Both edible (e. g. rapeseed, soybean or palm) and nonedible (e. g. Jatropha) oilseeds can be used for bio­diesel production. Biodiesel production is mainly achieved from soybean in the USA, rapeseed (or sun­flower in lower quantities) in Europe and palm oil in South-East Asian countries. Biodiesel production could be also achieved using nonconventional resources including microbial oil produced by yeast, fungi and algae (Meng et al., 2009). The continuous growth of bio­diesel production coincides with proportional produc­tion of by-products streams. The main by-product is glycerol that is generated during transesterification of triglycerides with predominantly methanol leading to the production of fatty acid methyl esters and glycerol (10%, w/w). It has been estimated that by 2021, the share of biodiesel production from vegetable oils will increase and the worldwide biodiesel production from such oils is projected to reach approximately 30 x 106 t (Anonymous, 2012c). This means that approximately 3 x 106 t of glycerol will be available for chemical and biopolymer production. Crude glycerol streams produced from biodiesel plants have purities in the range of 77—90% (w/w) (Mothes et al., 2007). The main impurities in crude glycerol are water, methanol, residual fatty acids and corresponding esters, and salts (NaCl or K2SO4) in varying proportions depending on the extent of glycerol purification (Mothes et al., 2007). Purification methods for glycerol have been proposed (Chatzifragkou and Papanikolaou, 2012) for the removal of impurities and salts after biodiesel production but they seem to be rather unprofitable, especially for small industries. Novel uses of glycerol involve both green chemical conversions and microbial bioconversions. Glycerol represents an easily assimilated carbon source for many microorganisms. Crude glycerol has been eval­uated as carbon source for various microbial bioconver­sions, such as 1,3-propanediol, citric acid, ethanol, succinic acid, propionic acid, microbial oil and PHAs (Koutinas et al., 2007a; da Silva et al., 2009; Koutinas and Papanikolaou, 2011; Sarris et al., 2011).

Biodiesel production from oilseeds leads to the pro­duction of oilseed meals as a valuable by-product stream. Oilseed meal is the protein — and carbohydrate — rich fraction that remains after the extraction of oil. The main conventional commercial outlet for oilseed meals is as animal feed. In the period 2012—2021, bio­diesel production from edible vegetable oils will still rely mainly on rapeseed and sunflower. However, bio­diesel production from palm oil is projected to increase twofold (Anonymous, 2012c). Based on recent estimates, approximately 315 x 106 t of oilseed meals are expected to be produced by 2021, corresponding to an increase up to 23% based on current production capacities (Anony­mous, 2012c). Future biodiesel industries could be con­verted into novel biorefineries through valorization of crude glycerol and oilseed meal streams leading to the production of biodiesel, chemicals, food and feed ingre­dients and biopolymers such as PHAs.

Ashby et al. (2004, 2011) evaluated the production and properties of PHAs accumulated by the bacterial strains Pseudomonas oleovorans NRRL B-14682 and Pseu­domonas corrugata 388 cultivated on crude glycerol. Ashby et al. (2011) reported that the molecular weight of PHB was decreased with increasing methanol concen­tration in crude glycerol. Mothes et al. (2007) and Garcia et al. (2013) evaluated the effect of NaCl and K2SO4 on PHA production during fermentation with the bacterial strains Paracoccus denitrificans, C. necator JMP 134 and C. necator DSMZ 545. These salts are present in crude glycerol depending on the catalyst (NaOH or KOH) employed during transesterification of triglycerides. The inhibition caused by NaCl on PHA production is more pronounced at significantly lower concentrations than K2SO4. Mothes et al. (2007) reported that bioreactor fermentations with C. necator JMP 134 cultivated on crude glycerol and inorganic chemicals as additional nu­trients could lead to the production of PHB contents up to 70% (w/w). Crude glycerol has also been employed in bioreactor fermentations for the production of PHB us­ing the bacterial strain C. necator DSM 545 leading to 50% (w/w) PHB content and 1.1 g/l h PHB productivity (Cavalheiro et al., 2009). Tanadchangsaeng and Yu (2012) stressed that the productivity (around 0.92 g/l) of glyc­erol fermentation to PHB synthesis is lower than the one achieved from glucose. Crude glycerol could be also combined with other carbon sources that could be used as precursors for the production of PHA co­polymers (Cavalheiro et al., 2012). The production of P(3HB-co-4HB-co-3HV) was reported when C. necator DSM 545 was cultivated on crude glycerol, propionic acid (stimulator of 4HB accumulation and 3HV precur­sor) and g-butyrolactone (4HB precursor). In all studies presented above, inorganic chemicals were used as nutrient supplements.

Apart from fermentation efficiency of PHA produc­tion, it is also crucial to assess the properties of the poly­mer produced and the associated production cost. Tanadchangsaeng and Yu (2012) reported that although the thermal and physical properties of the PHB pro­duced from glycerol is similar to the one produced from glucose, the molecular weight of the glycerol — derived homopolymer is lower than the molecular weight of the PHB produced from glucose. Posada et al. (2011) presented a comparative technoeconomic evaluation of PHB production from crude glycerol using two different bacterial strains, C. necator and B. megate — rium, and three different downstream separation strate­gies. Fermentation of C. necator resulted in the production of 81.6 g/l of which 57.1 g/l was PHB, significantly higher than B. megaterium. The most cost — competitive process involved PHB production in fed — batch fermentations with C. necator followed by PHB separation and purification with heat pretreatment, enzymatic alkaline digestion, centrifugation, washing, evaporation, and spray drying. Posada et al. (2011) re­ported also that glycerol purification to 98% (w/w) con­tributes approximately 6% of the overall PHB production cost, thereby slightly affecting the total cost. In this study, it was concluded that the PHB production cost from crude glycerol could be as low as US$2/kg depending on the downstream process utilized.

PHA production from crude glycerol could be com­bined with the valorization of oilseed meals or residues remaining after extraction of microbial oil. For instance, rapeseed meal could be utilized for the production of various value-added fractions including protein iso­lates, carbohydrates, hulls, phenolic compounds and glucosinolates with various applications such as animal feed, pesticidal agent, bioactive proteins, glues and ad­hesives, paper coatings and ingredients for cosmetics among others (Anonymous, 2011; Egues et al., 2010). Another alternative application of oilseed meals is based on the production of complex nutrient supplements for fermentation processes including PHA production. In this way, commercial inorganic chemicals will be replaced improving the sustainability of the whole bio­refinery concept. Oilseed meals contain significant quantities of protein, minerals and other necessary nu­trients for microbial growth. Enzymatic hydrolysis of protein to amino acids and peptides, and phytic acid to phosphorus could provide a hydrolysate suitable for PHA production. Crude enzymes could be produced via solid-state fermentation employing appropriate fungal strains and oilseed meals as substrates (Wang et al., 2010; Kachrimanidou et al. 2013). Wang et al.

(2010) reported the production of a nutrient-rich hydro­lysate from rapeseed meal with a free amino nitrogen content of 2016.2 mg/l and inorganic phosphorus (IP) of 304 mg/l that was subsequently used successfully as nutrient supplement combined with glucose as car­bon source for the cultivation of Saccharomyces cerevisiae. Garcia et al. (2013) investigated the generation of a mi­crobial feedstock through hydrolysis of rapeseed meal, which was combined with crude glycerol as the sole fermentation medium for PHA production. Fed-batch fermentations resulted in a production of 10.9 g/l P(3HB-co-3HV) without addition of any precursor. The properties of the biopolymer produced were also exam­ined, leading to the conclusion that this bioprocess could be incorporated in rapeseed-based biodiesel plants contributing to the sustainability of biodiesel bio­refineries. Kachrimanidou et al. (2013) reported the uti­lization of sunflower meal for the production of nutrient-rich hydrolysates that could be subsequently supplemented with crude glycerol for the production of 9.9 g/l P(3HB-co-3HV) with a PHA content of 50% (w/w) in shake-flask fermentations using the microbial strain C. necator DSM 545 without addition of any precursor.

Figure 24.2 presents a biorefinery concept in which sunflower (or other oilseed) meal is utilized only for the production of fermentation feedstock involving pro­duction of crude enzymes via solid-state fermentation followed by hydrolysate production via enzymatic hy­drolysis. Preliminary bioreactor fermentations carried out in fed-batch mode at the Agricultural University of Athens with the microbial strain C. necator DSM 7237 cultivated on sunflower meal hydrolysate and crude glycerol lead to the production of more than 20 g/l

PHB with a PHB content of approximately 70% (w/w). However, this processing scheme does not take advan­tage of the full potential of sunflower meal that contains value-added ingredients that could be isolated contrib­uting in the development of a more sustainable bio­refinery approach.

Figure 24.3 presents a sunflower-based biorefinery where besides fermentation feedstock, sunflower meal is also used for the production of an antioxidant-rich stream and a protein isolate product. The sunflower seed is covered by the hull that could be removed before oil separation by mechanical pressing and solvent extraction in biodiesel production processes. The protein content in sunflower meals can be increased via dehul — ling and complete oil extraction. The composition of

sunflower meal is variable and is highly dependent on cultivation conditions, sunflower variety and the indus­trial process used for biodiesel production. Dehulling or partial dehulling of sunflower seeds could provide a by­product that could be used for the production of energy, hemicelluloses, organic amendment for the soils, and biomaterial (Anonymous, 2011). The sunflower meal that remains as a by-product after (partial) dehulling and complete oil extraction could be fractionated in three different fractions (i. e. a protein-rich fraction, a lignocellulose-rich fraction and a liquid fraction) by a simple sedimentation/flotation process based on the formation of an aqueous suspension (Bautista et al., 1990; Parrado et al., 1991). This separation is based on the different densities of major components in sunflower

meal. Subsequently, antioxidants can be removed from the protein-rich fraction, as well as from the lignocellu — losic fraction. The most important of the phenolic com­pounds found in sunflower is chlorogenic acid.

The protein isolate extracted from the protein-rich fraction, after treatment with acid and alkaline solutions, can be utilized for the production of biopolymers and edible films. Yust et al. (2003) improved protein extrac­tion from sunflower meal through treatment with alka — lase. Remaining fractions can be used as substrate in solid-state fermentation with a fungal strain of Asper­gillus oryzae, an efficient producer mostly of proteolytic enzymes. The solids at the end of solid-state fermenta­tion can be used as enzyme-rich medium for hydrolysis of macromolecules contained in remaining sunflower. The liquid fraction from sunflower meal fractionation can be used as suspension liquid in enzymatic hydroly­sis, aiming at the generation of a nutrient-rich supple­ment. At the end of hydrolysis, remaining solids are separated from the hydrolysate by centrifugation, and can be possibly used for combustion to generate heat or as a carbohydrate-rich resource for the production of hydrolysates for other fermentations. The nutrient — rich supplement has been used successfully for enhanced production of PHB. The advanced biorefinery concept results in the production of three products (an­tioxidants, protein isolate and PHB) from the same raw material presenting a high potential of improved pro­cess economics.