A bioprocess engineering approach for the production of hydrocarbons and fatty acids from green microalga under high cobalt concentration as the feedstock of high-grade biofuels.

Botryococcus braunii, a colonial green microalga which is well-known for its capacity to synthesize hydrocarbons, has significant promise as a long-term source of feedstock for the generation of biofuels. However, cultivating and scaling up B. braunii using conventional aqua-suspended cultivation systems remains a challenge. In this study, we optimized medium components and light intensity to enhance lipid and hydrocarbon production in a multi-cultivator airlift photobioreactor. BBM 3N medium with 200 µmol/m2/s light intensity and a 16 h light-8 h dark regimen yielded the highest biomass productivity (110.00 ± 2.88 mg/L/day), as well as the highest lipid and hydrocarbon content. Cultivation in a flat-panel bioreactor resulted in significantly higher biomass productivity (129.11 ± 2.74 mg/L/day), lipid productivity (32.21 ± 1.31 mg/L/day), and hydrocarbon productivity (28.98 ± 2.08 mg/L/day) compared to cultivation in Erlenmeyer flasks and open 20-L raceway pond. It also exhibited 20.15 ± 1.03% of protein content including elevated levels of chlorophyll a, chlorophyll b, and carotenoids. This work is noteworthy since it is the first to describe fatty acid and hydrocarbon profiles of B. braunii during cobalt treatment. The study demonstrated that high cobalt concentrations (up to 5 mg/L of cobalt nitrate) during Botryococcus culture affected hydrocarbon synthesis, resulting in high amounts of n-alkadienes and trienes as well as lipids with elevated monounsaturated fatty acids concentration. Furthermore, pyrolysis experiments on microalgal green biomass and de-oiled biomass revealed the lipid and hydrocarbon compounds generated by the thermal degradation of B. braunii that facilitate extra economical value to this system.

Transcriptomics aids in uncovering the metabolic shifts and molecular machinery of Schizochytrium limacinum during biotransformation of hydrophobic substrates to docosahexaenoic acid.

BACKGROUND: Biotransformation of waste oil into value-added nutraceuticals provides a sustainable strategy. Thraustochytrids are heterotrophic marine protists and promising producers of omega (ω) fatty acids. Although the metabolic routes for the assimilation of hydrophilic carbon substrates such as glucose are known for these microbes, the mechanisms employed for the conversion of hydrophobic substrates are not well established. Here, thraustochytrid Schizochytrium limacinum SR21 was investigated for its ability to convert oils (commercial oils with varying fatty acid composition and waste cooking oil) into ω-3 fatty acid; docosahexaenoic acid (DHA). RESULTS: Within 72 h SR21 consumed ~ 90% of the oils resulting in enhanced biomass (7.5 g L- 1) which was 2-fold higher as compared to glucose. Statistical analysis highlights C16 fatty acids as important precursors of DHA biosynthesis. Transcriptomic data indicated the upregulation of multiple lipases, predicted to possess signal peptides for secretory, membrane-anchored and cytoplasmic localization. Additionally, transcripts encoding for mitochondrial and peroxisomal ß-oxidation along with acyl-carnitine transporters were abundant for oil substrates that allowed complete degradation of fatty acids to acetyl CoA. Further, low levels of oxidative biomarkers (H2O2, malondialdehyde) and antioxidants were determined for hydrophobic substrates, suggesting that SR21 efficiently mitigates the metabolic load and diverts the acetyl CoA towards energy generation and DHA accumulation. CONCLUSIONS: The findings of this study contribute to uncovering the route of assimilation of oil substrates by SR21. The thraustochytrid employs an intricate crosstalk among the extracellular and intracellular molecular machinery favoring energy generation. The conversion of hydrophobic substrates to DHA can be further improved using synthetic biology tools, thereby providing a unique platform for the sustainable recycling of waste oil substrates.

Biosynthesis of microalgal lipids, proteins, lutein, and carbohydrates using fish farming wastewater and forest biomass under photoautotrophic and heterotrophic cultivation.

Biorefineries enable the circular, sustainable, and economic use of waste resources if value-added products can be recovered from all the generated fractions at a large-scale. In the present studies the comparison and assessment for the production of value-added compounds (e.g., proteins, lutein, and lipids) by the microalga Chlorella sorokiniana grown under photoautotrophic or heterotrophic conditions was performed. Photoautotrophic cultivation generated little biomass and lipids, but abundant proteins (416.66 mg/gCDW) and lutein (6.40 mg/gCDW). Heterotrophic conditions using spruce hydrolysate as a carbon source favored biomass (8.71 g/L at C/N 20 and 8.28 g/L at C/N 60) and lipid synthesis (2.79 g/L at C/N 20 and 3.61 g/L at C/N 60) after 72 h of cultivation. Therefore, heterotrophic cultivation of microalgae using spruce hydrolysate instead of glucose offers a suitable biorefinery concept at large-scale for biodiesel-grade lipids production, whereas photoautotrophic bioreactors are recommended for sustainable protein and lutein biosynthesis.

Valorization of waste forest biomass toward the production of cello-oligosaccharides with potential prebiotic activity by utilizing customized enzyme cocktails.

BACKGROUND: Production of value-added materials from lignocellulosic biomass residues is an emerging sector that has attracted much attention as it offers numerous benefits from an environmental and economical point of view. Non-digestible oligosaccharides represent a group of carbohydrates that are resistant to gastrointestinal digestion, and therefore, they are considered as potential prebiotic candidates. Such oligosaccharides can derive from the biomass cellulose fraction through a controlled enzymatic hydrolysis that eliminates the yield of monomers. RESULTS: In the present study, hydrolysis of organosolv-pretreated forest residues (birch and spruce) was tested in the presence of four cellulases (EG5, CBH7, CBH6, EG7) and one accessory enzyme (LPMO). The optimal enzyme combinations were comprised of 20% EG5, 43% CBH7, 22% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of birch and 35% EG5, 45% CBH7, 10% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of spruce, leading to 22.3% and 19.1 wt% cellulose conversion into cellobiose, respectively. Enzymatic hydrolysis was applied on scale-up reactions, and the produced oligosaccharides (consisted of > 90% cellobiose) were recovered and separated from glucose through nanofiltration at optimized temperature (50 °C) and pressure (10 bar) conditions, yielding a final product with cellobiose-to-glucose ratio of 21.1 (birch) and 20.2 (spruce). Cellobiose-rich hydrolysates were tested as fermentative substrates for different lactic acid bacteria. It was shown that they can efficiently stimulate the growth of two Lactobacilli strains. CONCLUSIONS: Controlled enzymatic hydrolysis with processive cellulases, combined with product recovery and purification, as well as enzyme recycling can potentially support the sustainable production of food-grade oligosaccharides from forest biomass.