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.

Nutrient removal and biomass production of marine microalgae cultured in recirculating aquaculture systems (RAS) water with low phosphate concentration.

This study was conducted to investigate the feasibility of microalgal biomass production and nutrient removal from recirculating aquaculture systems (RAS) water (RASW) with low phosphate concentration. For this purpose, Nannochloropsis oculata, Pavlova gyrans, Tetraselmis suecica, Phaeodactylum tricornutum, and their consortium were cultivated in RASW and RASW supplemented with vitamins (+V). Among them, N. oculata showed the maximum biomass production of 0.4 g/L in RASW. Vitamins supplementation significantly increased the growth of T. suecica from 0.16 g/L in RASW to 0.33 g/L in RASW + V. Additionally, T. suecica showed the highest nitrate (NO3-N) removal efficiency of 80.88 ± 2.08 % in RASW and 83.82 ± 2.08 % in RASW + V. Accordingly, T. suecica was selected for scaling up study of microalgal cultivation in RASW and RASW supplemented with nitrate (RASW + N) in 4-L airlift photobioreactors. Nitrate supplementation enhanced the growth of T. suecica up to 2.2-fold (day 15). The fatty acid nutritional indices in T. suecica cultivated in RASW and RASW + N showed optimal polyunsaturated fatty acids (PUFAs)/saturated fatty acid (SFAs), omega-6 fatty acid (n-6)/omega-3 fatty acid (n-3), indices of atherogenicity (IA), and thrombogenicity (IT)). Overall, the findings of this study revealed that despite low phosphate concentration, marine microalgae can grow in RASW and relatively reduce the concentration of nitrate. Furthermore, the microalgal biomass cultivated in RASW consisting of pigments and optimal fatty acid nutritional profile can be used as fish feed, thus contributing to a circular bioeconomy.

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.

Advances in Biological Wastewater Treatment Processes: Focus on Low-Carbon Energy and Resource Recovery in Biorefinery Context.

Advancements in biological wastewater treatment with sustainable and circularity approaches have a wide scope of application. Biological wastewater treatment is widely used to remove/recover organic pollutants and nutrients from a diverse wastewater spectrum. However, conventional biological processes face challenges, such as low efficiency, high energy consumption, and the generation of excess sludge. To overcome these limitations, integrated strategies that combine biological treatment with other physical, chemical, or biological methods have been developed and applied in recent years. This review emphasizes the recent advances in integrated strategies for biological wastewater treatment, focusing on their mechanisms, benefits, challenges, and prospects. The review also discusses the potential applications of integrated strategies for diverse wastewater treatment towards green energy and resource recovery, along with low-carbon fuel production. Biological treatment methods, viz., bioremediation, electro-coagulation, electro-flocculation, electro-Fenton, advanced oxidation, electro-oxidation, bioelectrochemical systems, and photo-remediation, are summarized with respect to non-genetically modified metabolic reactions. Different conducting materials (CMs) play a significant role in mass/charge transfer metabolic processes and aid in enhancing fermentation rates. Carbon, metal, and nano-based CMs hybridization in different processes provide favorable conditions to the fermentative biocatalyst and trigger their activity towards overcoming the limitations of the conventional process. The emerging field of nanotechnology provides novel additional opportunities to surmount the constraints of conventional process for enhanced waste remediation and resource valorization. Holistically, integrated strategies are promising alternatives for improving the efficiency and effectiveness of biological wastewater treatment while also contributing to the circular economy and environmental protection.

Ameliorating microalgal OMEGA production using omics platforms.

Over the past decade, the focus on omega (ω)-3 fatty acids from microalgae has intensified due to their diverse health benefits. Bioprocess optimization has notably increased ω-3 fatty acid yields, yet understanding of the genetic architecture and metabolic pathways of high-yielding strains remains limited. Leveraging genomics, transcriptomics, proteomics, and metabolomics tools can provide vital system-level insights into native ω-3 fatty acid-producing microalgae, further boosting production. In this review, we explore 'omics' studies uncovering alternative pathways for ω-3 fatty acid synthesis and genome-wide regulation in response to cultivation parameters. We also emphasize potential targets to fine-tune in order to enhance yield. Despite progress, an integrated omics platform is essential to overcome current bottlenecks in optimizing the process for ω-3 fatty acid production from microalgae, advancing this crucial field.

Furan Distribution as a Severity Indicator upon Organosolv Fractionation of Hardwood Sawdust through a Novel Ternary Solvent System.

Beech sawdust was treated with a ternary solvent system based on binary aqueous ethanol with partial substitution of ethanol by acetone at four different water contents (60, 50, 40, and 30%v/v). In addition to standard, i.e., noncatalyzed treatments, the application of inorganic acid in the form of 20 mm H2SO4 was evaluated. The various solvent systems were applied at 180 °C for 60 min. The obtained biomass fractions were characterized by standard biomass compositional methods, i.e., sugar monomer and oligomer contents, dehydration product contents of the aqueous product, and lignin, cellulose, and hemicellulose contents in isolated solid fractions. More advanced analyses were performed on the lignin fractions, including quantitative 13C NMR analyses, 1H-13C HSQC analysis, size exclusion chromatography, and pyrolysis-GC/MS, and the aqueous product, in the form of size exclusion chromatography and determination of total phenol contents. The picture emerging from the thorough analytical investigation performed on the lignin fractions is consistent with that resulting from the characterization of the other fractions: results point toward greater deconstruction of the lignocellulosic recalcitrance upon higher organic solvent content, replacing ethanol with acetone during the extraction, and upon addition of mineral acid. A pulp with cellulose content of 94.23 wt % and 95% delignification was obtained for the treatment employing a 55/30/15 EtOH/water/acetone mixture alongside 20 mm H2SO4. Furthermore, the results indicate the formation of two types of organosolv furan families during treatment, which differ in the substitution of their C1 and C5. While the traditional lignin aryl-ether linkages present themselves as indicators for process severity for the nonacid catalyzed systems, the distribution of these furan types can be applied as a severity indicator upon employment of H2SO4, including their presence in the isolated lignin fractions.

Ultrasound-controlled acidogenic valorization of wastewater for biohydrogen and volatile fatty acids production: Microbial community profiling.

The present study explored the influence of ultrasound on acidogenic fermentation of wastewater for the production of biohydrogen and volatile fatty acids/carboxylic acids. Eight sono-bioreactors underwent ultrasound (20 kHz: 2W and 4W), with an ultrasound duration ranging from 15 min to 30 days, and the formation of acidogenic metabolites. Long-term continuous ultrasonication enhanced biohydrogen and volatile fatty acid production. Specifically, ultrasonication at 4W for 30 days increased biohydrogen production by 3.05-fold compared to the control, corresponding to hydrogen conversion efficiency of 58.4%; enhanced volatile fatty acid production by 2.49-fold; and increased acidification to 76.43%. The observed effect of ultrasound was linked to enrichment with hydrogen-producing acidogens such as Firmicutes, whose proportion increased from 61.9% (control) to 86.22% (4W, 30 days) and 97.53% (2W, 30 days), as well as inhibition of methanogens. This result demonstrates the positive effect of ultrasound on the acidogenic conversion of wastewater to biohydrogen and volatile fatty acid production.

Covalently bound humin-lignin hybrids as important novel substructures in organosolv spruce lignins.

Organosolv lignins (OSLs) are important byproducts of the cellulose-centred biorefinery that need to be converted in high value-added products for economic viability. Yet, OSLs occasionally display characteristics that are unexpected looking at the lignin motifs present. Applying advanced NMR, GPC, and thermal analyses, isolated spruce lignins were analysed to correlate organosolv process severity to the structural details for delineating potential valorisations. Very mild conditions were found to not fractionate the biomass, causing a mix of sugars, lignin-carbohydrate complexes (LCCs), and corresponding dehydration/degradation products and including pseudo-lignins. Employing only slightly harsher conditions promote fractionation, but also formation of sugar degradation structures that covalently incorporate into the oligomeric and polymeric lignin structures, causing the isolated organosolv lignins to contain lignin-humin hybrid (HLH) structures not yet evidenced as such in organosolv lignins. These structures effortlessly explain observed unexpected solubility issues and unusual thermal responses, and their presence might have to be acknowledged in downstream lignin valorisation.

Role and importance of solvents for the fractionation of lignocellulosic biomass.

Lignocellulosic biomass is one of the most important renewable materials to replace carbon-based fossil resources. Solvent-based fractionation is a promising route for fractionation of biomass into its major components. Processing is governed by the employed solvent-systems properties. This review sheds light on the factors governing both dissolution and potential reactivities of the chemical structures present in lignocellulose, highlighting how proper understanding of the underlying mechanisms and interactions between solute and solvent help to choose proper systems for specific fractionation needs. Structural and chemical differences between the carbohydrate-based structural polymers and lignin require very different solvents capabilities in terms of causing and eventually stabilizing conformational changes and consequent activation of bonds to be cleaved by other active components in the. A consideration of potential depolymerization events during dissolution and energetic aspects of the dissolution process considering the contribution of polymer functionalities allow for a mapping of solvent suitability for biomass fractionation.

From Yeast to Biotechnology.

Yeasts are widely used in various sectors of biotechnology, from white (industrial) to red (medical) [...].

Salicornia dolichostachya organosolv fractionation: towards establishing a halophyte biorefinery.

Halophytes are a potential source of lignocellulosic material for biorefinery, as they can be grown in areas unsuitable for the cultivation of crops aimed at food production. To enable the viable use of halophytes in biorefineries, the present study investigated how different organosolv process parameters affected the fractionation of green pressed fibers of Salicornia dolichostachya. We produced pretreated solids characterized by up to 51.3% ± 1.7% cellulose, a significant increase from 25.6% ± 1.3% in untreated fibers. A delignification yield of as high as 60.7%, and hemicellulose removal of as high as 86.1% were also achieved in the current study. The obtained cellulose could be completely converted to glucose via enzymatic hydrolysis within 24 h. The lignin fractions obtained were of high purity, with sugar contamination of only 1.22% w/w and ashes below 1% w/w in most samples. Finally, up to 29.1% ± 0.4% hemicellulose was recovered as a separate product, whose proportion of oligomers to total sugars was 69.9% ± 3.0%. To the best of our knowledge, this is the first report in which Salicornia fibers are shown to be a suitable feedstock for organosolv biomass fractionation. These results expand the portfolio of biomass sources for biorefinery applications.

Unravelling Metagenomics Approach for Microbial Biofuel Production.

Renewable biofuels, such as biodiesel, bioethanol, and biobutanol, serve as long-term solutions to fossil fuel depletion. A sustainable approach feedstock for their production is plant biomass, which is degraded to sugars with the aid of microbes-derived enzymes, followed by microbial conversion of those sugars to biofuels. Considering their global demand, additional efforts have been made for their large-scale production, which is ultimately leading breakthrough research in biomass energy. Metagenomics is a powerful tool allowing for functional gene analysis and new enzyme discovery. Thus, the present article summarizes the revolutionary advances of metagenomics in the biofuel industry and enlightens the importance of unexplored habitats for novel gene or enzyme mining. Moreover, it also accentuates metagenomics potentials to explore uncultivable microbiomes as well as enzymes associated with them.

Effect of metals on the regulation of acidogenic metabolism enhancing biohydrogen and carboxylic acids production from brewery spent grains: Microbial dynamics and biochemical analysis.

The present study reports the mixed culture acidogenic production of biohydrogen and carboxylic acids (CA) from brewery spent grains (BSG) in the presence of high concentrations of cobalt, iron, nickel, and zinc. The metals enhanced biohydrogen output by 2.39 times along with CA biosynthesis by 1.73 times. Cobalt and iron promoted the acetate and butyrate pathways, leading to the accumulation of 5.14 gCOD/L of acetic and 11.36 gCOD/L of butyric acid. The production of solvents (ethanol + butanol) was higher with zinc (4.68 gCOD/L) and cobalt (4.45 gCOD/L). A combination of all four metals further enhanced CA accumulation to 42.98 gCOD/L, thus surpassing the benefits accrued from supplementation with individual metals. Additionally, 0.36 and 0.31 mol green ammonium were obtained from protein-rich brewery spent grain upon supplementation with iron and cobalt, respectively. Metagenomic analysis revealed the high relative abundance of Firmicutes (>90%), of which 85.02% were Clostridium, in mixed metal-containing reactors. Finally, a significant correlation of dehydrogenase activity with CA and biohydrogen evolution was observed upon metal addition.

Prospects and trends in bioelectrochemical systems: Transitioning from CO2 towards a low-carbon circular bioeconomy.

Resource scarcity and climate change are the most quested topics in view of environmental sustainability. CO2 sequestration through bioelectrochemical systems is an attractive option for fostering bioeconomy development upon several value-added products generation. This review details the state-of-the-art of bioelectrochemical systems for resource recovery from CO2 along with various biocatalysts capable of utilizing CO2. Two bioprocesses (photo-electrosynthesis and chemolithoelectrosynthesis) were discussed projecting their potential for biobased economy development from CO2. Significance of adopting circular strategies for efficient resource recycling, intensifying product value, integrations/interlinking of multiple process chains for the development of circular bioeconomy were discussed. Existing constrains as well as outlook for near establishment of circular bioeconomy from CO2 is presented by weighing fore-sighted plans with current actions. Need for developing CO2-based circular bioeconomy via innovative business models by analyzing social, technical, environmental and product related aspects are also discussed providing a roadmap of gaps to pursue for attaining practicality.

Heterotrophic Cultivation of the Cyanobacterium Pseudanabaena sp. on Forest Biomass Hydrolysates toward Sustainable Biodiesel Production.

Environmental pollution, greenhouse gas emissions, depletion of fossil fuels, and a growing population have sparked a search for new and renewable energy sources such as biodiesel. The use of waste or residues as substrates for microbial growth can favor the implementation of a biorefinery concept with reduced environmental footprint. Cyanobacteria constitute microorganisms with enhanced ability to use industrial effluents, wastewaters, forest residues for growth, and concomitant production of added-value compounds. In this study, a recently isolated cyanobacterium strain of Pseudanabaena sp. was cultivated on hydrolysates from pretreated forest biomass (silver birch and Norway spruce), and the production of biodiesel-grade lipids was assessed. Optimizing carbon source concentration and the (C/N) carbon-to-nitrogen ratio resulted in 66.45% w/w lipid content when microalgae were grown on glucose, compared to 62.95% and 63.79% w/w when grown on spruce and birch hydrolysate, respectively. Importantly, the lipid profile was suitable for the production of high-quality biodiesel. The present study demonstrates how this new cyanobacterial strain could be used as a biofactory, converting residual resources into green biofuel.

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.

Microbial genetic engineering approach to replace shark livering for squalene.

Squalene is generally sourced from the liver oil of deep sea sharks (Squalus spp.), in which it accounts for 40-70% of liver mass. To meet the growing demand for squalene because of its beneficial effects for human health, three to six million deep sea sharks are slaughtered each year, profoundly endangering marine ecosystems. To overcome this unsustainable practice, microbial sources of squalene might offer a viable alternative to plant- or animal-based squalene, although only a few microorganisms have been found that are capable of synthesizing up to 30% squalene of dry biomass by native biosynthetic pathways. These squalene biosynthetic pathways, on the other hand, can be genetically manipulated to transform microorganisms into 'cellular factories' for squalene overproduction.

Advances in cathode designs and reactor configurations of microbial electrosynthesis systems to facilitate gas electro-fermentation.

In gas fermentation, a range of chemolithoautotrophs fix single-carbon (C1) gases (CO2 and CO) when H2 or other reductants are available. Microbial electrosynthesis (MES) enables CO2 reduction by generating H2 or reducing equivalents with the sole input of renewable electricity. A combined approach as gas electro-fermentation is attractive for the sustainable production of biofuels and biochemicals utilizing C1 gases. Various platform compounds such as acetate, butyrate, caproate, ethanol, butanol and bioplastics can be produced. However, technological challenges pertaining to the microbe-material interactions such as poor gas-liquid mass transfer, low biomass and biofilm coverage on cathode, low productivities still exist. We are presenting a review on latest developments in MES focusing on the configuration and design of cathodes that can address the challenges and support the gas electro-fermentation. Overall, the opportunities for advancing CO and CO2-based biochemicals and biofuels production in MES with suitable cathode/reactor design are prospected.

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach.

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.