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Concentrating organic matter in sludge and converting it into methane through anaerobic bioconversion can improve resource recovery from domestic wastewater. Enhanced membrane coagulation (EMC) is highly efficient at concentrating organic matter, but residual coagulants (aluminum salts) can obstruct bioconversion by blocking microbial access. Limited research exists on evaluating EMC sludge bioconversion performance and addressing coagulant inhibition. This study proposes alkaline pre-fermentation to break down HO-Al-P backbones in coagulated sludge flocs, thereby improving hydrolysis and organic acid production for anaerobic digestion. Among the tested alkaline conditions (pH 9, pH 10, pH 11), pre-fermentation at pH 11 released the most organic matter (4710.0 mg/L SCOD), 20.4 times higher than without alkaline treatment. At pH 11, phosphate (61 mg/L PO43−–P) and organic acid production (2728.1 mg COD/L, with nearly 50% acetic acid) peaked, resulting in superior volatile solids removal (65.2%) and methane production (185.8 mL/g VS) during anaerobic digestion. Alkaline pre-fermentation favored alkali-tolerant bacteria such as Firmicutes and Actinobacteria, especially at pH 11, while neutrophilic Proteobacteria were suppressed. Trichococcus and Bifidobacterium, known acid producers, dominated under all conditions, with their abundance increasing at higher pH levels. Anaerobic digestion enriched fermentative bacteria like Chloroflexi and Synergistota (e.g., Thermovirga), especially in high pH reactors. Methanothrix, an acetoclastic methanogen, became the dominant methanogenic archaeon, indicating that methane production from EMC sludge primarily followed the acetoclastic methanogenesis pathway. Our findings demonstrate that alkaline pre-fermentation at pH 11 significantly enhances the hydrolysis efficiency of EMC sludge for methane recovery. [ABSTRACT FROM AUTHOR]

Simple Summary: The intestinal health problems of weaned piglets cause serious economic losses. Historically, antibiotics have been used to prevent and treat intestinal problems in weaned piglets. However, the prohibition of antibiotics makes us need to find a safer and effective solution strategy. A large number of studies have shown that the endocannabinoid system, as a lipid mediator signaling system widely distributed in the gastrointestinal tract, interacts with the intestinal microbiota and maintains intestinal homeostasis. In this study, we found that the endocannabinoid system is closely related to the abundance of Lactobacillus johnsonii through a piglet antibiotic model. It was further found that Lactobacillus johnsonii could improve intestinal health problems and alleviate piglet diarrhea. At the same time, its ability to regulate the endocannabinoid system was verified, and the correlation analysis found that its benefits are partly achieved through the participation of the endocannabinoid system. Probiotic intervention is a well-established approach for replacing antibiotics in the management of weaning piglet diarrhea, which involves a large number of complex systems interacting with the gut microbiota, including the endocannabinoid system; nevertheless, the specific role of the endocannabinoid system mediated by probiotics in the piglet intestine has rarely been studied. In this study, we used antibiotics (ampicillin) to perturb the intestinal microbiota of piglets. This resulted in that the gene expression of the intestinal endocannabinoid system was reprogrammed and the abundance of probiotic Lactobacillus johnsonii in the colon was lowered. Moreover, the abundance of Lactobacillus johnsonii was positively correlated with colonic endocannabinoid system components (chiefly diacylglycerol lipase beta) via correlation analysis. Subsequently, we administered another batch of piglets with Lactobacillus johnsonii. Interestingly, dietary Lactobacillus johnsonii effectively alleviated the diarrhea ratio in weaning piglets, accompanied by improvements in intestinal development and motility. Notably, Lactobacillus johnsonii administration enhanced the intestinal barrier function of piglets as evidenced by a higher expression of tight junction protein ZO-1, which might be associated with the increased level in colonic diacylglycerol lipase beta. Taken together, the dietary Lactobacillus johnsonii-mediated reprogramming of the endocannabinoid system might function as a promising target for improving the intestinal health of piglets. [ABSTRACT FROM AUTHOR]

Thermoplastic polyurethane (TPU) composites with eutectic gallium (Ga) and indium (In) (eGaIn) fillings of 0 wt%–75 wt% were prepared using the electrostatic spinning method. Field emission scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared (FTIR) spectroscopy were used to characterize the eGaIn NDs/TPU composites. To evaluate their X-ray shielding properties, Phy-X/PSD and WinXCom were employed to calculate the mass attenuation coefficients, linear attenuation coefficients, half-value layers, tenth value layers, mean free paths, and adequate atomic numbers of the eGaIn NDs/TPU composites. The SEM results indicated that the eGaIn nanodroplets were evenly distributed throughout the TPU fibers, and the flowable eGaIn was well-suited for interfacial compatibility with the TPU. A comparison of the eGaIn NDs/TPU composites with different content levels showed that the composite with 75 wt% eGaIn had the highest μm at all the evaluated energies, indicating a superior ability to attenuate X-rays. This non-toxic, lightweight, and flexible composite is a potential material for shielding against medical diagnostic X-rays. [ABSTRACT FROM AUTHOR]

To improve the potassium availability of feldspar at ordinary temperatures, the mechanical grinding and addition of sodium hydroxide/salts were employed to study the effects of mechanical activation and strong alkali addition on particle characteristics, water-soluble potassium, and the available potassium of feldspar. A laser particle size analyzer was utilized for the direct determination of particle size distribution (PSD) using ground samples. The Brunauer–Emmett–Teller (BET) method was employed for specific surface areas. X-ray diffraction (XRD) was employed for structural characterization, scanning electron microscopy (SEM) for morphology exploration, and energy dispersive spectroscopy (EDS) to determine the chemical composition of potassium feldspar powder. The results revealed that the mechanical activation of potassium feldspar could reduce the particle size and produce agglomerated nanoparticles in the later period. The addition of NaOH and sodium salt did not cause agglomeration, and NaOH dissolved the nanoparticles. The water-soluble potassium content of feldspar in each treatment increased during mechanical grinding, from 21.64 mg kg−1 to 1495.81 mg·kg−1, by adding NaOH 5% weight of potassium feldspar powder and to 3044.08 mg·kg−1 by adding NaOH 10% weight with effects different from those of mechanical shaking. By comparison, only 162.93 mg·kg−1 water-soluble potassium was obtained by adding NaOH 5% weight. The dissolved potassium in the former case was significantly higher than in the latter, and the addition of NaOH and sodium salts significantly enhanced the water-soluble potassium contents due to ion exchange. Furthermore, the addition of sodium hydroxide improved the water-soluble potassium due to its mechanochemical action on potassium feldspar. The mechanical energy changed the crystal structure of potassium feldspar, explaining the increase in available potassium. The addition of sodium salts did not promote change in the feldspar's structure, thereby did not raise the available potassium content. The reason for this was related to the mechanochemical action on sodium hydroxide and feldspar, which could promote the dissolution of fine particles, thereby incrementing the available potassium. [ABSTRACT FROM AUTHOR]

Simple Summary: Tannins are widely found in natural plants and have a wide range of biological activities. Here, we compared the effects of different sources of tannins on broilers and found that tannins from different sources have their own unique properties, so each tannin can have a significant impact on the growth of broilers. Acacia mearnsii tannin extract has a positive effect on the nutrient absorption of broilers, which can increase the abundance of Lactobacillus in the intestine and enhance nutrient absorption; Castanea sativa tannin has excellent antioxidant and anti-inflammatory properties and can be used as immune enhancement additives, but it has no effect on the digestion and absorption capacity of broilers; both Schinopsis lorenzii tannin extract and Caesalpinia spinosa tannin extract have significant hepatoprotective effects, so these two tannins can be used for liver protection in broilers. In this study, four plant tannins, including AT (Acacia mearnsii tannin, 68%), CT (Castanea sativa tannin, 60%), QT (Schinopsis lorenzii tannin, 73%) and TT (Caesalpinia spinosa tannin, 50%) were added to broiler diets for 42 days to evaluate and compare their effects on growth performance, antioxidant capacity, immune performance and gut microbiota in broilers. The results showed that the supplementation of five tannins could increase the production of T-AOC, GSH-Px, SOD and CAT and reduce the production of MDA in the serum of broilers (p < 0.01), but the antioxidant effect of the AT group was lower than that of the other three groups (p < 0.01). All four tannins decreased the level of the pro-inflammatory factor IL-1β and increased the level of the anti-inflammatory factor IL-10 (p < 0.01). CT, QT and TT decreased the levels of pro-inflammatory factors IL-6 and TNF-α (p < 0.01), while AT and CT increased the level of IL-2 in serum (p < 0.01). Supplementation with four tannins also increased the levels of IgG, IgM, IgA and sIgA in serum (p < 0.01) and the levels of ZO-1, claudin-1 and occludin in the jejunum (p < 0.01). The detection results of ALT and AST showed that CT, QT and TT decreased the concentrations of ALT and AST in serum (p < 0.01). The results of the gut microbiota showed that the abundance of Clostridia and Subdoligranulum increased, and the abundance of Oscillospiraceae decreased, compared to the control group after adding the four tannins to the diets (p > 0.05). In addition, CT, QT and TT decreased the abundance of Lactobacillus and increased the abundance of Bacteroides compared to the control group, while AT showed the opposite result (p > 0.05). Overall, our study shows that tannins derived from different plants have their own unique effects on broilers. AT and CT can promote broilers' growth better than other tannins, CT has the best ability to improve immune and antioxidant properties, and QT and TT have the best effect on broilers' liver protection. [ABSTRACT FROM AUTHOR]

The hypergraph offers a platform to study structural properties emerging from more complicated and higher-order than pairwise interactions among constituents and dynamical behavior, such as the spread of information or disease. Considering the higher-order interaction between multiple nodes in the system, the mathematical model of infectious diseases spreading on simple scale-free networks is extended to hypernetworks based on hypergraphs. A SIS propagation model based on reaction process strategy in a universal scale-free hypernetwork is constructed, and the theoretical and simulation analysis of the model is carried out. Using mean field theory, the analytical expressions between infection density and hypernetwork structure parameters as well as propagation parameters in steady state are given. Through individual-based simulation, the theoretical results are verified and the infectious disease spread process under the structure of the hypernetwork and simple scale-free network is compared and analyzed. It becomes apparent that infectious diseases are easier to spread on the hypernetworks, showing the clear clustering characteristics of epidemic spread. Furthermore, the influence of the hypernetwork structure and model parameters on the propagation process is studied. The results of this paper are helpful in further studying the propagation dynamics on the hypernetworks. At the same time, it provides a certain theoretical basis for the current COVID-19 prevention and control in China and the prevention of infectious diseases in the future. [ABSTRACT FROM AUTHOR]

The main substances of rice are starches, which vary their metabolism during storage. We conducted a series of tests including rice physicochemical properties, edible quality, starch content and chain length distribution along with starch structure variation to disclose the shift of rice quality by observing the changes of rice during storage. The results showed that: (1) the rice deterioration occurred as time passed, and the germination rate decreased from 70.8% to 29.4% during the storage; (2) fatty acid values increased significantly during long-term storage; (3) electrical conductivity increased as time passed; and (4) the two-year-storage rice showed significantly decreased viscosity and edible quality after sensory evaluation, decreased hardness and damaged surface area of starch granules as storage time passed. Additionally, the damaged surface area of starch granules increased with storage time. Fourier transform infrared spectroscopy (FTIR) showed that the short-range order and spiral degree of rice starch first decreased in the first year and then increased over the storage time. Furthermore, X-ray diffraction showed that the main starch of rice was A-type crystalline. Meanwhile, apparent amylose content increased from 31.00% to 33.85%, then decreased to 31.75%. The peak viscosity reduced from 2735.00 mPa·s to 2163.67 mPa·s and the disintegration value was brought down from 1377.67 mPa·s to 850.33 mPa·s. Based on the results, rice should not be stored for more than 2 years under suitable granary conditions to maintain it at a good quality. [ABSTRACT FROM AUTHOR]

The preparation of binary alloys by surfactant-assisted chemical reduction in aqueous solution at room temperature has become a hot topic. In this article low melting point tin/indium (Sn/In) nanoparticles are synthesized. The formation process of the alloy was studied. Scanning electron microscopy, energy spectrometry, and X-ray diffraction are used to determine the morphology, composition, and crystal structure of the nanoparticles. Study found that fully alloyed indium-tin nanoparticles can be obtained by wet chemical method and the main phases of indium-tin alloy are β-phase (In3Sn) and γ-phase (InSn4). However, the Sn phase appears at a low content of indium (40 wt%). When the content of indium increases to 45 (wt%), the tin phase disappears. In addition, the most important finding is that the composition of the indium-tin alloy can be changed by ratio control, and the content of In3Sn increases with the increase of indium content. The relative content of In3Sn attains a maximum when the content of indium increases to 60 (wt%). In contrast, the content of InSn4 decreases. Finally, differential scanning calorimetry measurements is performed to understand the melting behavior of the nanoparticles and low melting temperatures are achieved for a wide range of indium compositions (from 40% to 60%). The melting temperature is found to be in the range of 125–132 °C and it increased with increasing In3Sn (also the increase of indium content). This gives us a new understanding into the binary alloy nano-system and gives important information for the application of low temperature alloy solders. The choice of composition can be based on the corresponding melting point. [ABSTRACT FROM AUTHOR]

Simple Summary: Feeding with a diet high in rice often results in several adverse effects for animals, such as ruminal acidosis, the translocation of endotoxin into the bloodstream, gastrointestinal barrier impairment, liver injures, and inflammation. A high rice grain-based diet also provides excess energy for human beings, which usually induces obesity via an miRNA-based mechanism. Currently, whether high-rice diets affect spleen growth and related molecular events remains unknown. Therefore, we take goats as an artiodactyl model to investigate the effects of feeding a highly concentrated diet, based on rice grains, on the growth and microRNA expression profiles in the goat spleen. Here, we first report about the splenic microRNA profile in artiodactyls fed a highly concentrated diet based on rice. Our results shed light on the miRNA-based regulatory mechanism that contributes to the development of high calorie-induced metabolic diseases in mammals. Several researchers have testified that feeding with diets high in rice grain induces subacute ruminal acidosis and increases the risk of gastrointestinal inflammation. However, whether diets high in rice grain affect spleen growth and related molecular events remains unknown. Therefore, the present study was conducted to investigate the effects of feeding a high-concentrate (HC) diet based on rice on the growth and microRNA expression profiles in goat spleen. Sixteen Liuyang black goats were used as an artiodactyl model and fed an HC diet for five weeks. Visceral organ weight, LPS (lipopolysaccharide) concentration in the liver and spleen, and microRNA expression were analyzed. The results showed that feeding an HC diet increased the heart and spleen indexes and decreased the liver LPS concentration (p < 0.05). In total, 596 microRNAs were identified, and twenty-one of them were differentially expressed in the spleens of goats fed with the HC diet. Specifically, several microRNAs (miR-107, miR-512, miR-51b, miR-191, miR-296, miR-326, miR-6123 and miR-433) were upregulated. Meanwhile, miR-30b, miR-30d, miR-1468, miR-502a, miR-145, miR-139, miR-2284f, miR-101 and miR-92a were downregulated. Additionally, their target gene CPPED1, CDK6, CCNT1 and CASP7 expressions were inhibited (p < 0.05). These results indicated that the HC diet promoted the growth of the heart and spleen. The HC diet also regulated the expression of miR-326, miR-512-3p, miR-30b, miR-30d, miR-502a and their target genes (CPPED1, CDK6 and CCNT1) related to the enhancement of splenocyte proliferation. The HC diet also modulated the expression of miR-15b-5p, miR-1468 and miR-92a, related to the suppression of splenocyte apoptosis. [ABSTRACT FROM AUTHOR]

Simple Summary: Effects of a high-rice dietary proportion on the meat quality, gut microbiota and metabolites in small ruminants are rarely reported. Thus, the objective of this study was to evaluate the slaughtering characteristic and meat quality, acute phase reaction proteins (APRPs) in plasma and colonic microbiota and metabolites of goats subjected to a high-rice diet. After a 35-day period, sixteen goats received a high-rice diet (HR, 90% concentrate) or a control diet (55% concentrate). In summary, the results showed that the slaughter performance and meat quality were improved in the growing goats after being fed the HR diet. However, the HR diet induced an acute phase reaction and disturbed the gut microbiota to some extent, which increases the health risk to growing goats. Effects of a high-rice dietary proportion on the meat quality, acute phase reaction proteins (APRPs) and colonic microbiota and metabolites in goats are rarely reported. This study was designed to investigate the meat quality and metabolism in goats. Sixteen goats were equally divided into two groups and fed a control diet (Con, 55% concentrate) or a high-rice diet (HR, 90% concentrate) for five weeks. We found that the HR diet improved the slaughtering characteristic and meat quality but induced an acute phase reaction and decreased bacterial richness and diversity when compared to the control group. Furthermore, the levels of acetate, propionate and total VFA concentrations were higher in the colonic contents of the HR-fed goats than in those of the control group (p < 0.05). Meanwhile, the HR diet decreased the pH value, lactic acid concentration and increased the activity of amylase and lipopolysaccharide concentration in the colonic contents of goats (p < 0.05). The proportion of Oscillibacter increased while Phocaeicola and Christensenellaceae_R-7_group significantly decreased with the HR diet (p < 0.05). Collectively, the HR diet induced an acute phase reaction and altered the colonic bacterial community, which increases the health risk to growing goats. [ABSTRACT FROM AUTHOR]

Simple Summary: The effects of dietary hydrolyzed Chinese gallnut tannic acid (GCT) as a replacement for ZnO were investigated on weaned piglets. A total of 72 piglets (31 ± 1 day) were selected and divided randomly into two groups: a control group, with a basal diet of + 1600 mg/kg ZnO; and a treated group, with a basal diet of + 1899.5 mg/kg GCT. The diarrhea rate of piglets in the treated group declined on days 14–21 than in the control group. Additionally, we found GCT can reduce the crypt depth of the ileum and improve antioxidant capacity. High throughput sequencing showed that the GCT increased the richness of bacteria (Lachnospiraceae, Prevotella, and Lactobacillus amylovorus) associated with the degradation of cellulose and hemicellulose. These data indicate that 1899.5 mg/kg GCT could be an alternative for 1600 mg/kg ZnO in the diet of piglets. The effects of dietary hydrolyzed Chinese gallnut tannic acid(GCT) as a replacement for ZnO were investigated on weaned piglets. A total of 72 weaned piglets at 31 ± 1 day (six replicate pens per treatment with six piglets per pen) were selected and divided randomly into two groups: a control group, with a basal diet of + 1600 mg/kg ZnO; and a treated group, with a basal diet of + 1899.5 mg/kg GCT. Data analysis showed that the significance of average daily gain and average daily feed intake between the two groups was p = 0.731 and p = 0.799, respectively. Compared with the control group, the diarrhea rate of piglets in the treated group underwent no noticeable change on days 0–7 (p = 0.383) and 7–14 (p = 0.263), but decreased significantly on days 14–21 (p < 0.05). Additionally, we found GCT can reduce the crypt depth of the ileum and improve its antioxidant capacity (p < 0.05). High throughput sequencing showed that GCT increased the richness of the bacteria Lachnospiraceae (p = 0.005), Prevotella_2 (p = 0.046) and Lactobacillus amylovorus (p = 0.081), which are associated with the degradation of cellulose and hemicellulose. The study indicated that 1899.5 mg/kg GCT could be an alternative for 1600 mg/kg ZnO in the diet of piglets. [ABSTRACT FROM AUTHOR]

This work aimed to enrich a denitrifying bacterial community for economical denitrification via nitrite to provide the basic objects for enhancing nitrogen removal from wastewater. A sequencing batch reactor (SBR) with continuous nitrite and acetate feeding was operated by reasonably adjusting the supply rate based on the reaction rate, and at a temperature of 20 ± 2 °C, pH of 7.5 ± 0.2, and dissolved oxygen (DO) of 0 mg/L. The results revealed that the expected nitrite concentration can be achieved during the whole anoxic reaction period. The nitrite denitrification rate of nitrogen removal from synthetic wastewater gradually increased from approximately 10 mg/(L h) to 275.35 mg/(L h) over 12 days (the specific rate increased from 3.83 mg/(g h) to 51.80 mg/(g h)). Correspondingly, the chemical oxygen demand/nitrogen (COD/N) ratio of reaction decreased from 7.9 to 2.7. Both nitrite and nitrate can be used as electron acceptors for denitrification. The mechanism of this operational mode was determined via material balance analysis of substrates in a typical cycle. High-throughput sequencing showed that the main bacterial community was related to denitrification, which accounted for 84.26% in the cultivated sludge, and was significantly higher than the 2.16% in the seed sludge. [ABSTRACT FROM AUTHOR]

To improve the potassium availability of feldspar at ordinary temperatures, the mechanical grinding and addition of sodium hydroxide/salts were employed to study the effects of mechanical activation and strong alkali addition on particle characteristics, water-soluble potassium, and the available potassium of feldspar. A laser particle size analyzer was utilized for the direct determination of particle size distribution (PSD) using ground samples. The Brunauer-Emmett-Teller (BET) method was employed for specific surface areas. X-ray diffraction (XRD) was employed for structural characterization, scanning electron microscopy (SEM) for morphology exploration, and energy dispersive spectroscopy (EDS) to determine the chemical composition of potassium feldspar powder. The results revealed that the mechanical activation of potassium feldspar could reduce the particle size and produce agglomerated nanoparticles in the later period. The addition of NaOH and sodium salt did not cause agglomeration, and NaOH dissolved the nanoparticles. The water-soluble potassium content of feldspar in each treatment increased during mechanical grinding, from 21.64 mg kg -1 to 1495.81 mg·kg -1 , by adding NaOH 5% weight of potassium feldspar powder and to 3044.08 mg·kg -1 by adding NaOH 10% weight with effects different from those of mechanical shaking. By comparison, only 162.93 mg·kg -1 water-soluble potassium was obtained by adding NaOH 5% weight. The dissolved potassium in the former case was significantly higher than in the latter, and the addition of NaOH and sodium salts significantly enhanced the water-soluble potassium contents due to ion exchange. Furthermore, the addition of sodium hydroxide improved the water-soluble potassium due to its mechanochemical action on potassium feldspar. The mechanical energy changed the crystal structure of potassium feldspar, explaining the increase in available potassium. The addition of sodium salts did not promote change in the feldspar's structure, thereby did not raise the available potassium content. The reason for this was related to the mechanochemical action on sodium hydroxide and feldspar, which could promote the dissolution of fine particles, thereby incrementing the available potassium.

Low-energy cost wastewater treatment is required to change its current energy-intensive status. Although promising, the direct anaerobic digestion of municipal wastewater treatment faces challenges such as low organic content and low temperature, which require further development. The hydrolysis-aerobic system investigated in this study utilized the two well-proven processes of hydrolysis and aerobic oxidation. These have the advantages of efficient COD removal and biodegradability improvement with limited energy cost due to their avoidance of aeration. A pilot-scale hydrolysis-aerobic system was built for performance evaluation with actual municipal wastewater as feed. Results indicated that as high as 39-47% COD removal was achieved with a maximum COD load of 1.10 kg/m³·d. The dominant bacteria phyla included Proteobacteria (36.0%), Planctomycetes (15.4%), Chloroflexi (9.7%), Bacteroidetes (7.7%), Firmicutes (4.4%), Acidobacteria (2.5%), Actinobacteria (1.8%) and Synergistetes (1.3%), while the dominant genera included Thauera (3.42%) and Dechloromonas (3.04%). The absence of methanogens indicates that the microbial community was perfectly retained in the hydrolysis stage instead of in the methane-producing stage., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

High solid anaerobic digestion (HSAD) is a rapidly developed anaerobic digestion technique for treating municipal sludge, and has been widely used in Europe and Asia. Recently, the enhanced HSAD process with thermal treatment showed its advantages in both methane production and VS reduction. However, the understanding of the microbial community is still poor. This study investigated microbial communities in a pilot enhanced two-stage HSAD system that degraded waste activated sludge at 9% solid content. The system employed process "thermal pre-treatment (TPT) at 70 °C, thermophilic anaerobic digestion (TAD), and mesophilic anaerobic digestion (MAD)". Hydrogenotrophic methanogens Methanothermobacter spp. dominated the system with relative abundance up to about 100% in both TAD and MAD. Syntrophic acetate oxidation (SAO) bacteria were discovered in TAD, and they converted acetate into H₂ and CO₂ to support hydrogenotrophic methanogenesis. The microbial composition and conversion route of this system are derived from the high solid content and protein content in raw sludge, as well as the operational conditions. This study could facilitate the understanding of the enhanced HSAD process, and is of academic and industrial importance., Competing Interests: The authors declare no conflict of interest.