Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Immobilization of rough morphotype Mycolicibacterium neoaurum R for androstadienedione production.

OBJECTIVES: Enhance the androstadienedione (Androst-1,4-diene-3,17-dione, ADD) production of rough morphotype Mycolicibacterium neoaurum R by repeated-batch fermentation of immobilized cells. RESULTS: M. neoaurum R was a rough colony morphotype variant, obtained from the routine plating of smooth M. neoaurum strain CICC 21097. M. neoaurum R showed rougher cell surface and aggregated in broth. The ADD production of M. neoaurum R was notably lower than that of M. neoaurum CICC 21097 during the free cell fermentation, but the yield gap could be erased after proper cell immobilization. Subsequently, repeated-batch fermentation of immobilized M. neoaurum R was performed to shorten the production cycle and enhance the bio-production efficiency of ADD. Through the optimization of the immobilization carriers and the co-solvents for phytosterols, the ADD productivity of M. neoaurum R immobilized by semi-expanded perlite reached 0.075 g/L/h during the repeated-batch fermentation for 40 days. CONCLUSIONS: The ADD production of the rough-type M. neoaurum R was notably enhanced by the immobilization onto semi-expanded perlite. Moreover, the ADD batch yields of M. neoaurum R immobilized by semi-expanded perlite were maintained at high levels during the repeated-batch fermentation.

Magnetic biochar promotes the risk of mobile genetic elements propagation in sludge anaerobic digestion.

Mobile genetic elements (MGEs) mediated horizontal gene transfer is the primary reason for the propagation of antibiotic resistance genes in environment. The behavior of MGEs under magnetic biochar pressure in sludge anaerobic digestion (AD) is still unknown. This study evaluated the effects of different dosage magnetic biochar on the MGEs in AD reactors. The results showed that the biogas yield was highest (106.68 ± 1.16 mL g-1 VSadded) with adding optimal dosage of magnetic biochar (25 mg g-1 TSadded), due to it increased the microorganism's abundance involved in hydrolysis and methanogenesis. While, the total absolute abundance of MGEs in the reactors with magnetic biochar addition increased by 11.58%-77.37% compared with the blank reactor. When the dosage of magnetic biochar was 12.5 mg g-1 TSadded, the relative abundance of most MGEs was the highest. The enrichment effect on ISCR1 was the most significant, and the enrichment rate reached 158.90-214.16%. Only the intI1 abundance was reduced and the removal rates yield 14.38-40.00%, which was inversely proportional to the dosage of magnetic biochar. Co-occurrence network explored that Proteobacteria (35.64%), Firmicutes (19.80%) and Actinobacteriota (15.84%) were the main potential host of MGEs. Magnetic biochar changed MGEs abundance by affecting the potential MGEs-host community structure and abundance. Redundancy analysis and variation partitioning analysis showed that the combined effect of polysaccharides, protein and sCOD exhibited the greatest contribution (accounted for 34.08%) on MGEs variation. These findings demonstrated that magnetic biochar increases the risk of MGEs proliferation in AD system.

Heat-induced overexpression of the thermophilic lipase from Bacillus thermocatenulatus in Escherichia coli by fermentation and its application in preparation biodiesel using rapeseed oil.

Due to its simple, less by-product and environment friendly properties, enzymatic transesterification of oil with short-chain alcohol to biodiesel, fatty acid methyl esters (FAMEs) is considered to be a promising way of green production and has attracted much attention. In this study, FAMEs were synthesized by an enzymatic method with recombinant lipase as catalysts. A thermophilic Bacillus thermocatenulatus lipase 2 (BTL2) was overexpressed in Escherichia coli BL21(DE3) through relative and quantitative analysis using real-time quantitative PCR. The results suggested that the BTL2 gene was overexpressed in E. coli at the mRNA level, and the recombinant strain harboring a high-copy number vectors was selected and applied to fermentation to produce BTL2 with enzyme activity of 35.54 U/mg cells. The recombinant BTL2 solution exhibited excellent resistance to neutral pH, high temperature, and organic solvents after a certain treatment. Finally, the effects of enzymatic transesterification for preparing biodiesel were studied, using rapeseed oil as raw material, as well as BTL2 solution as catalysts, which resulted in 86.04% yield of FAMEs under 50°C for 36 h. The liquid BTL2 was directly used to prepare FAMEs at a higher temperature efficiently, making the thermophilic BTL2 had the potential application value in biodiesel reproduction subsequently.

Leaf reflectance and functional traits as environmental indicators of urban dust deposition.

BACKGROUND: How to quickly predict and evaluate urban dust deposition is the key to the control of urban atmospheric environment. Here, we focus on changes of plant reflectance and plant functional traits due to dust deposition, and develop a prediction model of dust deposition based on these traits. RESULTS: The results showed that (1) The average dust deposition per unit area of Ligustrum quihoui leaves was significantly different among urban environments (street (18.1001 g/m2), community (14.5597 g/m2) and park (9.7661 g/m2)). Among different urban environments, leaf reflectance curves tends to be consistent, but there were significant differences in leaf reflectance values (park (0.052-0.585) > community (0.028-0.477) > street (0.025-0.203)). (2) There were five major reflection peaks and five major absorption valleys. (3) The spectral reflectances before and after dust removal were significantly different (clean leaves > dust-stagnant leaves). 695 ~ 1400 nm was the sensitive range of spectral response. (4) Dust deposition has significant influence on slope and position of red edge. Red edge slope was park > community > street. After dust deposition, the red edge position has obviously "blue shift". The moving distance of the red edge position increases with the increase of dust deposition. The forecast model of dust deposition amount established by simple ratio index (y = 2.517x + 0.381, R2 = 0.787, RMSE (root-mean-square error) = 0.187. In the model, y refers to dust retention, x refers to simple ratio index.) has an average accuracy of 99.98%. (5) With the increase of dust deposition, the specific leaf area and chlorophyll content index decreased gradually. The leaf dry matter content, leaf tissue density and leaf thickness increased gradually. CONCLUSION: In the dust-polluted environment, L. quihoui generally presents a combination of characters with lower specific leaf area, chlorophyll content index, and higher leaf dry matter content, leaf tissue density and leaf thickness. Leaf reflectance spectroscopy and functional traits have been proved to be effective in evaluating the changes of urban dust deposition.

Algae-Derived Bioactive Molecules for the Potential Treatment of SARS-CoV-2.

The recently emerged COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has adversely affected the whole world. As a significant public health threat, it has spread worldwide. Scientists and global health experts are collaborating to find and execute speedy diagnostics, robust and highly effective vaccines, and therapeutic techniques to tackle COVID-19. The ocean is an immense source of biologically active molecules and/or compounds with antiviral-associated biopharmaceutical and immunostimulatory attributes. Some specific algae-derived molecules can be used to produce antibodies and vaccines to treat the COVID-19 disease. Algae have successfully synthesized several metabolites as natural defense compounds that enable them to survive under extreme environments. Several algae-derived bioactive molecules and/or compounds can be used against many diseases, including microbial and viral infections. Moreover, some algae species can also improve immunity and suppress human viral activity. Therefore, they may be recommended for use as a preventive remedy against COVID-19. Considering the above critiques and unique attributes, herein, we aimed to systematically assess algae-derived, biologically active molecules that could be used against this disease by looking at their natural sources, mechanisms of action, and prior pharmacological uses. This review also serves as a starting point for this research area to accelerate the establishment of anti-SARS-CoV-2 bioproducts.

Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield.

Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield in long-term and large-scale bioproduction.

Polyethylenimine-based nanovector grafted with mannitol moieties to achieve effective gene delivery and transfection.

Polyethylenimine (PEI), a kind of cationic non-viral gene delivery vector, is capable of stable and efficient transgene expression for gene delivery. However, low transfection efficiency in vivo along with high toxicity limited the further application of gene therapy in the clinic. To enhance gene transfection performance and reduce cytotoxicity of polyethylenimine, branched polyethylenimine-derived cationic polymers BPEI25 k-man-S/L/M/H with different grafting degree with mannitol moieties were prepared and the transfection efficiency was evaluated. Among them, BPEI25 k-man-L showed the best transfection efficiency, lower toxicity, and significantly enhanced long-term systemic transgene expression for 96 h in vivo even at a single-dose administration. The results of cellular uptake mechanism and western-blot experiments revealed that the mannitol modification of BPEI25 k induced and up-regulated the phosphorylation of caveolin-1 and thus enhanced the caveolae-mediated cellular uptake. This class of gene delivery system highlights a paradigmatic approach for the development of novel and safe non-viral vectors for gene therapy.

Enhance transgene responses through improving cellular uptake and intracellular trafficking by bio-inspired non-viral vectors.

BACKGROUND: Gene therapy remains a significant challenge due to lots of barriers limiting the genetic manipulation technologies. As for non-viral delivery vectors, they often suffer insufficient performance due to inadequate cellular uptake and gene degradation in endosome or lysosome. The importance of overcoming these conserved intracellular barriers is increasing as the delivery of genetic cargo. RESULTS: A surface-functionalized non-viral vector involving the biomimetic mannitol moiety is initiated, which can control the cellular uptake and promote the caveolae-mediated pathway and intracellular trafficking, thus avoiding acidic and enzymatic lysosomal degradation of loaded gene internalized by clathrin-mediated pathway. Different degrees of mannitol moiety are anchored onto the surface of the nanoparticles to form bio-inspired non-viral vectors and CaP-MA-40 exhibits remarkably high stability, negligible toxicity, and significantly enhanced transgene expression both in vitro and in vivo. CONCLUSIONS: This strategy highlights a paradigmatic approach to construct vectors that need precise intracellular delivery for innovative applications.

Engineering Corynebacterium glutamicum Mutants for 3-Methyl-1-butanol Production.

3-Methyl-1-butanol (3MB) is a promising biofuel that can be produced from 2-ketoisocaproate via the common L-leucine biosynthesis pathway. Corynebacterium glutamicum was chosen as a host bacterium because of its strong resistance to isobutanol. In the current study, several strategies were designed to overproduce 3MB in C. glutamicum through a non-fermentation pathway. The engineered C. glutamicum mutant was obtained by silencing the pyruvate dehydrogenase gene complex (aceE) and deleting the lactic dehydrogenase gene (ldh), followed by mutagenesis with diethyl sulfate (DES) and selection with Fmoc-3-4-thiazolyl-L-alanine (FTA). The mutant could produce 659 mg/L of 3MB after 12 h of incubation. To facilitate carbon flux to 3MB biosynthesis, the engineered recombinant was also constructed without branched-chain acid aminotransferase (ilvE) activity by deleting the ilvE gene. This recombinant could produce 697 mg/L of 3MB after 12 h of incubation.

Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 µmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

Water-saving analysis on an effective water reuse system in biodiesel feedstock production based on Chlorella zofingiensis fed-batch cultivation.

The micralgae-based biofuel obtained from dairy wastewater (DWW) is considered a promising source of energy. However, this process consumes water due to the concentration of wastewater being normally too high for some micoralgae cultivation, and dilution is always needed. In this work, the cultivation of microalgae has been examined in non-recirculated water (NR) and recirculated water systems (R). The growth of Chlorella zofingiensis and the nutrient removal of DWW have been recorded. The comparison indicates the R had a little more advantage in biomass and lipid output (1.55, 0.22 g, respectively) than the NR (1.51, 0.20 g, respectively). However, the total chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorus (TP) removals of the R were lower than those of the NR system during the culture. The highest removal of total COD, TKN, and TP were 85.05%, 93.64%, and 98.45%, respectively. Furthermore, no significant difference has been observed in the higher heating value and lipid content of the biomass of the R and NR. The results show the R can save 30% of the total water input during the culture. All above results indicate the R system has great potential in industry.

Current progress and future perspective of microalgae biomass pretreatment using deep eutectic solvents.

Pretreatment process is considered as the most important step for effective microalgae biomass refining and has gained more interest since last decades. However, the main obstacles to commercialize microalgae products are recalcitrant cell wall and lack of cost-effective, green, and sustainable pretreatment approaches. Till now, various microalgae pretreatment approaches have been applied prior to extraction steps to enhance the accessibility of solvent inside the cells. However, high energy consumption and the hazardousness of solvents are considerable problem for these pretreatment methods. In this regard, deep eutectic solvents are recognized as sustainable and green solvents possessing great potential for microalgae biomass processing due to their low toxicity, low cost, biodegradability, easy recycling, and reuse. This article provides the fundamentals of DES composition, synthesis, properties, and the current advances in the application of microalgae biomass process.

Pretreatment of shrimp shells with an acidic deep eutectic solvent system for chitin extraction and its enhanced performance as a carrier for immobilized lipase.

Existing methods for chitin extraction usually produce substantial waste, which poses ecological hazards. Natural deep eutectic solvent (NADES) offers a promising one-step pretreatment alternative, replacing the resource-intensive demineralization (DM) and deproteinization (DP) process. Hence, in this study, the influence of various acidic NADES, on achieving a simplified one-step DM and DP in the chitin extraction process was investigated. The study yielded chitin with 87.73 % purity, and microstructural analysis showed that NADES pretreatment minimally affected chitin quality without deacetylation. In addition, chitin extracted using choline chloride-oxalic acid as a carrier displayed excellent performance in the immobilization of Geobacillus thermocatenulatus lipase 2 (GTL2) because of obvious Ca2+ activation effect. This process contributed to enhancement of immobilized enzyme activity. The immobilized GTL2 showed excellent hydrolytic capabilities, with its highest activity reaching 547.80 ± 20.62 U/mg, significantly better than the five commercial lipases that exhibited <40 % of the enzyme activity. Furthermore, the hydrolytic capacity of immobilized GTL2 was notably high for 4-nitrophenyl butyrate, measuring 935.47 ± 51.60 U/mg. This study provided a constructive approach for the one-step pretreatment of shrimp shells with organic acid-based NADES to isolate and purify chitin and its potential application as an immobilized carrier to enhance enzyme activity.

Solid-state fermentation of corn straw using synthetic microbiome to produce fermented feed: The feed quality and conversion mechanism.

Straw is a typical biomass resource which can be converted into high nutritional value feed via microbial fermentation. The degradation and conversion of straw using a synthetic microbial community (SMC-8) was functionally investigated to characterise its nitrogen conversion and carbon metabolism. Four species of bacteria were found to utilise >20 % of the inorganic nitrogen within 15 h, and the ratio of the diameter of fungal transparent circles (D) to the diameter of the colony (d) of the four fungal species was >1. Solid-state fermentation of corn straw increased the total amino acid (AA) content by 41.69 %. The absolute digestibility of fermented corn straw dry weight (DW) and true protein was 34.34 % and 45.29 %, respectively. Comprehensive analysis of functional proteins revealed that Aspergillus niger, Trichoderma viride, Cladosporium cladosporioides, Bacillus subtilis and Acinetobacter johnsonii produce a complex enzyme system during corn straw fermentation, which plays a key role in the degradation of lignocellulose. This study provided a new insight in utilizing corn straw.

Improving Microbial Cell Factory Performance by Engineering SAM Availability.

Methylated natural products are widely spread in nature. S-Adenosyl-l-methionine (SAM) is the secondary abundant cofactor and the primary methyl donor, which confer natural products with structural and functional diversification. The increasing demand for SAM-dependent natural products (SdNPs) has motivated the development of microbial cell factories (MCFs) for sustainable and efficient SdNP production. Insufficient and unsustainable SAM availability hinders the improvement of SdNP MCF performance. From the perspective of developing MCF, this review summarized recent understanding of de novo SAM biosynthesis and its regulatory mechanism. SAM is just the methyl mediator but not the original methyl source. Effective and sustainable methyl source supply is critical for efficient SdNP production. We compared and discussed the innate and relatively less explored alternative methyl sources and identified the one involving cheap one-carbon compound as more promising. The SAM biosynthesis is synergistically regulated on multilevels and is tightly connected with ATP and NAD(P)H pools. We also covered the recent advancement of metabolic engineering in improving intracellular SAM availability and SdNP production. Dynamic regulation is a promising strategy to achieve accurate and dynamic fine-tuning of intracellular SAM pool size. Finally, we discussed the design and engineering constraints underlying construction of SAM-responsive genetic circuits and envisioned their future applications in developing SdNP MCFs.

A novel integrated approach employing Desertifilum tharense BERC-3 for efficient wastewater valorization and recycling for developing peri-urban algae farming system.

Peri-urban environments are significant reservoirs of wastewater, and releasing this untreated wastewater from these resources poses severe environmental and ecological threats. Wastewater mitigation through sustainable approaches is an emerging area of interest. Algae offers a promising strategy for carbon-neutral valorization and recycling of urban wastewater. Aiming to provide a proof-of-concept for complete valorization and recycling of urban wastewater in a peri-urban environment in a closed loop system, a newly isolated biocrust-forming cyanobacterium Desertifilum tharense BERC-3 was evaluated. Here, the highest growth and lipids productivity were achieved in urban wastewater compared to BG11 and synthetic wastewater. D. tharense BERC-3 showed 60-95% resource recovery efficiency and decreased total dissolved solids, chemical oxygen demand, biological oxygen demand, nitrate nitrogen, ammonia nitrogen and total phosphorus contents of the water by 60.37%, 81.11%, 82.75%, 87.91%, 85.13%, 85.41%, 95.87%, respectively, making it fit for agriculture as per WHO's safety limits. Soil supplementation with 2% wastewater-cultivated algae as a soil amender, along with its irrigation with post-treated wastewater, improved the nitrogen content and microbial activity of the soil by 0.3-2.0-fold and 0.5-fold, respectively. Besides, the availability of phosphorus was also improved by 1.66-fold. The complete bioprocessing pipeline offered a complete biomass utilization. This study demonstrated the first proof-of-concept of integrating resource recovery and resource recycling using cyanobacteria to develop a peri-urban algae farming system. This can lead to establishing wastewater-driven algae cultivation systems as novel enterprises for rural migrants moving to urban areas.

High-Level Production of Recombinant Lipase by Fed-Batch Fermentation in Escherichia coli and Its Application in Biodiesel Synthesis from Waste Cooking Oils.

The enzymatic production of biodiesel from waste cooking oils (WCOs) offers a green and sustainable solution for the liquid fuel manufacture as well as waste resource recovery. In present study, liquid lipase was used to simplify the catalysis process, thereby reducing biodiesel production costs. An engineered Escherichia coli expressing Geobacillus thermocatenulatus lipase 2 (GTL2) was screened at an enzyme activity of 6.96 U/mg, after evaluating the propagating stability of the recombinant plasmids exceeding 86.11%. Through the beneficial feeding strategy and effective pH control, high-level production of GTL2 by fed-batch fermentation was achieved with an enzyme activity of 434.32 U/mg, which was almost 62 times that of shake flask fermentation. In addition, liquid GTL2 was used to prepare fatty acid methyl esters (FAMEs) using WCOs. The effects of the reaction time, catalyst loading, temperature, and methanol-to-oil molar ratio on FAMEs production using WCOs were explored, and a maximum FAMEs yield of 96.62% was achieved under optimized conditions. These results indicate that liquid GTL2 is a promising biocatalyst for efficient utilization of WCOs in the synthesis of biodiesel and provide a novel enzymatic process for biodiesel reducing the cost of production.

Produce D-allulose from non-food biomass by integrating corn stalk hydrolysis with whole-cell catalysis.

D-allulose is a high-value rare sugar with many health benefits. D-allulose market demand increased dramatically after approved as generally recognized as safe (GRAS). The current studies are predominantly focusing on producing D-allulose from either D-glucose or D-fructose, which may compete foods against human. The corn stalk (CS) is one of the main agricultural waste biomass in the worldwide. Bioconversion is one of the promising approach to CS valorization, which is of significance for both food safety and reducing carbon emission. In this study, we tried to explore a non-food based route by integrating CS hydrolysis with D-allulose production. Firstly we developed an efficient Escherichia coli whole-cell catalyst to produce D-allulose from D-glucose. Next we hydrolyzed CS and achieved D-allulose production from the CS hydrolysate. Finally we immobilized the whole-cell catalyst by designing a microfluidic device. Process optimization improved D-allulose titer by 8.61 times, reaching 8.78 g/L from CS hydrolysate. With this method, 1 kg CS was finally converted to 48.87 g D-allulose. This study validated the feasibility of valorizing corn stalk by converting it to D-allulose.

Determination of active sites on the synthesis of novel Lewis acidic deep eutectic solvent catalysts and kinetic studies in microalgal biodiesel production.

Experimental and theoretical considerations for kinetic modeling of the transesterification reaction of microalgae lipids into biodiesel were investigated using Lewis acid deep eutectic solvents (DESs) as a catalyst. The acid sites involved in the reaction were characterized using acetonitrile as a probe to understand the mechanism. DES ChCl-SnCl2 (choline chloride-tin ii chloride) showed higher catalytic activity in transesterification due to its higher acidity compared to DES ChCl-ZnCl2 (choline chloride-zinc chloride). This was illustrated by geometric optimization of the DES structures through density functional theory (DFT) which showed that the metal centers furthest from the choline moiety are the most acidic and the bond lengths of Sn-Cl were between 2.56 and 2.77 Å, and were greater than the Zn-Cl bond lengths from 2.30 to 2.48 Å, making the ChCl-SnCl2 DES more acidic and more suitable for the biodiesel production. The fatty acid methyl ester (FAME) conversion from microalgae lipid was 36.75 mg g-1 under ideal conditions (6 molar ratio methanol-lipid with 8 vol% DES dosage in methanol at 140 °C for 420 min). The activation energy is found to be 36.3 kJ mol-1 based on the pseudo-first-order reaction, in addition, the DES catalyst (ChCl-SnCl2) drove the reaction chemically and did not show mass transfer limitation. Information from this study can help to advance the development of an efficient and environmentally friendly industrial biodiesel production technology.