Metabolic Remodulation of Chassis and Corn Stover Bioprocessing to Unlock 3-Hydroxypropionic Acid Biosynthesis from Agrowaste-Derived Substrates.

Embracing the principles of sustainable development, the valorization of agrowastes into value-added chemicals has nowadays received significant attention worldwide. Herein, Escherichia coli was metabolically rewired to convert cellulosic hydrolysate of corn stover into a key platform chemical, namely, 3-hydroxypropionic acid (3-HP). First, the heterologous pathways were introduced into E. coli by coexpressing glycerol-3-P dehydrogenase and glycerol-3-P phosphatase in both single and fusion (gpdp12) forms, making the strain capable of synthesizing glycerol from glucose. Subsequently, a glycerol dehydratase (DhaB123-gdrAB) and an aldehyde dehydrogenase (GabD4) were overexpressed to convert glycerol into 3-HP. A fine-tuning between glycerol synthesis and its conversion into 3-HP was successfully established by 5'-untranslated region engineering of gpdp12 and dhaB123-gdrAB. The strain was further metabolically modulated to successfully prevent glycerol flux outside the cell and into the central metabolism. The finally remodulated chassis produced 32.91 g/L 3-HP from the cellulosic hydrolysate of stover during fed-batch fermentation.

Sustainable bio-manufacturing of D-arabitol through combinatorial engineering of Zygosaccharomyces rouxii, bioprocess optimization and downstream separation.

Biosynthesis of D-arabitol, a high value-added platform chemical, from renewable carbon sources provides a sustainable and eco-friendly alternative to the chemical industry. Here, a robust brewing yeast, Zygosaccharomyces rouxii, capable of naturally producing D-arabitol was rewired through genome sequencing-based metabolic engineering. The recombinant Z. rouxii obtained by reinforcing the native D-xylulose pathway, improving reductive power of the rate-limiting step, and inhibiting the shunt pathway, produced 73.61% higher D-arabitol than the parent strain. Subsequently, optimization of the fermentation medium composition for the engineered strain provided 137.36 g/L D-arabitol, with a productivity of 0.64 g/L/h in a fed-batch experiment. Finally, the downstream separation of D-arabitol from the complex fermentation broth using an ethanol precipitation method provided a purity of 96.53%. This study highlights the importance of D-xylulose pathway modification in D-arabitol biosynthesis, and pave a complete and efficient way for the sustainable manufacturing of this value-added compound from biosynthesis to preparation.

A comprehensive review of recent advances in the characterization of L-rhamnose isomerase for the biocatalytic production of D-allose from D-allulose.

D-Allose and D-allulose are two important rare natural monosaccharides found in meager amounts. They are considered to be the ideal substitutes for table sugar (sucrose) for, their significantly lower calorie content with around 80 % and 70 % of the sweetness of sucrose, respectively. Additionally, both monosaccharides have gained much attention due to their remarkable physiological properties and excellent health benefits. Nevertheless, D-allose and D-allulose are rare in nature and difficult to produce by chemical methods. Consequently, scientists are exploring bioconversion methods to convert D-allulose into D-allose, with a key enzyme, L-rhamnose isomerase (L-RhIse), playing a remarkable role in this process. This review provides an in-depth analysis of the extractions, physiological functions and applications of D-allose from D-allulose. Specifically, it provides a detailed description of all documented L-RhIse, encompassing their biochemical properties including, pH, temperature, stabilities, half-lives, metal ion dependence, molecular weight, kinetic parameters, specific activities and specificities of the substrates, conversion ratio, crystal structure, catalytic mechanism as well as their wide-ranging applications across diverse fields. So far, L-RhIses have been discovered and characterized experimentally by numerous mesophilic and thermophilic bacteria. Furthermore, the crystal forms of L-RhIses from E. coli and Stutzerimonas/Pseudomonas stutzeri have been previously cracked, together with their catalytic mechanism. However, there is room for further exploration, particularly the molecular modification of L-RhIse for enhancing its catalytic performance and thermostability through the directed evolution or site-directed mutagenesis.

Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose.

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

Biocatalytic gateway to convert glycerol into 3-hydroxypropionic acid in waste-based biorefineries: Fundamentals, limitations, and potential research strategies.

Microbial conversion of bioenergy-derived waste glycerol into value-added chemicals has emerged as an important bioprocessing technology due to its eco-friendliness, feasible technoeconomics, and potential to provide sustainability in biodiesel and bioethanol production. Glycerol is an abundant liquid waste from bioenergy plants with a projected volume of 6 million tons by 2025, accounting for about 10% of biodiesel and 2.5% of bioethanol yields. 3-Hydroxypropionic acid (3-HP) is a major product of glycerol bioconversion, which is the third largest biobased platform compound with expected market size and value of 3.6 million tons/year and USD 10 billion/year, respectively. Despite these biorefinery values, 3-HP biosynthesis from glycerol is still at an immature stage of commercial exploitation. The main challenges behind this immaturity are the toxic effects of 3-HPA on cells, the distribution of carbon flux to undesirable pathways, low tolerance of cells to glycerol and 3-HP, co-factor dependence of enzymes, low enzyme activity and stability, and the problems of substrate inhibition and specificity of enzymes. To address these challenges, it is necessary to understand the fundamentals of glycerol bioconversion and 3-HP production in terms of metabolic pathways, related enzymes, cell factories, midstream process configurations, and downstream 3-HP recovery, as discussed in this review critically and comprehensively. It is equally important to know the current challenges and limitations in 3-HP production, which are discussed in detail along with recent research efforts and remaining gaps. Finally, possible research strategies are outlined considering the recent technological advances in microbial biosynthesis, aiming to attract further research efforts to achieve a sustainable and industrially exploitable 3-HP production technology. By discussing the use of advanced tools and strategies to overcome the existing challenges in 3-HP biosynthesis, this review will attract researchers from many other similar biosynthesis technologies and provide a common gateway for their further development.

Magnetic/electric field intervention on oil-rich filamentous algae production in the application of acrylonitrile butadiene styrene based wastewater treatment.

Globally, the release of acrylonitrile-butadienestyrene (ABS) wastewater from numerous industries is a serious concern. Recently, oil-rich filamentous algae Tribonema sp has been grown utilizing toxic but nutrient-rich ABS effluent. Here, Tribonema sp. was cultivated under intervention of different magneto-electric combinatory fields (MCFs) (control, 0.6 V/cm, 1 h/d-1.2 V/cm, 1 h/d-0.6 V/cm, and 1 h/d-1.2 V/cm). Results showed MCF (1 h/d-0.6 V/cm) intervention increased the biomass by 9.7% (2.4 g/L) combined with high removal efficiencies (95% and 99%) of ammonium nitrogen and total phosphorus. The chemical oxygen demand (COD) removal rate increased to 82%, 6% higher than the control. Moreover, MCF of 1 h/d-0.6 V/cm significantly increased lipid and carbohydrate by 7.71% and 4.73% respectively. MCF increased premium fatty acid content such as palmitic acid (C16:0), myristic acid (C14: 0), and hexadecenoic acid (C16:1). MCF intervention also supported a diverse microbial flora, offering a favorable solution for ABS wastewater treatment.

Activity measurement of arylsulfatase and ß-glucuronidase in activated sludge: HPLC-based versus classical spectrophotometric method.

Arylsulfatase and ß-glucuronidase are two important enzymes in wastewater and surface water, which play important roles on cleavage of sulfate/glucuronide estrogens. In this work, a high-performance liquid chromatography (HPLC)-based new method was firstly established for arylsulfatase/ß-glucuronidase with determination of p-nitrophenyl sulfate (pNPS)/p-nitrophenyl-ß-D-glucuronide (pNPG). The limits of detections (LODs) of the developed method for pNPS and pNPG were 0.164 and 0.098 µM, respectively. Intraday and interday reproducibility expressed as relative standard deviation (RSD) values of retention times and peak areas was 0.39%-3.68% and 0.23%-4.74%, respectively. The respective recovery efficiencies of this HPLC-based method spiking at three different concentrations for p-nitrophenol (pNP), pNPS, and pNPG in activated sludge were 76.5%-88.1%, 79.2%-93.1%, and 84.2%-96.1%, with RSD below 3.9%. The HPLC-based method was finally applied to estimate the enzyme activity of arylsulfatase/ß-glucuronidase in one activated sludge system and along which the classical spectrophotometric method was also evaluated. Compared with the classic spectrophotometric analytical method, the HPLC-based new method could simultaneously measure arylsulfatase/ß-glucuronidase one time, which was convenient and time-saving. Moreover, the developed method could effectively avoid possible underestimation that the spectrophotometric method might encounter. PRACTITIONER POINTS: A new HPLC-based method for activity estimation of arylsulfatase and ß-glucuronidase was developed. The HPLC-based method can simultaneously estimate enzyme activity of both arylsulfatase and ß-glucuronidase. The HPLC-based method can avoid possible underestimation that spectrophotometric method may encounter.

Biomass production of carbohydrate-rich filamentous microalgae coupled with treatment and nutrients recovery from acrylonitrile butadiene styrene based wastewater: Synergistic enhancement with low carbon dioxide supply strategy.

This study attempted to remove acrylonitrile and acetophenone from simulated acrylonitrile butadiene styrene (ABS) based wastewater while recovering nitrogen and phosphorus using the carbohydrate-rich filamentous microalgae Tribonema sp.. Results showed that typical acetophenone and acrylonitrile presented significant inhibitory effect on Tribonema sp. growth and co-metabolism of CO2 improved the tolerance of Tribonema sp. to toxic pollutants. The microalgae biomass increased by 34.47% (3.16 g/L) and 58.17% (3.97 g/L) via supplementing 2% CO2 in the 100 mg/L acrylonitrile and acetophenone groups, respectively. The filamentous microalga was rich in carbohydrates and its productivity was further enhanced by 32.52% and 70.34%, respectively, in 100 mg/L acrylonitrile and acetophenone groups with 2% CO2 supplement. The synergistic CO2 supply strategy effectively enhanced the biomass production of filamentous microalgae, and moreover, improved the treatment efficiency of ABS based wastewater simulated by acetophenone or acrylonitrile addition, while at same time enhanced the recovery of nitrogen and phosphorus nutrients.

Improvement of betalains stability extracted from red dragon fruit peel by ultrasound-assisted microencapsulation with maltodextrin.

Natural betalains can be potential food additives because of their antioxidant activities, but they have poor thermal stability. In this study, betalains were extracted from red dragon fruit peel, and then encapsulated with maltodextrin by ultrasound method to increase the physicochemical properties of betalains microcapsules. The encapsulation efficiency of the betalains was above 79%, and the particle size and Zeta potential values were 275.46 nm and -29.01 mV, respectively. Compared to the control sample, onset temperature and DPPH free radical scavenging of betalains microcapsules under the modest ultrasound treatment (200 W, 5 min) was increased by 1.6 °C and 12.24%, respectively. This increase could be due to the ability of ultrasonification to create interactions between maltodextrin and betalains (as evidenced by FT-IR). Therefore, modest ultrasound treatment can be used for microcapsulation to improve the stability of betalains, and then expand the application of betalains in heat processed food field.

Effect of ultrasonication on the metabolome and transcriptome profile changes in the fermentation of Ganoderma lucidum.

Development of an efficient liquid fermentation method is helpful for food and pharmaceutical applications. This study investigated the effect of ultrasonication on the liquid fermentation of Ganoderma lucidum, a popular edible and medical fungi. Significant changes at both metabolic and transcriptional levels in mycelia were induced by ultrasound treatment. Compared with the control, 857 differential metabolites were identified (578 up- and 279 down-regulated metabolites), with more metabolites biosynthesis after sonication; 569 differentially expressed genes (DEGs) (267 up- and 302 down-) and 932 DEGs (378 up- and 554 down-) were identified in ultrasound-treated samples with recovery time of 0.5 and 3 h, respectively. Furthermore, 334 DEGs were continuously induced within the recovery time of 3 h, indicating the lasting influence of sonication on mycelia. The DEGs and differential metabolites were mainly involved in pathways of carbohydrate, energy metabolism, amino acids, terpenoids biosynthesis and metabolism and membrane transport, suggesting that ultrasound induced multifaceted effects on primary and secondary metabolism. Ultrasonication enhanced the triterpenoids production of G. lucidum (34.96 %) by up-regulating the expression of terpenoids synthase genes. This study shows that the application of ultrasound in liquid fermentation of G. lucidum is an efficient approach to produce more metabolites.

Modification of wheat straw to improve the caproate production in a cell immobilized system.

Wheat straw is a favorable cell carrier in the caproate fermentation system, yet its smooth surface limits the biofilm formation. In this study, the modification of wheat straw was conducted using three different chemical methods and the influence of its modified surface on the caproate fermentation was investigated. Results showed that the sodium hydroxide was the optimum reagent for modification of wheat straw, where both the external and internal surfaces were effectively modified, resulting in 34.4% increased specific surface area. The highest caproate production of 21.1 g/L was obtained in fed-batch fermentation, which was ascribed to the formation of a thick biofilm on the modified carrier. Moreover, the crystallinity index of the carrier increased during the fed-batch fermentation, implying that the modified wheat straw was a stable matrix for cell immobilization. This study provides an effective way for efficient caproate production through modification of wheat straw.

Immobilization of Clostridium kluyveri on wheat straw to alleviate ammonia inhibition during chain elongation for n-caproate production.

Biosynthesis of n-caproate from waste streams rich in acetate and ethanol through chain elongation has offered a potentially sustainable way for future production of liquid biofuels. However, most of the waste streams that fit with the purpose (e.g., digestate) are also rich in ammonium which at high concentration may cause toxic effects on the bioconversion process. This study aims to develop a robust, efficient, and cost-effective chain elongation process with high caproate productivity and tolerance to high ammonia concentration, through immobilization of Clostridium kluyveri on biomass particles as immobilization material. The threshold ammonia concentration for suspended cells cultivation was 2.1 g/L, while it was higher than 5.0 g/L for the wheat straw immobilized system. The caproate production process was dependent on the selected carriers and was performing in the order of: wheat straw > grass straw > saw dust. The biofilm immobilized on the wheat straw showed good reuse capability for caproate production under high ammonia concentration. Moreover, the lag phase for caproate production was shortened from 72 to 30 h after 8 times reuse. These results proved that caproate production and tolerance of chain elongation to ammonia toxicity could be enhanced via cell immobilization. This study offers insight into future development of efficient and cost-effective chain elongation system for production of caproate and other value-added products.

Microstructure and Mechanical Properties of the As-Cast and As-Homogenized Mg-Zn-Sn-Mn-Ca Alloy Fabricated by Semicontinuous Casting.

In this paper, a new type of low-cost Mg-3.36Zn-1.06Sn-0.33Mn-0.27Ca (wt %) alloy ingot with a diameter of 130 mm and a length of 4800 mm was fabricated by semicontinuous casting. The microstructure and mechanical properties at different areas of the ingot were investigated. The microstructure and mechanical properties of the alloy under different one-step and two-step homogenization conditions were studied. For the as-cast alloy, the average grain size and the second phase size decrease from the center to the surface of the ingot, while the area fraction of the second phase increases gradually. At one-half of the radius of the ingot, the alloy presents the optimum comprehensive mechanical properties along the axial direction, which is attributed to the combined effect of relatively small grain size, low second-phase fraction, and uniform microstructure. For the as-homogenized alloy, the optimum two-step homogenization process parameters were determined as 340 °C × 10 h + 520 °C × 16 h. After the optimum homogenization, the proper size and morphology of CaMgSn phase are conducive to improve the microstructure uniformity and the mechanical properties of the alloy. Besides, the yield strength of the alloy is reduced by 20.7% and the elongation is increased by 56.3%, which is more favorable for the subsequent hot deformation processing.

Mixotrophic Chlorella sp. UJ-3 cultivation in the typical anaerobic fermentation effluents.

The growth of mixotrophic Chlorella sp. UJ-3 cultivated in the three typical anaerobic fermentation effluents was investigated in this paper. The results showed that the microalgae grew best under intermediate light intensity for all the types of fermentation effluents. The butyrate type fermentation effluents induced the fastest growth rate for Chlorella sp. UJ-3, with a maximal cell concentration of 3.8×107 cells/mL. Under intermediate light intensity, the volatile fatty acids (VFAs) were almost depleted on the fifth day of the cultivation for all the three types of fermentation systems. The ratios of chlorophyll a/b were all increased for the three systems, indicating enhanced energy-capturing capability of the microalgae for photosynthesis after the VFAs were depleted. The highest lipid content was 25.4%dwt achieved in the butyrate type fermentation, and the fatty acid compositions were found to be considerably different for these three types of fermentation systems.

Effects of ultrasonic treatment on the maturation of Zhenjiang vinegar.

High-quality vinegars are traditionally produced by aging them in barrels or bottles. However, these processes are very time-consuming. To accelerate of Zhenjiang vinegar maturation, the ultrasound was used to treat the steeped vinegar. Results showed that, the optimum ultrasonic power, time and ethanol addition for aging vinegar were determined to be 50W/100mL, 75min and 0.75% (V/V), respectively. Under the optimum experimental conditions, the total amino acid of fresh vinegar decreased from 1082.259mg/100mL to 871.045mg/100mL. Several volatile components increased significantly, such as the total esters, aldehydes and heterocyclic. Total non-volatile organic acids increased from 202.59mg/10mL to 233.87mg/10mL. The changes of above-mentioned components develop towards the direction of mature vinegar. Coupling the HS-SPME/GC-MS analysis data with Principal Components Analysis, ultrasonic treatment vinegar was determined to be equivalent to 2-3years of natural aged Zhenjiang vinegar. This study has showed that ultrasound is promising not only in shortening the aging time and lowering costs for the vinegar-making industry, but also in producing fine vinegar.

Influence of fluid dynamics on anaerobic digestion of food waste for biogas production.

To enhance the stability and efficiency of an anaerobic process, the influences of fluid dynamics on the performance of anaerobic digestion and sludge granulation were investigated using computational fluid dynamics (CFD). Four different propeller speeds (20, 60, 100, 140 r/min) were adopted for anaerobic digestion of food waste in a 30 L continuously stirred tank reactor (CSTR). Experimental results indicated that the methane yield increased with increasing the propeller speed within the experimental range. Results from CFD simulation and sludge granulation showed that the optimum propeller speed for anaerobic digestion was 100 r/min. Lower propeller speed (20 r/min) inhibited mass transfer and resulted in the failure of anaerobic digestion, while higher propeller speed (140 r/min) would lead to higher energy loss and system instability. Under this condition, anaerobic digestion could work effectively with higher efficiency of mass transfer which facilitated sludge granulation and biogas production. The corresponding mean liquid velocity and shear strain rate were 0.082 m/s and 10.48 s-1, respectively. Moreover, compact granular sludge could be formed, with lower energy consumption. CFD was successfully used to study the influence of fluid dynamics on the anaerobic digestion process. The key parameters of the optimum mixing condition for anaerobic digestion of food waste in a 30 L CSTR including liquid velocity and shear strain rate were obtained using CFD, which were of paramount significance for the scale-up of the bioreactor. This study provided a new way for the optimization and scale-up of the anaerobic digestion process in CSTR based on the fluid dynamics analysis.

Biogas by semi-continuous anaerobic digestion of food waste.

The semi-continuous anaerobic digestion of food waste was investigated in 1-L and 20-L continuously stirred tank reactors (CSTRs), to identify the optimum operation condition and the methane production of the semi-continuous anaerobic process. Results from a 1-L digester indicated that the optimum organic loading rate (OLR) for semi-continuous digestion is 8 g VS/L/day. The corresponding methane yield and chemical oxygen demand (COD) reduction were 385 mL/g VS and 80.2 %, respectively. Anaerobic digestion was inhibited at high OLRs (12 and 16 g VS/L/day), due to volatile fatty acid (VFA) accumulation. Results from a 20-L digester indicated that a higher methane yield of 423 mL/g VS was obtained at this larger scale. The analysis showed that the methane production at the optimum OLR fitted well with the determined kinetics equation. An obvious decrease on the methane content was observed at the initial of digestion. The increased metabolization of microbes and the activity decrease of methanogen caused by VFA accumulation explained the lower methane content at the initial of digestion.

Batch and semi-continuous anaerobic digestion of food waste in a dual solid-liquid system.

To avoid the inhibition from both of waste oil and high concentrations of cationic elements, anaerobic digestion of food waste in a dual solid-liquid (ADSL) system was examined in this present paper. Results from batch test indicated that a higher methane yield could be obtained in the ADSL system. The methane yield of food solid waste (FSW), food liquid waste (FLW) and raw food waste (RFW) were 643, 659 and 581 mL/g-VS, respectively. In semi-continuous anaerobic digestion, the optimum organic loading rates (OLR) for FSW, FLW and RFW were 9, 4 and 7 g-VS/L/d, respectively. The total methane production of RFW and ADSL systems, based on 1 kg-VS(RFW), were 405 and 460 L, respectively, indicating that the methane production increased by 13.6% in the ADSL system. The optimum C/N ratio, redistribution of metal element and lower content of waste oil in FSW explain the higher methane production.

The anaerobic co-digestion of food waste and cattle manure.

This study assessed the anaerobic co-digestion of food waste and cattle manure, in order to identify the key parameters that determine the biogas and methane yield. Results of both batch and semi-continuous tests indicated that the total methane production is enhanced in co-digestion, with an optimum food waste (FM) to cattle manure (CM) ratio of 2. At this ratio, the total methane production in batch tests was enhanced by 41.1%, and the corresponding methane yield was 388 mL/g-VS. In the semi-continuous mode, the total methane production in co-digestion, at the organic loading rate (OLR) of 10 g-VSFW/L/d, increased by 55.2%, corresponding to the methane yield of 317 mL/g-VS. Addition of cattle manure enhanced the buffer capacity (created by NH4+ and VFAs), allowing high organic load without pH control. The C/N ratio and the higher biodegradation of lipids might be the main reasons for the biogas production improvement.