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The present study demonstrates the influence of substrate flow rate on continuous biohydrogen and volatile fatty acids production from acidogenic fermentation of cheese whey in a tubular biofilm reactor. Three tubular bioreactors with a varied substrate flow rate of (2 mL/min, 5 mL/min, and 8 mL/min) were examined for 75 days. At 8 mL/min flow rate, the biohydrogen evolution was higher (3.88 mL H 2 /h), while its conversion efficiency was lower compared to 5 and 2 mL/min flow rate. The formation of volatile fatty acids and ammonium was also influenced by substrate flow rates. The volatile fatty acids production was slightly higher at 2 mL/min (12.74 ± 2.42 gCOD/L) and 5 mL/min (18.09 ± 2.01 gCOD/L) while, decreasing at 8 mL/min (11.85 ± 0.78 gCOD/L). Substrate flow rate significantly affected the pattern and composition of volatile fatty acids showing higher acetic acid, butyric and propionic acid production of 4.72 ± 1.46 gCOD/L (2 mL/min) 10.41 ± 0.91 gCOD/L (5 mL/min) and 1.78 ± 0.13 gCOD/L (5 mL/min). Continuous substrate input maintained the pH in the reactor due to replacement with fresh substrate, thereby controlling feedback inhibition and boosting metabolite production. Hydrogen-producing Firmicutes on the biofilm confirmed the pivotal role of the microbial community's significant contribution to converting waste to bioenergy. Overall, the present results support the use of a continuous operation mode for large-scale biohydrogen production. However, to ensure the efficacy of the system using waste or wastewater, low substrate flow rates are recommended. [Display omitted] • Hydrogen output is directly and yield inversely proportional to substrate flow rate. • Biohydrogen conversion efficiency is greater at lower substrate flow rates. • Continuous operation mode does not require additional buffers to regulate the pH. • Continuous operation mode enables consistent metabolite production. [ABSTRACT FROM AUTHOR]

CO 2 Capture Utilization and Storage (CCUS) is a fundamental strategy to mitigate climate change, and carbon sequestration, through absorption, can be one of the solutions to achieving this goal. In nature, carbonic anhydrase (CA) catalyzes the CO 2 hydration to bicarbonates. Targeting the development of novel biotechnological routes which can compete with traditional CO 2 absorption methods, CA utilization has presented a potential to expand as a promising catalyst for CCUS applications. Driven by this feature, the search for novel CAs as biocatalysts and the utilization of enzyme improvement techniques, such as protein engineering and immobilization methods, has resulted in suitable variants able to catalyze CO 2 absorption at relevant industrial conditions. Limitations related to enzyme recovery and recyclability are still a concern in the field, affecting cost efficiency. Under different absorption approaches, CA enhances both kinetics and CO 2 absorption yields, besides reduced energy consumption. However, efforts directed to process optimization and demonstrative plants are still limited. A recent topic with great potential for development is the CA utilization in accelerated weathering, where industrial residues could be re-purposed towards becoming carbon sequestrating agents. Furthermore, research of new solvents has identified potential candidates for integration with CA in CO 2 capture, and through techno-economic assessments, CA can be a path to increase the competitiveness of alternative CO 2 absorption systems, offering lower environmental costs. This review provides a favorable scenario combining the enzyme and CO 2 capture, with possibilities in reaching an industrial-like stage in the future. [Display omitted] • CO 2 capture by absorption can be enhanced by utilizing CA. • Improvement techniques produce CAs more adapted to likewise industrial conditions. • Biomimetic and CA-assisted amine absorption show superior CO 2 capture efficiency. • CA-assisted accelerated weathering is an emerging approach for enhanced CO 2 capture. [ABSTRACT FROM AUTHOR]

A peroxidase from the thermophilic fungus Myceliophthora thermophila that belongs to ascomycete Class II based on PeroxiBase classification was functionally expressed in methylotrophic yeast Pichia pastoris . The putative peroxidase from the genomic DNA was successfully cloned in P. pastoris X-33 under the transcriptional control of the alcohol oxidase ( AOX1 ) promoter. The heterologous production was greatly enhanced by the addition of hemin with a titer of 0.41 U mL −1 peroxidase activity at the second day of incubation. The recombinant enzyme was purified to homogeneity (50 kDa) and characterized using a series of phenolic substrates that indicated similar characteristics with those of generic peroxidases. In addition, the enzyme was found thermostable, retaining its activity for temperatures up to 60 °C after eight hours incubation. Moreover, the enzyme displayed remarkable H 2 O 2 stability, retaining more than 80% of its initial activity after 24 h incubation in 5000-fold molar excess of H 2 O 2 . The ability of the peroxidase to polymerize catechol at high superoxide concentrations, together with its high thermostability and substrate specificity, indicate a potential commercial significance of the enzyme. [ABSTRACT FROM AUTHOR]

Growing evidence points to a link between specific fatty acids ingested through the diet and human health. Chain length, saturation degree, and position of double bonds in fatty acids determine their effect in humans. Omega-3 and omega-6 fatty acids have been recognized for their contribution to the prevention and/or treatment of diabetes, cancer, visual impairment, cardiovascular diseases, as well as neurological and musculoskeletal disorders. Humans cannot synthesize these fatty acids in sufficient amounts and need to absorb them through the diet. Oleaginous microalgae constitute a promising, sustainable source of such fatty acids, as they can accumulate up to 85% of lipids on a cell dry weight basis. The present review summarizes the potential of oleaginous microalgae as a convenient, economical, and sustainable source of polyunsaturated fatty acids, and explores their beneficial role in human health. The growing prevalence of cardiovascular diseases and changing dietary preferences are driving the increasing demand for microbial omega-3 fatty acids. Following the COVID-19 pandemic, the importance of a healthy immune system has further strengthened the market for omega-3 fatty acids. • Microalgae considered as alternative source of fish-free omega-3 fatty acids. • PUFA synthesized autotrophically, mixotrophically and heterotrophically. • Omega-3 fatty acids for their role in human health is elaborated. • Metabolic pathway for PUFA synthesis in microorganisms is described. • Current market scenario of fish-free essential fatty acids is highlighted. [ABSTRACT FROM AUTHOR]

Abstract: The ability of sweet sorghum to be utilized as feedstock for ethanol production at high initial dry material concentration was investigated. Sweet sorghum, after being dried, was liquefacted employing commercial cellulase solution Celluclast® 1.5L, in order submerged fermentation to be permitted under high-solids concentrations. The presence of a separate enzymatic liquefaction step at 350 kg m−3 initial DM enhanced both ethanol production and productivity by 29.76% and 250%, respectively. Response surface methodology, based on the central composite design was applied to explore the combined effect of liquefaction duration and enzyme loading in order liquefaction conditions to be optimized. When the optimum conditions were tested using an enzyme load of 8.32 FPU g−1 of dry material for 8.6 h at 50 °C, high productivity (3.0 kg m−3 h−1) and final ethanol production (62.5 kg m−3) were achieved. [Copyright &y& Elsevier]

Abstract: The ability of sweet sorghum bagasse to be utilized as feedstock for ethanol production at high initial dry matter concentration was investigated. In order to achieve high enzymatic hydrolysis yield, a hydrothermal pretreatment prior to liquefaction and saccharification was applied. Response surface methodology had been employed in order to optimize the pretreatment step, taking into account the yield of cellulose hydrolysis. Liquefaction of the pretreated bagasse was performed at a specially designed liquefaction chamber at 50°C for either 12 or 24h using an enzyme loading of 10FPU/g·DM and 18% DM. Fermentation of liquefacted bagasse was not affected by liquefaction duration and leaded to an ethanol production of 41.43g/L and a volumetric productivity of 1.88g/Lh. The addition of extra enzymes at the start up of SSF enhanced both ethanol concentration and volumetric productivity by 16% and 17% after 12 and 24h saccharification, respectively. [Copyright &y& Elsevier]

Abstract: Mushrooms comprise a vast source of new pharmaceutical and nutraceutical products. Submerged fermentation of the mycelial form of mushroom-producing fungi is promising for efficient production of their biomass and active metabolites. The aim of this study was the isolation and identification of bioactive metabolites derived from the edible mushroom Pleurotus ostreatus biomass, produced by submerged fermentation in a batch stirred tank bioreactor. The dichloromethane extract was fractioned by medium pressure liquid chromatography (MPLC) and Sephadex LH-20 column chromatography. For extracting efficiently the phenolic compounds of the methanolic extract, an adsorption-desorption process, using XAD-4 type resin, was performed. The pure compounds were elucidated with 1D/2D NMR-spectroscopic analyses, NMR data comparisons, and chemical correlations combined with GC/MS- LC/MS experiments. The compounds afforded by the dichloromethane extract were identified as linoleic acid (1), oleic acid (2), stearic acid (3), palmitic acid (4) and their corresponding methyl esters (5-8, respectively), benzoic acid (9), trans 3, 4-dihydro-3, 4, 8-trihydroxynapthalen-1(2H)-one (10), 4-hydroxy- benzaldehyde (11), indolo-3-carboxylic acid (12) and uracil (13). The investigation of the methanolic extract led to the isolation of 3-formyl-pyrrole (14), 4-hydroxy-benzoic acid (15), uridine (16), nicotinic acid (17) and nicotinamide (18). Based on existing literature data, all these compounds exhibit valuable biological properties. The information obtained is considered fundamental for further investigation of the P. ostreatus fermentation process on an industrial scale for enhanced bioactive metabolite production. [Copyright &y& Elsevier]

Abstract: Two xylanases were purified to electrophoretic homogeneity from the thermophilic fungus Sporotrichum thermophile grown in a submerged liquid culture using wheat straw as carbon source. The enzymes, StXyn1 and StXyn2, have molecular masses of 24kDa and 48kDa, respectively, and are optimally active at pH 5 and at 60°C. Both enzymes displayed remarkable stability up to 50°C for 1h, exhibiting a half-life of 60min (StXyn1) and 115min (StXyn2) at 60°C. Biochemical characterization of the two xylanases against poly- and oligosaccharides indicated that StXyn1 and StXyn2 hydrolytic profiles match those of xylanase family 11 and family 10, respectively. LC–MS/MS analysis provided peptide mass and sequence information that assisted the identification of the corresponding xylanase genes from the S. thermophile genome and the classification of the two purified StXyn1 and StXyn2 as a family GH11 and GH10 endo-1,4-β-xylanases, respectively. [Copyright &y& Elsevier]

Microbial Electrosynthesis (MES) is emerging as a promising technology for the decarbonization of the economy. The use of CO 2 as a feedstock, i.e. through carbon capture and utilization, can create attractive business opportunities for the production of third generation biofuels. Achieving highly efficient electron transfer from the cathode to microbes is one of the main challenges hindering the development of MES reactors. It is, therefore, essential to improve biofilm growth for enhanced electron-transfer during bioconversion. In this study we show the first use of 3D bio-printing for microbial electrosynthesis systems, creating a "synthetic biofilm", containing Sporomusa ovata. The synthetic biofilm greatly improves the acetate production rate while drastically decreasing start-up time. Using H-cell reactors, poised at −0.8 V vs Ag/AgCl, with a synthetic biofilm printed on a carbon cloth electrode, an average acetate productivity of 47±5.1 g day−1 m−2 (0.31±0.55 g L−1 day−1) with a maximum productivity of 104 g day1 m−2 (0.68 g L−1⋅day−1) was achieved. This is an order of magnitude higher than typical S. ovata production rates, and 2–3 fold higher than reactors using specialized cathodes. Start-up of MES reactors typically require days, whereas a high production rate was achieved almost directly after the start-up (±40 h) using the synthetic biofilm. Cyclic voltammetry data showed H 2 formation occurred at much higher potentials than in the control reactors, (approx. −550 mV vs. −950 mV for controls). Imaging showed that the synthetic biofilm allowed for dense growth of S. ovata cells at the cathode, increasing electron transfer efficiency and potentially improving the bio-catalyzation of H 2. [Display omitted] [ABSTRACT FROM AUTHOR]

The increasing environmental impact of fuels and chemicals from non-renewable fossil-based resources, as well as possible depletion of the latter, has spurred interest in sustainable alternatives. Fuels and chemicals can be produced in a carbon-neutral manner from renewable feedstock via biological processes. Short-and medium-chain fatty acids (SCCA and MCCA, respectively) have potential applications in the food, pharmaceutical, chemical, and biofuel industries. Microbial production of valuable elongated MCCA from SCCA requires an electron donor. The present study investigated the influence of ethanol as an electron donor for the mixed microbial fermentation of SCCA (C2-C5) and MCCA (C6) from brewery spent grains as feedstock. Chain elongation of SCCA to MCCA was evaluated under different ethanol concentrations (3, 6, 9, 12, 15, and 18 g/L) and compared with a non-ethanol control. Acidogenic fermentation successfully converted brewery spent grains to SCCA, reaching 19.66 gCOD/L (15 g/L ethanol supplementation) along with bio-hydrogen production of 41%. Accumulated SCCA were elongated to MCCA in a reverse β oxidation pathway to 9.1 gCOD/L of caproic acid (9 g/L ethanol). Ethanol consumption displayed a good correlation with MCCA formation, confirming the chain elongation capability of mixed cultures. Volatile solids were reduced by more than 70%. Continuous hydrolysis of the substrate with the release of sugars points to the beneficial role of mixed culture fermentation for the production of renewable fuels and chemicals. [Display omitted] • Carboxylic acids and biohydrogen production from BSG by acidogenic fermentation. • Ethanol as electron donor was examined for chain elongation of SCCA to MCCA. • Mixed culture was effective and robust degrading untreated BSG with resource recovery. • 9 g/L ethanol dose was optimum with highest caproic production of 9.1 gCOD/L. [ABSTRACT FROM AUTHOR]

Abstract: The influence of aeration on the fermentation of a mixture of glucose and xylose by the naturally xylose fermenting fungus Fusarium oxysporum was studied in batch cultivations. The aeration level had considerable influence on the co-metabolism of glucose and xylose. Under anaerobic conditions xylose consumption was limited and xylitol was the main product of xylose metabolism. When the artificial electron acceptor acetoin was added xylose consumption increased by 95% with a significant improvement of ethanol yield from 0.96 to 1.52mol/mol, mainly due to a 72% reduction of xylitol excretion. The profile of the phosphorylated intermediates during the fermentation suggests that a high glycolytic flux occurred during the oxygen-limited conditions, characterized by high efflux of glyceraldehyde-3-P (G3P) and fructose-6-P (F6P) from Pentose Phosphate Pathway (PPP) to Embden-Meyerhof-Parnas Pathway (EMP) resulting in the higher ethanol production. An accumulation of sedoheptulose-7-P (S7P) was also observed indicating a block in the PPP which affects the production of NADPH and results in the production of acetate and the reduced xylose consumption. [Copyright &y& Elsevier]

Long-chain polyunsaturated fatty acids comprising omega (ω)-3 and ω-6 are essential fatty acids with myriad health benefits. Marine microalgae, such as Phaeodactylum, Nannochloropsis , and Crypthecodinium , have emerged as a potential source of ω-3 fatty acids, which comprise 50–70% of their total fatty acid content. Although there is a plethora of studies on bioprocess optimization, the system-level information of these microalgae remains restricted to model microalgae. Omics studies have moonlighted the bioprocess optimization by identifying the presence of CAZymes, xylose isomerase, and other pathways for utilization of unconventional carbon sources, such as glycerol. Similarly, transcriptomics and metabolomics studies have aided understanding of the effect of various abiotic factors, enabling the unraveling of several molecular mechanisms for ω-3 fatty acid production. 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. [ABSTRACT FROM AUTHOR]

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. Squalene is an isoprenoid hydrocarbon naturally synthesized via the mevalonate (MVA) or methyl-D-erythritol 4-phosphate (MEP) pathways and is the key precursor for cholesterol biosynthesis. Microbial sources are alternatives to plant and animal sources; however, genetic manipulation of the native squalene biosynthesis pathway can turn microorganisms into a cell factories for squalene production at industrial scale. Overexpression of MEP or MVA pathway structural genes has been used to boost metabolic flux towards squalene synthesis. Downregulation or knockout of genes responsible for the conversion of squalene to sterols can enhance the overall titer of squalene in yeast. Squalene synthesis is higher in thraustochtrids than in any other known microbe; however, further improvement in squalene titer in these marine protists by genetic manipulation of native biochemical pathways remains a challenge. [ABSTRACT FROM AUTHOR]