Valorisation of rapeseed meal for microbial astaxanthin production Department of Food and Nutritional Sciences Valorisation of rapeseed meal for microbial astaxanthin production Zuharlida Tuan Harith, A. Chatzifragkou, D. Charalampopoulos University of Reading

Food processing industry Out of date fruits/vegetables BIOREFinery concept Agriculture waste Food processing industry Municipal waste Out of date fruits/vegetables Bioprocessing bioplastics biofuel Pharmaceuticals Cosmetics Biorefining is the sustainable processing of biomass into a spectrum of marketable products and energy” The biorefinery concept embraces a wide range of technologies able to separate biomass resources (wood, grasses, corn…) into their building blocks (carbohydrates, proteins, triglycerides…) which can be converted to value added products, biofuels and chemicals. A biorefinery is a facility (or network of facilities) that integrates biomass conversion processes and equipment to produce transportation biofuels, power, and chemicals from biomass. This concept is analogous to today’s petroleum refinery, which produces multiple fuels and products from petroleum. Francesco Cherubini (2010) Feed Chemicals

Rapeseed cultivation Brassica napus L UK is the 3rd largest producer after Germany and France 700, 000 ha of UK land used for rapeseed cultivation annually Major oilseed cultivated in the EU Products: oil, biodiesel, engine lubricants Brassica napus L

Rapeseed meal (RSM) Value Production: 39.67 Mt per annum Price: $226/ton Current applications Feed Fertiliser Compost Opportunity Protein source Microbial conversion

RSM composition Rapeseed meal – rich in protein, cellulose, hemicellulose Need to be hydrolysed into assimilable, simple sugars and amino acids Lignocellulosic biomass has three major components: cellulose, hemicellulose and lignin. Cellulose (C6H10O6)n has a strong molecular structure made by long chains of glucose molecules (C6 sugar). Hemicellulose (C5H8O5)n is a relatively amorphous component that is easier to break down with chemicals and/or heat than cellulose; it contains a mix of C6 and C5 sugars. It is the second main component of lignocellulosic biomass (20–40% of total feedstock dry matter). Lignin (C9H10O2(OCH3)n), is essentially the glue that provides the overall rigidity to the structure of plants and trees and is made of phenolic polymers. While cellulose and hemicellulose are polysaccharides that can be hydrolyzed to sugars and then fermented to ethanol, lignin cannot be used in fermentation processes, but it may be useful for other purposes (chemical extraction or energy generation). Lignin (15–25% of total feedstock dry matter) is the largest non-carbohydrate fraction of lignocellulose. Basic plant cell wall structure

One of most important carotenoids Market – projection 1.1 billion USD in 2020 (MRPD, 2015) Found in salmon, crustaceans, trouts, flamingo – sourced through diet Sources: Haematococcus pluvialis; Xanthophyllomyces dendrorhous; synthetically produced via Wittig reaction & Grignard reaction Shrimp shells from shrimp processing waste Benefits: antioxidant , Vitamin A precursor, food colourant

The Yeast Aim of study Xanthophyllomyces dendrorhous DSMZ 5626 Previously known as Phaffia rhodozyma Major producer of astaxanthin-90% of total carotenoids Aim of study To investigate the feasibility of microbial astaxanthin production by Xanthophyllomyces dendrorhous DSMZ 5626 using rapeseed meal hydrolysates as fermentation nutrients.

Research scheme

GROWTH OFX. dendrorhous on refined sugars Carbon source (30 g/l) Time (h) Biomass (g/l) Ast (mg/l) Y Ast/x (mg/g) Y Ast/s (mg/g) µmax (h-1) Cellobiose 120 13.3 (0.2) 4.24 (0.22) 319.70 (11.8) 0.14 (0.01) 0.19 Xylose 112 12.8 (0.2) 3.76 (0.19) 294.67 (10.1) 0.15 (0.01) 0.17 Fructose 11.1 (0.0) 3.47 (0.02) 312.38 (2.07) 0.13 (0.01) Arabinose 115 10.7 (1.2) 1.87 (0.02) 174.75 (1.23 0.07 (0.01) 0.11 Galactose 13.3 (1.3) 0.57 (0.04) 46.51 (3.23) 0.12 (0.01) 0.12 Glucose 117 9.13 (0.6) 2.32 (0.03) 255 (15) 0.08 (0.01) 0.16 Glycerol 88 8.55 (0.2) 2.88 (0.01) 337 (5.0) 0.16 (0.01) 0.13

GROWTH OFX. dendrorhous on refined sugars B Specific growth rate, µ (h-1) of X. dendrorhous when cultivated using different initial concentrations of (A) glucose and (B) glycerol.

Rapeseed MeAL COMPOSITION

Enzymatic hydrolysis of Rapeseed MeAL Effect of different enzyme concentrations on sugar release after 24 h of reaction using Viscozyme (A), pectinase (B), Acellerase 1500 (C )and cellulase (D)

X. dendrorhous GROWTH PROFILE IN BIOREACTOR X. dendrorhous cultivated in pectinase-derived rapeseed meal hydrolysate

Rapeseed MeAL hydrolysates as substrates A. Batch flask: RSM hydrolysate Time (h) Biomass (g/l) Ast (mg/l) YAst/x (mg/g) EtOHmax Viscozyme L 115 3.07 ± 0.07 0.4 ± 0.03 156 ± 11.75 3.8 ± 0.13 Cellulase 14.63 ± 0.64 1.10 ± 0.18 71 ± 11.4 6.92 ± 0.00 Pectinase 120 25.83 ± 1.52 6.71 ± 0.44 258 ± 1.83 - Acellerase 118 8.2 ± 0.01 2.69 ± 0.05 332 ± 12 2.02 ± 0.15 B. 2-L batch bioreactor: RSM hydrolysates Time (h) Biomass (g/l) Ast (mg/l) YAst/x (mg/g) Cellulase 120 25.3 ± 0.28 7.66 ± 0.04 302.70 ± 1.69 Pectinase 39.75 ± 0.07 10.78 ± 0.15 271.12 ± 4.23 Acellerase 112 14.3 ± 0.85 3.59 ± 0.14 250.93 ± 5.23

conclusions Rapeseed meal is a rich by-product that can be valorised for the production of high value pigments via microbial conversion. Screening of enzymes is essential in determining the hydrolysis rate of rapeseed meal. The production of microbial pigments using renewable biomass residues is feasible. Its optimisation could replace the domination of synthetically produced astaxanthin.

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