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.

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.

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.

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.

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.

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.

Pretreatment of Luzhou distiller's grains for feed protein production using crude enzymes produced by a synthetic microbial consortium.

Chinese distillers' grains (CDGs) have low fermentation efficiency due to the presence of lignocellulosic components, such as rice husk. In this study, a microbial consortium synthesized was used based on the "functional complementarity" principle to produce lignocellulolytic crude enzyme. The crude enzyme was used to hydrolyze CDGs. After enzymatic hydrolysis, lignocellulose was damaged to varying degrees and the crystallinity decreased. Subsequently, the feed protein was produced using yeast through two pathways. The results showed that the crude enzyme produced by the microbial consortium (comprising Trichoderma reesei, Aspergillus niger, and Penicillium) exhibited excellent enzymatic efficiency, yielding 27.88%, 19.64%, and 10.88% of reducing sugar, cellulose, and hemicellulose. The true protein content of CDGs increased by 53.49% and 48.35% through the first and second pathways, respectively. Notably, the second pathway demonstrated higher economic benefits to produce feed protein. This study provides a pathway for high-quality utilization of CDGs.

Construction of a synthetic microbial community based on multiomics linkage technology and analysis of the mechanism of lignocellulose degradation.

The efficient degradation of lignocellulose is a bottleneck for its integrated utilization. This research performed species analysis and made functional predictions in various ecosystems using multiomics coupling to construct a core synthetic microbial community with efficient lignocellulose degradation function. The synthetic microbial community was employed to degrade corn straw via solid-state fermentation. The degradation mechanisms were resolved using proteomics. The optimum culture conditions included 10% inoculum level (w/v), 4% nitrogen source ratio and a fermentation time of 23 d. Under these conditions, the degradation rates of cellulose, hemicellulose, and lignin were 34.91%, 45.94%, and 23.34%, respectively. Proteomic analysis revealed that lignin 1,4-ß-xylanase, ß-xylosidase and endo-1,4-ß-xylanase were closely related to lignocellulose degradation. The metabolic pathways involved in lignocellulose degradation and the functional roles of eight strains were obtained. The synthesis of a microbial community via multiomics linkage technology can effectively decompose lignocellulose, which is useful for their further utilization.

Employing Cationic Kraft Lignin as Additive to Enhance Enzymatic Hydrolysis of Corn Stalk.

A water-soluble cationic kraft lignin (named JLQKL50), synthesized by combining quaternization and crosslinking reactions, was used as an additive to enhance the enzymatic hydrolysis of dilute-alkali-pretreated corn stalk. The chemical constitution of JLQKL50 was investigated by Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR) and 13C NMR spectroscopy, and elemental analysis. The enzymatic hydrolysis efficiency of corn stalk at solid content of 10% (w/v) was significantly improved from 70.67% to 78.88% after 24 h when JLQKL50 was added at a concentration of 2 g/L. Meanwhile, the enzymatic hydrolysis efficiency after 72 h reached 91.11% with 10 FPU/g of cellulase and 97.92% with 15 FPU/g of cellulase. In addition, JLQKL50 was found capable of extending the pH and temperature ranges of enzymatic hydrolysis to maintain high efficiency (higher than 70%). The decrease in cellulase activity under vigorous stirring with the addition of JLQKL50 was 17.4%, which was much lower than that (29.7%) without JLQKL50. The addition of JLQKL50 reduced the nonproductive adsorption of cellulase on the lignin substrate and improved the longevity, dispersity, and stability of the cellulase by enabling electrostatic repulsion. Therefore, the enzymatic hydrolysis of the corn stalk was enhanced. This study paves the way for the design of sustainable lignin-based additives to boost the enzymatic hydrolysis of lignocellulosic biomass.

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.

Engineering an efficient whole-cell catalyst for d-allulose production from glycerol.

d-Allulose has many health-benefiting properties and therefore sustainably applied in food, pharmaceutical, and nutrition industries. The aldol reaction-based route is a very promising alternative to Izumoring strategy in d-allulose production. Remarkable studies reported in the past cannot get rid of by-product formation and costly purified enzyme usage. In the present study, we explored the glycerol assimilation by modularly assembling the d-allulose synthetic cascade in Escherichia coli envelop. We achieved an efficient whole-cell catalyst that produces only d-allulose from cheap glycerol feedstock, eliminating the involvement of purified enzymes. Detailed process optimization improved the d-allulose titer by 1500.00%. Finally, the production was validated in 3-L scale using a 5-L fermenter, and 5.67 g L-1 d-allulose was produced with a molar yield of 31.43%.

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.

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.

Assessment of the Tolerance of a Chlorophyte Desmodesmus to CuO-NP for Evaluation of the Nanopollution Bioremediation Potential of This Microalga.

Broad application of CuO nanoparticles (CuO-NP) for industrial and household purposes leads to a continuous increase in their discharge to, and, hence, ever-increasing environmental hazards for aquatic ecosystems. Microalgae-based technologies hold promise for bioremediation of diverse hazardous micropollutants (HMP), including NP, from wastewater. In this study, we tested the ability of the green microalga Desmodesmus sp. to accumulate CuO-NP or their components. We also assessed the tolerance of this microalga to the environmentally relevant concentrations of CuO-NP. Using scanning electron microscopy, we demonstrated that the average size of CuO-NP was 50-100 nm, and their purity was confirmed with elemental composition analysis. Tests of the colloidal suspensions of CuO-NP showed that the hydrodynamic diameter of CuO-NP and their aggregates was below 100 nm. Flow cytometry analysis showed that CuO-NP at a concentration of 100 µg L-1 slightly inhibited the viability of microalgae cells and led to an increase in their oxidative stress. The assessment of the condition of photosystem II showed that CuO-NP exert a multifaceted effect on the photosynthetic apparatus of Desmodesmus sp., depending on the concentration of and the exposure to the CuO-NP. Desmodesmus sp. turned to be relatively tolerant to CuO-NP. In addition, the ICP-MS method revealed increased bioaccumulation of copper by microalgae cells in the experimental groups. The outcomes of this study indicate that the Desmodesmus sp. has a significant potential for bioremoval of the copper-based nanostructured HMP from an aquatic environment.

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.

Coupling with in-situ electrochemical reactive chlorine species generation and two-phase partitioning method for enhanced microalgal biodiesel production.

Microalgal-based biofuel production is of great significance in alleviating energy crisis and achieving carbon neutrality. However, the excessive costs and high solvent consumption in lipids extraction from microalgal obstruct the widespread application of biodiesel in practice. Reported herein is the construction of facile strategy for lipids extraction via electrocatalytic pretreatment and a subsequent two-phase partitioning method. Electrocatalytic pretreatment method adopts the solar as power source and avoids the drying of microalgal biomass, in favor of carbon neutrality requirement. During this process, eco-friendly electrode with high specific surface area could contribute to the sufficient generation of reactive chlorine species (RCS), facilitating the outflows of intracellular lipid. As a result, assisted with two-phase partitioning method, a satisfied performance of lipid recovery (86.72 %) was obtained. Notably, compared with traditional solvent method, two-phase partitioning method greatly reduced the dosage of organic solvent, which is an economical or environmental technique.

Research Note: The gut microbiota varies with dietary fiber levels in broilers.

It is generally accepted the gut microbiota have a profound effect on the nutrition, health, and production in poultry. To deeply understand the gut microbiota composition with the dietary fiber level in broilers, we evaluated the cecal microbiota profiles feeding on different dietary fiber level with alfalfa as additive in Dahen broilers based on 16S rRNA gene sequencing and gas chromatography. As a result, the gut microbiota diversity was greatly accelerated with the dietary fiber level. The dietary fiber stimulated the growth of many intestinal communities such as Rikenellaceae RC9 gut group, Faecalibacterium, Prevotellaceae UCG 001 and Ruminococcaceae UCG 014, and led to an altered microbial function such as Carbohydrate metabolism and Genetic information processing. Meanwhile, we found the genera Anaerofilum and Dielma were significantly correlated with the production of short chain fatty acids (SCFAs). All these results provide a reference for the broilers gut microbiota changes with different dietary fiber level. The key role of the altered microbiota with the dietary fiber may mediate beneficial effects in broiler production, which also reflect the substantial potential of dietary fiber level in poultry.

Water-plasma-enhanced and phase-separation-assisted extraction of microalgal lipid for biodiesel production.

Traditional methods for lipid extraction from microalgal biomass usually involve harsh reaction conditions or the use of contaminant reagents, which lead to enormous energy consumption and wastage. Hence, a novel strategy was presented, which combined water-plasma and three-phase partitioning (TPP) techniques. Benefiting from its unique advantages such as rapid and low cost, water-plasma strategy can disrupt microalgal cell wall and can thus greatly affect lipid extraction. As a result, assisted with the TPP method, excellent performance lipid recovery (74.34%) was obtained at 200 W in 10 min. The performance was superior to that achieved through cell disruption via water-plasma pretreatment. Importantly, the whole process of lipid extraction prevented the drying of microalgal biomass, contributing to reduced energy consumption in large-scale biodiesel production. Moreover, the high fatty acids content suggested that the extracted lipids are great potential candidate for biodiesel production.

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.