*3.1. Materials*

Paper sludge was derived from Norway spruce (*Picea abies*) as residual fibre from a sulphite process (Domsjö Fabriker AB refinery, Örnsköldsvik, Sweden). All the chemicals and substrates (Carboxymethyl cellulose (CMC-Na), Avicel, *p*-nitrophenyl-glucopyranoside (*p*NPG) and *p*-nitrophenyl-cellobioside (*p*NPC)) were purchased from Sigma Aldrich (St. Louis, MI, USA) unless stated otherwise. The 1, 4-β-<sup>d</sup>-cellopentaitol was obtained from Megazyme™ (Ireland). The enzyme cocktails (Celluclast® 1.5 L, Cellic CTec2 ® and Viscozyme ® L) were supplied by Novozymes A/S (Denmark). Amicon Pro Centrifugal filters (10 K) were purchased from MERCK (South Africa).

#### *3.2. Paper Sludge Moisture Content Determination*

Exactly 10 g of the paper sludge was added to a dry pre-weighed aluminium weighing boat and placed in an oven at 50 ◦C until a constant weight was achieved. The *% moisture content* of the paper sludge was calculated as follows:

$$\% \text{ moisture content} = \frac{\text{biomass wet weight} - \text{biomass dry weight}}{\text{biomass dry weight}} \times 100\% \tag{1}$$

#### *3.3. Paper Sludge Chemical Composition*

The paper sludge was characterised using a modified sulphuric acid method by the National Renewable Energy Laboratory (NREL) (Golden, CO, USA) [48]. After the two-step acid hydrolysis, the biomass was filtered with the filtrate analysed with regards to acid-soluble lignin (ASL) and monosaccharide content using UV–V is spectroscopy and HPAEC, respectively, while the solid residue was set aside for Klason lignin and ash content estimation. The mass of the solid residue was measured after drying overnight at 105 ◦C to estimate the Klason lignin content. The mass of the solid residue used for Klason lignin was ignited in a mu ffle furnace at 525 ◦C for 6 h, cooled in a desiccator, and weighed to determine the ash content of the paper sludge.

#### *3.4. Paper Sludge Crystallinity Index (CrI)*

The crystallinity of paper sludge was determined by X-ray di ffraction using Cu K radiation (1.5405 Å, nickel filter) on a Bruker D8 ® Discover (Bruker, United Kingdom) equipped with a proportional counter. The sample was scanned from 2θ of 10 to 40◦ with a step size of 0.02◦. The determination time was 0.02◦ per second. The crystallinity index (*CrI*) was then defined as follows:

$$\text{CrI} = \frac{I\_{002} - I\_{am}}{I\_{002}} \times 100\tag{2}$$

where *I*002 and *Iam* are the intensities of di ffraction at 2θ 22.6◦ (crystalline portion) and 18.9◦ (amorphous portion), respectively.

#### *3.5. Simon's Staining (SS) of Paper Sludge*

The accessibility of cellulose contained in paper sludge to cellulases was assessed using a modified version of Simon's Stain technique with Direct Orange 15 (DO) dye as an adsorbent as described previously [49]. An increase in the DO dye absorption was proportional to increased cellulose accessibility [50]. SS (mg/g pulp) represents the amount of DO dye absorbed by paper sludge.

#### *3.6. Media, Yeast Strains and Culture Conditions*

The laboratory *S. cerevisiae* Y294 strain was used as a parental strain for the constitutive expression of the di fferent cellulase genes. The *S. cerevisiae* Y294 [*Tr*EGII], Y294 [*Te*CBHI], Y294 [*Cl*CBHIIb] and Y294 [*Pc*BGLIIB] strains express the recombinant mono-component cellulase genes which produced the *Trichoderma reesei* endoglucanase II (*Tr*Cel5A) [12], the *Talaromyces emersonii* cellobiohydrolase I (*Te*Cel7A) with a carbohydrate binding module (CBM) attached to its carboxyl terminal (Tecbh1-TrCBM-C [11,12], the *Chrysosporium lucknowensis* cellobiohydrolase II (*Cl*Cel6A) [11] and *Phanerochaeta chrysospoprium* β-glucosidase (*Pc*BGL1B) [25], hereafter referred to as EGII, CBHI, CBHII and BGL, respectively.

SC-URA growth medium was prepared in double strength (13.4 g/<sup>L</sup> yeas<sup>t</sup> nitrogen base without amino acids (BD-Diagnostic Systems, Sparks, MD, USA), 20 g/<sup>L</sup> glucose, and 3 g/<sup>L</sup> yeas<sup>t</sup> synthetic dropout medium supplements). The aerobic cultivation of *S. cerevisiae* Y294 strains was performed

on a rotary shaker (200 rpm) at 30 ◦C in 500 mL Erlenmeyer flasks containing 100 mL medium. The supernatant of the individual strains was harvested after 72 h of cultivation through centrifugation for 10 min at 4000× *g*.

#### *3.7. Preparation of Partially Purified Enzymes*

Approximately 15 mL of crude cellulase supernatants were ultra-filtrated using an Amicon Ultra-15 Centrifugal device with a 10 kDa cut o ff membrane. The samples were centrifuged at 4000× *g* for 20 min at 4 ◦C using a swinging-bucket rotor in the Heraeus Megafuge 1.0R (ThermoFischer Scientific, Waltham, MA, USA). The 10 kDa retentate (approximately 0.5 mL) were diluted to 1.5 mL using 50 mM sodium citrate bu ffer (pH 5) and stored at 4 ◦C.

#### *3.8. Protein Content and Purity Determination*

The protein content of the yeast-derived cellulases was determined by the Bradford method with bovine serum albumin (BSA) used as a suitable protein standard [51]. To determine purity, protein samples were subsequently separated by electrophoresis on denaturing 12% SDS-PAGE gels according to a protocol described previously [52]. Coomassie Brilliant Blue R-250 staining was used to visualise the gels.
