LO 1 ,   J .  GÓME Z ‐HE RNÁND E Z 1 ,  B .  MAYAN S 12 ,   R .  AN TÓN 1 ,  C .   E S CO L Á S T I CO 2 ,  C .  GARC I A ‐D E LGADO 3 E .   E YMAR 1

1Department of Agricultural Chemistry and Food Sciences, University Autónoma of Madrid, 28049 Madrid, Spain2Department of Organic and Biorganic Chemistry. National Distance Education University (UNED)

3 Institute of Natural Resources and Agrobiology of Salamanca (IRNASA) Spain

Ligninolytic fungiCharacteristics• 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 fungiCharacteristics• 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

Geneticalmodification

Mushroom cultivation straw

Compost

Deinkingsludgefrom

paper mill

Enzyme production is influence by:

Moisture

C/N ratio

pH (4.5 optimum for fungi growth)

Microbial competition

Objectives and experimental designTo obtain an organic material from agricultural or industrial wastes to revitalize the mycelia remnant in spentmushroom 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)

Chemicalcharacterization

• 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 AnalyzerElemental 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 organic 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 oCfor 90 min

washing

• Filtered (42 nm) and washed with3 mL of metahnol

Extraction

• 3 times with3 mL of hexane

Drying

• N2 sparging

Analysis

• Redisolvedwith 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

OxidationABTS (2,2’‐azino‐bis (3‐

ethylbenzothiazoline‐6‐sulphonic acid) to ABTS+*

Mn2+ to Mn3+ ABTS in presence of H2O2

λ (nm) 420 270 310

Reactives880 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 of H2O2 0.1 mM and 10 µL of sample 

Enzymaticextraction

30 mL of Tris‐HCl0.1 M pH 7.5 buffer + 1.5 g of sample

Washed in water‐ice (4oC) bath for60 min under

agitation (160 rpm)

Centrifugation for10 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 4.0O‐alkyl (60‐110) 42.9 59.4 81.1 52.4

Aromatic (110‐140) 5.8 7.7 2.4 11.2O‐aryl (140‐160) 4.9 6.1 2.9 5.3

Carboxyl (160‐190) 9.4 4.9 3.0 2.8C 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-alkylfraction belongs to celluloseand hemicelluloseWS

Peak at 173 ppm is associated to decarboxylic C raises with the compostmaturation rate GC more maturationrate than the SM

e3

Slide 10

e3 Poner characterization of solid wastesenrique.eymar@uam.es; 14/6/2018

Wastes characteristics

GC SM WS PWC (%) X 14.2 23.7 43.8 23.0N (%) X 1.5 2.5 0.4 0.4C/N X 9.5 9.5 109.5 57.5

Cu (mg/Kg) X 10.90 50.29 1.15 195.90Mn (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

e1MBMR2

Slide 11

e1 qué significa la columna X? Aquyí sin que cuentes nada no veo el efecto de la esterilización que pones en el títuloenrique.eymar@uam.es; 14/6/2018

MBMR2 Significa no esterilizadoMARIA BEGOÑA MAYANS RIVILLA; 14/6/2018

Sterilization effect in the wastes

GC SM WS PWC (%) X 14.2 23.7 43.8 23.0N (%) X 1.5 2.5 0.4 0.4C/N X 9.5 9.5 109.5 57.5

Cu (mg/Kg) X 10.90 50.29 1.15 195.90Mn (mg/Kg) X 428.9 400.3 47.86 63.78

pHE 7.58  7.17  5.55 b 8.01X 7.65  7.20 5.82 a 7.76

EC (dS/m)E 3.24 7.47 a 3.53 a 0.40X 3.38 6.99 b 2.84 b 0.44

WSOC (mg/kg)E 584.6 a 580.5 a 2347.3 a 304.4X 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

e2MBMR1

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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 PWC (%) X 14.2 23.7 43.8 23.0N (%) X 1.5 2.5 0.4 0.4C/N X 9.5 9.5 109.5 57.5

Cu (mg/Kg) X 10.90 50.29 1.15 195.90Mn (mg/Kg) X 428.9 400.3 47.86 63.78

pHE 7.58  7.17  5.55 b 8.01X 7.65  7.20 5.82 a 7.76

EC (dS/m)E 3.24 7.47 a 3.53 a 0.40X 3.38 6.99 b 2.84 b 0.44

WSOC (mg/kg)E 584.6 a 580.5 a 2347.3 a 304.4X 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 PWC (%) X 14.2 23.7 43.8 23.0N (%) X 1.5 2.5 0.4 0.4C/N X 9.5 9.5 109.5 57.5

Cu (mg/Kg) X 10.90 50.29 1.15 195.90Mn (mg/Kg) X 428.9 400.3 47.86 63.78

pHE 7.58  7.17  5.55 b 8.01X 7.65  7.20 5.82 a 7.76

EC (dS/m)E 3.24 7.47 a 3.53 a 0.40X 3.38 6.99 b 2.84 b 0.44

WSOC (mg/kg)E 584.6 a 580.5 a 2347.3 a 304.4X 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

Ergosterol content

‐ Sterilization: No significant differences

‐ Fungi: P. ostreatus > A. bisporus

‐ Waste: In both fungi GC > WS > SM > PW

MBMR3MBMR4

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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

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Laccase Activ

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a) b)

Ligninolytic activitiesLaccase

Sterilized No sterilized

‐ Under non‐sterile conditionslaccase activities were slightlylower than in sterilizedconditions.

‐ WS and PW were the mosteffective wastes to preservethe laccase activity of bothfungi.

‐ Laccase activity was notrelated to ergosterol.

‐ Cu concentration could havesome incidence in theexpression of laccase in PW

Mn Peroxidase

‐ Behaviour in the no sterilizedamendments was similar to thesterilized treatments but with lowervalues.

‐ P.ostreatus with sterilized straw trendsto maintain its enzymatic activity overtime, both laccase and MnP.

‐ VP activity was lower than laccase andMnP

a) Sterilized b) No sterilized

Selection of the most effective treatment

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 theiractivity over time

Peroxidases activities triplicateother treatments

acid pH

High C/N rate

Selection of the most effective treatment

The values of no sterile treatments (X) were lower than the sterile treatment (E). This fact suggested that theamendments should be incubated for at least 14 days in this condition before their application.

P.ostreatus A.bisporus

Enzymatic activity ‐ Persisted over time ‐ ↑ Peroxidase

↑ LaccaseandMnP

Ergosterol ↑More competive and vigorous

ConclusionsWS was the best waste for bioremediation purposes because of its capability of enzymatic stimulation which was also themost persistent over time.

Regardless the specie, PW was the waste which caused more enhanced stimulation of laccase activity due to its high Cucontent, 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 onA.bisporus.

P.ostreatus was competitive and adequate to bioremediation the waste factor had also influence in the enzymatic activity.

Aplications

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)