Raw Glycerol Based Medium for DHA and Lipids Production, Using the Marine Heterotrophic Microalga Crypthecodinium cohnii

Crude glycerol, a biodiesel industry byproduct, and corn steep liquor (CSL) derived from a starch industry, were used as carbon and nitrogen sources, respectively, for lipid production, using the heterotrophic microalga C. cohnii grown in a bench bioreactor, in a batch culture. The maximum biomass concentration, lipid content and lipid productivity attained were 5.34 g/L, 24.6% (w/w Dry Cell Weight-DCW) and 0.016 g L−1 h−1, respectively. Flow cytometry analysis was used to evaluate the impact of these substrates on the microalgae cells. A high proportion of intact cells with enzymatic (esterases) activity (>50%) was present throughout the cultivation time course. These results indicate that crude glycerol and CSL can be used in the medium formulation for DHA and lipid production using this microalga, which reduce the process costs in an expected maximum of 84%.

Production of a β-Glucosidase-Rich Cocktail From Talaromyces Amestolkiae Using Raw Glycerol: Its Role for Lignocellulose Wastes Valorization

As β-glucosidases represent the major bottleneck for industrial degradation of plant biomass, great efforts are being devoted both to discover novel and robust versions of these enzymes, as well as to develop efficient and inexpensive ways to produce them. In this work, raw glycerol from chemical production of biodiesel was tested as carbon source for the fungus Talaromyces amestolkiae with the aim of producing enzyme cocktails rich in this activity. Approximately 11 U/mL β-glucosidase were detected in these cultures, constituting the major cellulolytic activity. Proteomic analysis revealed BGL-3 as the most abundant protein and the main β-glucosidase. This enzyme crude was successfully used to supplement a basal commercial cellulolytic cocktail (Cellu-clast 1.5L) for saccharification of pretreated wheat straw, corroborating that even hardly exploitable industrial wastes, such as glycerol, can be used as secondary raw materials to produce valuable enzymatic preparations in a framework of circular economy

Rhodotorula Strains Isolated from Seawater That Can Biotransform Raw Glycerol into Docosahexaenoic Acid (DHA) and Carotenoids for Animal Nutrition

Due to the overexploitation of industrial fisheries, as the principal source of fish oil, as well as the increasing replacement of synthetic pigments for animal nutrition, we need to find sustainable sources for these essential nutrient productions. Marine Rhodotorula strains NCYC4007 and NCYC1146 were used to determine the biosynthesis of docosahexaenoic acid (DHA) and carotenoids by biotransforming raw glycerol, a waste product of biodiesel. To evaluate the presence of inhibitory substances in raw glycerol, both strains were also grown in the presence of analytical grade glycerol and glucose as the main carbon source separately. With raw glycerol, NCYC4007 showed a significant correlation between DHA production and intracellular phosphorous concentrations. NCYC1146, a new Rhodotorula strain genetically described in this work, can produce canthaxanthin but only when glycerol is used as a main carbon source. Then, NCYC4007 could synthesize DHA as a phospholipid, and the production of canthaxanthin depends on the kind of carbon source used by NCYC1146. Finally, malate dehydrogenase activity and glucose production can be used as a proxy of the metabolisms in these marine Rhodotorula. This is the first evidence that marine Rhodotorula are capable of synthesizing DHA and canthaxanthin using an alternative and low-cost source of carbon to potentially scale their sustainable production for animal nutrition.

Fed-Batch mcl- Polyhydroxyalkanoates Production in Pseudomonas putida KT2440 and ΔphaZ Mutant on Biodiesel-Derived Crude Glycerol

Crude glycerol has emerged as a suitable feedstock for the biotechnological production of various industrial chemicals given its high surplus catalyzed by the biodiesel industry. Pseudomonas bacteria metabolize the polyol into several biopolymers, including alginate and medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs). Although P. putida is a suited platform to derive these polyoxoesters from crude glycerol, the attained concentrations in batch and fed-batch cultures are still low. In this study, we employed P. putida KT2440 and the hyper-PHA producer ΔphaZ mutant in two different fed-batch modes to synthesize mcl-PHAs from raw glycerol. Initially, the cells grew in a batch phase (μmax 0.21 h–1) for 22 h followed by a carbon-limiting exponential feeding, where the specific growth rate was set at 0.1 (h–1), resulting in a cell dry weight (CDW) of nearly 50 (g L–1) at 40 h cultivation. During the PHA production stage, we supplied the substrate at a constant rate of 50 (g h–1), where the KT2440 and the ΔphaZ produced 9.7 and 12.7 gPHA L–1, respectively, after 60 h cultivation. We next evaluated the PHA production ability of the P. putida strains using a DO-stat approach under nitrogen depletion. Citric acid was the main by-product secreted by the cells, accumulating in the culture broth up to 48 (g L–1) under nitrogen limitation. The mutant ΔphaZ amassed 38.9% of the CDW as mcl-PHA and exhibited a specific PHA volumetric productivity of 0.34 (g L–1 h–1), 48% higher than the parental KT2440 under the same growth conditions. The biosynthesized mcl-PHAs had average molecular weights ranging from 460 to 505 KDa and a polydispersity index (PDI) of 2.4–2.6. Here, we demonstrated that the DO-stat feeding approach in high cell density cultures enables the high yield production of mcl-PHA in P. putida strains using the industrial crude glycerol, where the fed-batch process selection is essential to exploit the superior biopolymer production hallmarks of engineered bacterial strains.

Production of a β-glucosidase-Rich Cocktail from Talaromyces Amestolkiae using Raw Glycerol: Its Role for Lignocellulose Wastes Valorization

Background: As β-glucosidases represent the major bottleneck for industrial degradation of plant biomass, great efforts are being devoted both to discover novel and robust versions of these enzymes, as well as to develop efficient and inexpensive ways to produce them. In this work, raw glycerol from chemical production of biodiesel was tested as carbon source for the fungus Talaromyces amestolkiae with the aim of producing enzyme cocktails rich in this activity. Results: When using raw glycerol as sole carbon source, approximately 11 U/mL β-glucosidase were detected in these cultures, constituting the major cellulolytic activity. Besides, it was detected that the enzymatic production started when glycerol was completely depleted, which implicates that it was produced under carbon starvation stimuli. Proteomic analysis of the produced crudes revealed BGL-3 as the most abundant protein and the main b-glucosidase. This enzymatic cocktail was successfully used to supplement a basal commercial cellulolytic cocktail (Celluclast 1.5L) for saccharification of different pretreated wheat straw, and improving the yield that the commercial preparation can reach alone.Conclusions: This study corroborates that even hardly exploitable industrial wastes, such as glycerol, can be used by Talaromyces amestolkiae as carbon sources to produce very valuable enzymatic preparations for the production of biofuels and other bioproducts in a framework of circular economy.

Effect of Four Novel Bio-Based DES (Deep Eutectic Solvents) on Hardwood Fractionation

Using the basic principle of construction between a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD), four bio-based deep eutectic solvents (DESs) were prepared in a 1:2 molar ratio of HBA:HBD. 2,3-Dihydroxypropyl-1-triethylammonium chloride ([C9H22N+O2]Cl−) was synthesized from raw glycerol and used as an HBA. Lactic acid, urea, pure glycerol, and ethylene glycol were selected as HBD. Attempts to prepare DESs, using citric acid and benzoic acid as HBDs, were unsuccessful. All these DESs were characterized using FTIR and NMR techniques. Besides, physicochemical parameters such as pH, viscosity, density, and melting point were determined. The behavior of these DES to fractionate olive pomace was studied. Lignin recovery yields spanned between 27% and 39% (w/w) of the available lignin in olive pomace. The best DES, in terms of lignin yield ([C9H22N+O2]Cl− -lactic acid), was selected to perform a scale-up lignin extraction using 40 g of olive pomace. Lignin recovery on the multigram scale was similar to the mg scale (38% w/w). Similarly, for the holocellulose-rich fractions, recovery yields were 34% and 45% for mg and multi-gram scale, respectively. Finally, this DES was used to fractionate four fruit pruning samples. These results show that our novel DESs are alternative approaches to the ionic liquid:triethylammonium hydrogen sulfate and the widely used DES: choline chloride:lactic acid (1:10 molar ratio) for biomass processing.