Hydrolysate analysis

3.2.7.1 Reducing sugars

Reducing sugars were measured using an automated robotic platform (Tecan 200 Liquid Handling Robot) using the high-throughput 3-methyl-2-benzothiazolinonehydrazone (MBTH) assay developed by Gomez et al., (2010). Briefly, a 96 well plate (1.2 ml, VWR) was set up with liquid samples (diluted in 25 mM sodium acetate buffer (pH 4.5)) alongside a series of standards containing 0-200 nmol D-glucose in 75 μl MilliQ water. Into a 96 well PCR plate the autosampler pipetted 75 μl of each sample and standard, 25 μl of NaOH (1 N), 50 μl MBTH reagent (0.21 mg/ml MBTH, 0.7 mg/ml DTT, freshly prepared), 100 μl oxidising reagent (0.5% FeNH4(SO4)2, 0.5% Sulfamic acid and 0.25 N HCl) and 500 μl MilliQ water. Plates were incubated at 60°C for 20 minutes in a PCR machine and then left to cool and develop at RT for 24 hours. Samples were quantified by measuring absorbance at 620 nm. Absorbance readings were plotted for the standards and sugar concentrations of the samples were then calculated based on the line of best fit equation. Two replicate plates were prepared for each iteration of the assay.

𝑺𝑮 =

𝛒_{𝒘𝒂𝒕𝒆𝒓}

SG Specific Gravity of the fluid or substance

ρ

substance density of the fluid or substance, [kg/m

3 ]

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3.2.7.2 Monosaccharides and oligosaccharides

The concentrations of monosaccharides and oligosaccharides in the hydrolysate were quantified by high-performance anion exchange chromatography (HPAEC) as follows: The sterile filtered hydrolysate described in 3.2.2 was serially diluted (1:1500 for monosaccharide analysis and 1:200 for oligosaccharide analysis) in Milli-Q H2O and then 200 µl was filtered through a 0.45 µm filter into tapered HPAEC vials (Dionex). Samples were analysed by HPAEC on an ICS-3000 PAD system with an electrochemical gold electrode using a CarboPac PA20 analytical column (3x150 mm, Dionex) and guard column (3x30 mm, Dionex). Identification and quantification of monosaccharides and oligosaccharides was carried out by comparing retention times and integrated peak areas of the samples to equimolar standard mixtures analysed during the same run under the same conditions. The monosaccharide standard mixture contained: L-fucose, L-arabinose, L-rhamnose, D-galactose, D-glucose, D-xylose, D-mannose, D-galacturonic acid and D- glucuronic acid. The oligosaccharide standard mixture contained: glucose, cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose.

3.2.7.3 Metals

Metal levels in the hydrolysate and the residual solids left over after hydrolysis were analysed in triplicate by ICP-MS as described in 2.2.2.6. Note that the residual solids were dried and ground to powder with a mortar and pestle before digestion. The digestion step was not required for the aqueous hydrolysate samples as the metals were already soluble.

3.2.7.4 Marker inhibitors

Inhibitory compounds commonly found in lignocellulosic hydrolysates were analysed in the OMSW fibre hydrolysate. Furfural, 5-hydroxymethylfurfural (5-HMF), vanillin and levulinic acid were measured by ultra-performance liquid chromatography (UPLC) with mass spectrometric detection (MS). Samples were chromatographically separated on a Waters Acquity I-Class System with VanGuard pre-column with C18 frit (Waters) and a BEH C18 column (100x2.1 mm, 1.7 μm, Waters). All runs were carried out with a gradient (min/%B = 0/16, 2.5/16, 2.8/100, 2.9/100, 3.3/16, 4/16) of solvent A (5% MeOH, 0.1% acetic acid) and solvent B (0.1% acetic acid in MeOH). Injection volume was 2 μl with a flow rate of 0.5 ml/min at 45°C. MS was carried out on a Thermo Endura Triple Quad with HESI positive ion source and single reaction monitoring (SRM) with one transition for each compound (compound, precursor m/z/productm/z: levulinic acid, 99.12/71.22; HMF,

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109.09/81.15; furfural, 97.12/69.22; vanillin, 153.05/93.11). Data was analysed with Thermo Xcalibur 4.0.27.10 software.

A range of organic acids, including acetic, butyric, formic, heptanoic, hexanoic, isobutyric isovaleric, 4-methylvaleric, propionic and valeric acid were measured in the OMSW fibre hydrolysate by gas chromatography (GC) with flame-ionization detection (FID). OMSW fibre hydrolysate was prepared for analysis in triplicate by acidifying 1 ml with 7.5 µl concentrated orthophosphoric acid (Sigma-Aldrich) and run on the GC-FID in parallel alongside a volatile free acid standard (CRM46975, Sigma-Aldrich). The GC-FID set up consisted of a Nukol column (30 m x 0.25 mm, I.D 0.25 µm (24107)) with a helium carrier (30 psi) and liquid injection. Detectors and injectors were operated at 200°C. Temperature was increased from 75-150°C (10°C/min), 150-200°C (20°C/min) and held for 10 minutes.

3.2.7.5 Nutrients

Essential nutrients (nitrogen, phosphate and sulphate) were analysed using commercial testing kits that rely on enzymatic or chemical reactions followed by quantification by spectrophotometry.

Phosphate and sulphate were quantified using standard Hach-Lange Kits designed for water quality testing, in conjunction with a Hach-Lange HT200S High Temperature Thermostat (used to heat samples according to manufacturer’s instructions) and a Hach- Lange DR3900 Spectrophotometer (for automatic test quantification). Total Phosphate and orthophosphate were measured by Hach-Lange LCK350 Phosphate Kit (detection range: 60-60 mg/L PO43- _{and 2-20 mg/L PO4-P), based on the phosphomolybdenum blue assay in} which phosphate reacts with Mo6+ _{under acidic conditions to produce a blue product that} is colorimetrically quantified. Sulphate was measured by Hach-Lange LCK153 Sulphate Kit (detection range: 40-150 mg/L SO42-_{), based on the reaction of barium chloride ions with} sulphate to produce insoluble barium sulphate which can be quantified as a change in turbidity.

Nitrogenous compounds were quantified using enzymatic assay kits by Megazyme. First Ammonia, Urea and L-Arginine (L-Arg) were measured according to manufacturer’s instructions by L-Arginine/Urea/Ammonia Kit (K-LARGE), then primary amino nitrogen (PAN) was measured by Primary Amino Nitrogen Kit (K-PANOPA) according to manufacturer’s instructions. Results from both kits were used to calculate the total Yeast Available Nitrogen (YAN), defined as the total concentration of nitrogen in a fermentation

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that is accessible to the Brewer’s Yeast Saccharomyces cerevisiae. YAN is calculated from total ammonia, urea, L-arginine and PAN, as shown in Equation 3.3.

Equation 3.3: Calculating Yeast Available Nitrogen (YAN)

𝑌𝐴𝑁_{𝑡𝑜𝑡𝑎𝑙} = 1000 × [𝐴𝑚𝑚𝑜𝑛𝑖𝑎 × 14.01 17.03 + 𝑈𝑟𝑒𝑎 × 28.02 60.06 + 𝐿−𝐴𝑟𝑔 × 28.02 174.21 ] + 𝑃𝐴𝑁

Total YAN (YANtotal) is calculated as mg of nitrogen per Litre (mg/L) and the values of each nitrogenous compound (ammonia, urea, L-Arg) are given in g/L. PAN is given in mg/L. Note that each ammonium ion contributes one nitrogen atom (14.01 g/mol), each urea ion contributes two, and each L-arginine contributes three. However, only two nitrogen atoms are counted for L-arginine because its primary amino group is already accounted for as part of the PAN (measured separately).