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Development of Microbial and Enzymatic Processes for Valorisation of Industrial Side Streams for the Production of Microbial Lipids and Esters

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Development of Microbial and Enzymatic Processes for Valorisation of Industrial Side Streams for the Production of Microbial Lipids and Esters Agricultural University of Athens Department of Food Science and Human Nutrition Laboratory of Food Process Engineering Development of microbial and enzymatic processes for valorisation of industrial side streams for the production of microbial lipids and esters A thesis submitted for the degree of Doctor of Philosophy by Erminta L. Tsouko ATHENS 2019 SUPERVISOR Doctor Apostolos Koutinas Agricultural University of Athens Department of Food Science and Human Nutrition Laboratory of Food Process Engineering Development of microbial and enzymatic processes for valorisation of industrial side streams for the production of microbial lipids and esters A thesis submitted for the degree of Doctor of Philosophy by Erminta L. Tsouko ATHENS 2019 SUPERVISOR Doctor Apostolos Koutinas ii DOCTORAL DISSERTATION Development of microbial and enzymatic processes for valorisation of industrial side streams for the production of microbial lipids and esters Thesis committee Thesis supervisor Dr. Apostolos Koutinas, Assistant Professor, Agricultural University of Athens Thesis co-supervisors Serafeim Papanikolaou, Associate Professor, Agricultural University of Athens Ioannis Kookos, Professor, University of Patra Other members Ioanna Mantala, Associate Professor, Agricultural University of Athens Vasiliki Evaggeliou, Associate Professor, Agricultural University of Athens Nikolaos Stoforos, Professor, Agricultural University of Athens Denise M.G Freire, Professor, Federal University of Rio de Janeiro, Brazil iii Γεωπονικό Πανεπιστήμιο Αθηνών Τμήμα Επιστήμης Τροφίμων και Διατροφής του Ανθρώπου Εργαστήριο Μηχανικής και Επεξεργασίας Τροφίμων Ανάπτυξη μικροβιακών και ενζυμικών διεργασιών για την αξιοποίηση παράπλευρων βιομηχανικών ρευμάτων προς παραγωγή μικροβιακών ελαίων και βιογενών εστέρων ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ ΕΡΜΙΝΤΑ Λ. ΤΣΟΥΚΟ ΑΘΗΝΑ 2019 ΕΠΙΒΛΕΠΩΝ ΚΑΘΗΓΗΤΗΣ Απόστολος Κουτίνας, Αναπληρωτής Καθηγητής Γ.Π.Α iv ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ Ανάπτυξη μικροβιακών και ενζυμικών διεργασιών για την αξιοποίηση παράπλευρων βιομηχανικών ρευμάτων προς παραγωγή μικροβιακών ελαίων και βιογενών εστέρων ΕΠΙΒΛΕΠΩΝ ΚΑΘΗΓΗΤΗΣ Απόστολος Κουτίνας (Αναπληρωτής Καθηγητής, Γ.Π.Α) ΕΠΤΑΜΕΛΗΣ ΕΞΕΤΑΣΤΙΚΗ ΕΠΙΤΡΟΠΗ Απόστολος Κουτίνας (Αναπληρωτής Καθηγητής Γ.Π.Α) Σεραφείμ Παπανικολάου (Αν. Καθηγητής Γ.Π.Α) Ιωάννης Κούκος (Καθηγητής Πανεπιστημίου Πατρών) Ιωάννα Μαντάλα (Αν. Καθηγήτρια Γ.Π.Α) Νικόλαος Στοφόρος (Καθηγητής, Γ.Π.Α) Βασιλική Ευαγγελίου (Αν. Καθηγήτρια, Γ.Π.Α) Denise M.G Freire (Professor, Federal University of Rio de Janeiro, Brazil) v ABSTRACT This doctoral dissertation focused on the valorisation of renewable resources including liquid and solid industrial waste and by-products streams for the biotechnological production of value-added products. Their potential utilisation as raw materials for the chemical industry was evaluated. Extraction of phenolic compounds from by-product streams of palm oil production was assessed in terms of total phenolic content and antioxidant activity index. Phenolics incorporation into various edible oils aimed to prolong their shelf life. The first part of this thesis demonstrated the production of a generic nutrient-rich feedstock using by-product streams from palm oil production as a substitute for commercial fermentation supplements. Solid-state fermentations of palm kernel cake (PKC) and palm- pressed fiber (PPF) were conducted in tray bioreactors and a rotating drum bioreactor by the fungal strain Aspergillus oryzae for the production of crude enzymes. The production of protease was optimized (319.3 U/g) at an initial moisture content of 55%, when PKC was used as the sole substrate. The highest free amino nitrogen (FAN) production (5.6 mg/g) obtained via PKC hydrolysis using the crude enzymes produced via solid-state fermentation was achieved at 50 °C. Three initial PKC concentrations were tested in hydrolysis experiments, leading to total Kjeldahl nitrogen to FAN conversion yields up to 27.9%. Sequential solid-state fermentation followed by hydrolysis, was carried out in the same rotating drum bioreactor, leading to the production of 128.8 U/g of protease activity during fermentation and 201.5 mg/L of FAN during hydrolysis. Microbial oil production was successfully achieved with the oleaginous yeast strain Lipomyces starkeyi DSM 70296 cultivated on the produced PKC hydrolysate mixed with commercial carbon sources, including glucose, xylose, mannose, galactose, and arabinose. A sequential part of the thesis, dealt with the extraction of phenolic compounds from various side streams derived from palm oil production including PKC, PPF, palm kernel shells (PKS) and empty fruit bunches (EFB). Among these streams, PKC showed the highest total phenolic content with a value of 5.2 mg of gallic acid equivalents (GAE)/g of dry sample, whereas the EFB presented the lowest value (1.8 mg GAE/g dry sample). The extraction time as well as the liquid-to-solid ratio were investigated to optimize the phenolic extraction. PKC exhibited the highest total phenolic content (5.4 mg GAE/g dry sample) with a liquid- to-solid ratio of 40:1 during 20 minutes of extraction. The main phenolic compounds of the extracts deriving from all by-product streams were also identified and quantified employing HPLC-DAD. Catechin and 4-hydroxybenzoic acid were detected in all samples. The i addition of 0.8% of PKC extract increased more than 50 % the induction time of sunflower oil. A subsequent objective of this study was the valorisation of various protein-rich agricultural residues for the production of microbial oil-based polyol esters with lubrication properties. Palm kernel cake was initially used as substrate to evaluate lipid production via solid state fermentations using the fungal strains Cunninghamella echinulata, Mortierella ramanniana sp., Mortierella isabellina and Thamnidium elegans. Based on fermentation efficiency and fatty acid composition, C. echinulata was selected for further optimisation of fermentative conditions (i.e. temperature, initial moisture content, ratio of palm kernel cake and palm pressed fiber). Under the optimum conditions, microbial oil production in tray fermentations was 129.1 mg/g of fermented solids (gfs), with a γ-linolenic content of 5.3% (w/w). Cotton seed cake and castor seed cake were also evaluated for microbial lipid production in tray fermentations. The highest biomass production (304.1 mg/gfs) with a lipid content of 40.1% (w/w) was achieved when cotton seed cake was used. Subsequently, microbial lipid production using cotton seed cake was also evaluated in packed bed bioreactors, leading to a fungal biomass production of 320.5 mg/gfs with an intracellular lipid content of 37.7% (w/w). The microbial oil was extracted from the fermented solids and used for polyol ester production catalyzed by Lipomod 34MDP in a solvent free system. The highest conversion yield of 80% was achieved after 2 h reaction. The produced esters were characterized via Nuclear Magnetic Resonance analysis. The present study showed the perspective of valorising agricultural residues into value added oleochemicals with potential use as biolubricants. Next, spent sulphite liquor (SSL), the major waste stream of pulp and paper industry, was valorized for the production of microbial oil rich in γ-linolenic acid (GLA) by five oleaginous fungi of the genus Mortierella sp., Cunninghmamella sp. and Thamnidium sp. The aim was the utilization of fungal oil as substrate for the enzymatic synthesis of isopropyl and 2-ethylhexanol esters employing a solvent-free system catalyzed by commercial lipases. The reaction conditions evaluated, were temperature (30 - 70 ○C), microbial oil to alcohol molar ratio (1:2.84 - 1:4) and biocatalyst amount (5 - 20%, w/w). Under the optimized conditions, subsequent transesterifications were conducted using lipid classes obtained via fractionation of microbial oil, including neutral lipids (NL), glycolipids and sphingolipids (GL+SL) and phospholipids (PL). Results showed that the presence of GL+SL and PL fractions negatively affected the enzymatic synthesis of the esters. Transesterification of the NL fraction with isopropanol and 2-ethylhexanol led to the maximum conversion yield of ii 80.2% and 73.8%, respectively. Subsequently, the conversion yield was maximized (90% and 80% for isopropyl and 2-ethylhexyl esters respectively) by the utilization of hydrolyzed microbial oil. The reuse and stability of biocatalyst in 6 repeated reactions as well as the physicochemical properties of the produced esters were also evaluated using the hydrolyzed microbial oil. The present study demonstrates the perspective of utilizing renewable resources for the production of specialty fatty acid esters rich in GLA with potent application in food, cosmetics and oleochemicals industries. The last part of the thesis was related to the exploitation of SSL as the sole carbon source for the production of microbial oil. Initially, the effect of different C/FAN ratios and lignosulphonates (LS) concentrations on microbial oil production and fatty acid composition by Lipomyces starkeyi, Cunnighamella echinulata and Mortierella isabellina was evaluated in shake flasks fermentations. The highest lipid and total dry weight (TDW) production of L. starkeyi was 11.2 g/L and 2.8 g/L, respectively, using at C/FAN ratio of 173. In fed-batch fermentation, L. starkeyi showed the highest lipid production (40.9 g/L) at a C/FAN ratio of 33. In shake flask fermentations of M. isabellina, biomass production and microbial oil content were highly inhibited when 10 g/L and 50 g/L
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