IN-SITU XYLANASE PRODUCTION USING AGRO-INDUSTRIAL RESIDUES

FOR LIGNOCELLULOSIC ETHANOL 4-CIAB - 4th IBEROAMERICAM CONGRESS ON BIOREFINERIES 24-28 October 2018, Jaen, Spain

Susana Marques1, Ekaterina Vorobieva1, João Tavares1, Nuno Faria1,2, César Fonseca3 & Francisco Gírio1

1 Bioenergy Unit, Laboratório Nacional de Energia e Geologia, I.P., Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal, francisco.girio@lneg.pt 2 Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Univ. Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

3Section for Sustainable Biotechnology, Department of Chemistry and Bioscience, Aalborg University, A. C. Meyers Vænge 15, 2450 Copenhagen SV, Denmark

On-site production of cellulase-free (FPase < 0.2 U/mL) hemicellulases exhibiting high levels of

xylanase activities was successfully achieved with M. antarcticus and M. aphidis using extrudates

In-House Hemicellulases were validated for selective xylan hydrolysis of BS and SCC extrudates

The supplementation of Cellic CTec2 with these in-house Hemicellulases allowed high yields

(close to 80%) for hydrolysis of cellulose and xylan on BS and SCC extrudates at high solids

loadings – up to 33% (w/v) for SCC

Acknowledgements

This work has been co-funded by the European Commission - Horizon 2020 Program under Grant agreement reference

no. 654362 (BABET-REAL5 Project).

The authors acknowledge the BABET-REAL5 partners CIEMAT, INPT and UNAM for providing pre-treated samples.

Reference

[1] Faria NT, Santos M, Ferreira C, Marques S, Ferreira FC, Fonseca C (2014) “Conversion of cellulosic materials into glycolipid

biosurfactants, mannosylerythritol lipids, by Pseudozyma spp. under SHF and SSF processes”, Microb Cell Factories, 13: 155.

Background and Aim Enzymes cost still constitutes one of the major barriers to commercial implementation of

lignocellulosic ethanol. Therefore, search for more efficient and cost-effective enzymes is

desirable. Given the market availability of efficient commercial cellulases, focus should be given to

novel effective xylanases to boost hemicellulose conversion.

In the present work, two strains of Moesziomyces spp. - M. antarcticus and M. aphidis,

previously reported [1] as efficient xylanase producers - have been used to produce

hemicellulases using agro-industrial residues as inexpensive inducing substrates. Cellulase-

free xylanases were produced by both strains using Barley Straw (BS), Sweet Corn Cobs (SCC)

and Blue Agave Bagasse (BAB) pretreated through mild alkaline extrusion. The performance of

the in-house hemicellulases was assessed, in supplementation of Cellic CTec2, on the hydrolysis

of the same substrates under high solids loadings.

Experimental

SUBSTRATE

Barley Straw (BS), Sweet Corn Cobs (SCC) and Blue Agave Bagasse (BAB) previously pre-treated through

mild alkaline extrusion

HEMICELLULASES PRODUCTION

Strains: Moesziomyces antarcticus PYCC 5048T (former Pseudozyma antarctica)

Moesziomyces aphidis PYCC 5535T (former Pseudozyma aphidis)

Cultivation conditions: T=28C; 140 rpm; Inoculum (10% v/v) 48h-growth on 40 g/L Glucose

Basal culture medium: MgSO4 0.3 g/L, KH2PO4 0.3 g/L, NaNO3 3 g/L, Yeast extract 1 g/L

ENZYMATIC HYDROLYSIS

Initial extrudate loading: 5-33.3% (w/v) (oven-dried weight basis)

Enzyme Formulation: - Cellic CTec2 (1-50 FPU/gextrudate) supplemented with In-House Hemicellulase or

NS22002 (20-250 U Xylanase/gextrudate); commercial enzymes kindly provided by Novozymes A/S

Stirring: 150 rpm; T=50ºC; pH 5.5 (0.05 M sodium citrate buffer); 0.02% sodium azide; Time=72 hours

Hydrolysis Yield: based on the sugars - glucose (G) or xylose (X) - released from the corresponding

polysaccharide (glucan or xylan) in the initial substrate

ANALYTICAL ASSAYS

Xylanase Activity: with beechwood Xylan (Megazyme); T=50ºC; pH 5.5; expressed in international units (U)

b-Xylosidase Activity: with p-NPXp (Sigma); T=50ºC; pH 5.5; expressed in international units (U)

Cellulase (FPase) Activity: with Whatman #1 filter paper; T=50ºC; pH 5.5; expressed in international units (U)

Protein: with PierceTM BCA (Bicinchoninic acid) Protein Assay Kit

HPLC: with Bio-Rad Aminex HPX-87H column (eluent - H2SO4 5 mM; flow rate - 0.4 mL/min; T=50ºC)

(characterization by HPLC using Aminex HPX-87H (BIO-RAD) column,

following 2-steps complete acid hydrolysis)

1. PRODUCTION OF EXTRACELLULAR HEMICELLULASES Microorganism Inducer Substrate [Xyl]equivalents

(g/L)

Xylanase

Activity

(U/mL)

Xylanase/

b-Xylosidase

(ratio)

[Protein]

(mg/L)

M. antarcticus Xylose 10 13.8 2.4 > 104 1339

20 73.4 2.9 > 104 916

BS extrudate 10 (37 gDM/L) 73.0 3.7 29 4552

20 (74 gDM/L) 14.5 1.7 248 9129

SCC extrudate 10 (34 gDM/L) 99.6 0.6 173 3377

20 (68 gDM/L) 101.0 8.7 95 7028

BAB extrudate 10 (48 gDM/L) 62.3 3.7 107 1838

20 (96 gDM/L) 6.8 0.2 9 4085

M. aphidis Xylose 10 31.7 2.4 > 103 659

20 81.4 7.0 > 103 1172

BS extrudate 10 (37 gDM/L) 105.3 3.2 59 4124

20 (74 gDM/L) 163.3 0.7 76 8347

SCC extrudate 10 (34 gDM/L) 112.6 8.3 209 3328

20 (68 gDM/L) 210.6 3.4 280 7006

BAB extrudate 10 (48 gDM/L) 78.9 8.4 142 1736

20 (96 gDM/L) 17.9 2.2 32 3723

High levels of extracellular

xylanase activities

M. aphidis > M. antarcticus

SCC > BS > BAB > Xylose

as C-inducers

2. ASSESSMENT OF IN-HOUSE HEMICELLULASES

2a. HYDROLYSIS of BS EXTRUDATE boosted by IN-HOUSE HEMICELLULASES

Hydrolysis at increasing solids loadings (5-15% (w/v)) of BS Extrudate with Cellic CTec2 at 15 FPU/gextrudate

supplemented with extracellular extracts of M. antarcticus (=) and M. aphidis (ll) grown on Xylose (Xyl), Barley Straw

(BS), Sweet Corn Cobs (SCC) or Blue Agava Bagasse (BAB) extrudates bench-marking against commercial

hemicellulase NS22002 (NS) at a dosage of 100 U Xylanase/gextrudate

2b. HYDROLYSIS of SCC EXTRUDATE boosted by IN-HOUSE HEMICELLULASES PRODUCED BY M. aphidis

Hydrolysis at increasing solids loadings (15-33% (w/v)) of SCC Extrudate with Cellic CTec2 at dosages of 5, 10, 15 or 50 FPU/gextrudate supplemented with

extracellular extracts of M. aphidis grown on Barley Straw (BS) or Sweet Corn Cobs (SCC) extrudates

bench-marking against commercial hemicellulase NS22002 (NS) at dosages of 20-250 U Xylanase/gextrudate

Conversion to Xylose is boosted by Hemicellulases

Higher effect with Hemicellulases in-situ produced using the same SCC extrudate

Xylose Yield increases with Hemicellulase and Cellic CTec2 dosages

In-House Hemicellulases promote no significant effect on conversion to Glucose

due to absence of Cellulase activity, contrarily to NS2202 exhibiting Cellulase activity

Glucose Yield increases with Cellic CTec2 dosage

Conversion to Xylose is boosted by Hemicellulases

Higher effect with Hemicellulases in-situ produced using the same BS extrudate

Effect more noticeable for Hemicellulases produced by M. aphidis

In-House Hemicellulases produced by M. aphidis on BS extrudate (even lacking

Cellulase activity) promote a slight effect on conversion to Glucose

comparable to the effect of NS2202 (exhibiting Cellulase activity)