Prosopis juliflora (Pj) as an energy feedstock. Proximate, ultimate and biochemical characterization of Pj pods were conducted, and thermal behaviour and calorific values were determined. Cellulose and hemicellulose were isolated and characterized by reliable methods. The overall characterization has revealed the Pj pods as a potential feedstock for bioenergy. The collected Pj pods contain (% w/w) moisture 7.89 ± 0.002, volatile matter 87.67 ± 0.002, ash 0.21 ± 0.002, fixed carbon 4.23 ± 0.002 with a calorific value of 17.62 kg/MJ. The CHNS content was (w/w %) carbon 41.77, nitrogen 3.58, sulfur 26.3 and hydrogen 6.55. The biochemical composition analysis yields (% w/w) on a dry basis; cellulose 26.6 ± 0.18, hemicellulose 30.86 ± 0.27, lignin 4.71 ± 0.12, protein 11.63 ± 0.12 and starch 1.1 ± 0.06 and extractives 30.56 ± 0.008. The isolated cellulose and hemicellulose were analyzed and confirmed by CP/MAS &1H NMR, FTIR, TG-DSC, SEM, XRD, and TGA. The present results revealed that the tested biomass, Prosopis juliflora, could be used as a feedstock in biorefinery for bioenergy.The production of renewable and sustainable biofuels using inevitable wastes is a promising alternative to the alarming depletion of fossil fuels. Significantly, the sustainable biorefinery of lignocellulosic waste, as an alternative fuel source, is a prognosticating approach to tackle many agricultural/forestry residues and offers a circular economy as well as environmental benefits. But, the heterogeneity of lignocellulosic biomass is one of the major bottlenecks in lignocellulosic biorefinery. Thus the characterization of lignocellulosic biomass is essential to understanding the feedstock's nature, composition and suitability for biofuel production. The present study taps evergreen spiny non-edible pods of  Energy demand is anticipated to increase by around 50% over the next 20 years, with predictions of 778 EJ by 20352. But the exhaustion of fossil fuels and the subsequent increase in greenhouse gas emissions have become urgent problems3. These harmful greenhouse gases also harm human health because they cause cancer4. Thus finding an alternative energy source that can satisfy energy needs also be environmentally benign is vital4. In contrast to other environmentally friendly energy sources, including wind, solar, geothermal, marine, and hydropower, biomass may directly produce fuel and chemicals5.The foundation of modern civilization, energy is essential to most human activities. Modernizing our lives and cities would not have been possible without energy6. The energy from the lignocellulosic biomass is greatly interesting since it is abundantly available, technically feasible and economically viable7. Several technologies, including gasification, combustion, pyrolysis, enzymatic hydrolysis and fermentation, are employed to turn biomass into fuel and platform chemicals3. Thermochemical processes often demand a large amount of energy, including a solvent or catalyst. In contrast, the biochemical approach has a protracted time and has less effective at breaking down the resistant biomass3. Various lignocellulosic biomass like sugarcane bagasse8, berry waste2, jatropha seeds9, sunflower straw10, sorghum stem11, rice straw12, waste peach pulp13, wheat straw14, corn stover15 and so forth have been studied for the effective production of biofuels.Lignocellulosic biomass comes in various wastes, including woody, aquatic detritus, farm manure and byproducts, and agricultural wastes16. Cellulose is the primary polymer that contains long chains of cellobiose units, a renewable resource that is safe for the environment, inexpensive, non-toxic, biodegradable, and biocompatible17. Hemicellulose comprises monosaccharides such as mannose, galactose, arabinose, glucose, rhamnose, xylose and uranic acid18. Lignin is a high molecular weight, complex structure containing cross-linked phenolic monomers that provide cell wall rigidity, impermeability and resistance against microbial attack19.The lignocellulosic feedstock contains majorly cellulose 40–50%, hemicellulose 25–35% and lignin 15–20%4. Nevertheless, there is a constant demand for environment-friendly disposal or refinery of various lignocellulosic biomass20. From the standpoint of waste valorization, the fractionation of this lignocellulosic residue into cellulose, hemicellulose, and lignin is the primary step for producing a variety of products with a higher economic value, such as biofuel, cosmetics, biodegradable films, paper, and plastic materials, while minimising the associated environmental impacts19.The lignocellulosic biomass usage in biorefineries for biofuels offers reduced greenhouse gas emissions, zero waste generation and significant sustainability in the circular bioeconomy21. By recycling trash using pyrolysis to create biochar, energy, and value-added goods, agricultural and animal waste disposal can lessen its negative environmental effects22. Biochar production from lignocellulose is simple, reliable, technically feasible and commercially viable23. Using biochar as a feed for animal growth reduces methane formation and enhances anaerobic digestion23.One of the most promising technologies for converting lignocellulosic biomass into high-value products like biochar, syngas, and bio-oil without oxygen is pyrolysisProsopis juliflora pods, for its suitability as bioenergy feedstock. The Prosopis juliflora (Pj) plant is a drought-tolerant, evergreen tree with drooping branches of the Leguminosae family as an energy stock24. The native of the Pj pods to arid and semi-arid regions of the world. Pj plants have been colonized in many countries, such as North America, Africa and Asia. Pj plants have flexible branches, a green–brown, a twisted stem, and curving multi-seeded pods with a hardened pericarp and flattened25. Pj plants can survive in a very harsh and poor soil environment, control erosion, improve soil fertility, and reduce soil salinity in drylands. Due to invasive properties, Pj plants grow as much as 9 m in length and 50 cm in diameter. The fruit is a non-dehiscent curved pod of 10–15 cm in length, 0.8–1.0 cm in width, 2–4 mm thick, and contains a hard endocarp, soft and heavy mesocarp and woody endocarp with many seeds24. The pods generated by the Prosopis species are legume pods containing high levels of carbohydrates, protein, and starch used as animal feed for the past decades. Prosopis species are made up of exocarps, fibrous endocarps and fleshy mesocarps. The nutritional value of a plant might vary depending on the phenological stage, species, plant portion, world region, and growing climatic conditions26.The present study aims to exploit one such lignocellulosic biomass, 27 as biopesticides28, gums for antioxidants29, extracts for antiplasmodial activity30, seed powder for the removal of Pb (II)31 and all parts of the plant for antibacterial activity32. Pj plants have higher carbon and less hydrogen content, leading to the best carbon-effective ratio for biorefinery. Pj pods were used as a raw material in a few fermentations to produce cellulolytic enzymes33, xylanase34, oxytetracycline35, ethanol36, bio-oil37 and biohydrogen38 and biochar production under pyrolysis39. To the best of our knowledge, the structural characterization of Pj pods was not yet reported.Pj plant has been investigated for several applications, leaf extract to treat sore eyes, sore throat, diarrhoea, and open wounds40. The physicochemical properties of biomass help design the biomass conversion process, including feeding, conversion, sorting of intermediate products, and collection of products. Biomass physical, chemical and thermal properties are related to the gasification process, and the knowledge of biomass composition is also essential for solid waste management41. Therefore, the present study deals with the proximate, ultimate, biochemical and thermal properties of the Prosopis juliflora (Pj) pods to aid the selection of Pj pods as a feedstock for bioenergy and value-added products.The systemic characterization of biomass is essential for biofuel production35.Pj pods  were collected from Pudukkottai, Tamil Nadu, India, as per the ASTM International standards—ASTM E1757-1. The plant biomass was identified by botanist Dr Sam Aldrin, SASTRA Deemed University, India. The raw pods were collected from the same region for the entire study to prevent changes due to different nutritional content. The collected pods were washed completely with tap water and dried under sunlight. The dried pods were ground to achieve a 0.4–0.1 mm particle size using an ASTM sieve40. Essential biofuel quality can be determined by proximate and ultimate analysis42.Proximate analysis of Pj pod powder was conducted to estimate the moisture, volatile matter, ash content and fixed carbon40.The conventional oven-dry method (NREL) was used to estimate the moisture content according to ASTM E1756-01. Briefly, 10 g of Pj pods powder was weighed in the crucible and heated in a hot air oven at 105 °C for 4 h. Pj pods powder was removed from the crucible and allowed to cool in a desiccator. The moisture content of Pj pods was calculated using Eq.  content was estimated as the difference in weight of moisture, volatile matter and ash content from the initial biomass. The fixed carbon content of Pj pods was calculated using Eq. 40.3\docu40.The elemental composition of Pj pod powder, including carbon, nitrogen, oxygen, hydrogen and sulfur, was determinedCHNS analysis was carried out using CHNS Elemental analyser model Elemental Vario EL III Germany, analysed at CECRI Karaikkudi, India.43.X-ray fluorescence spectrophotometry was used to estimate the mineral composition of the Pj pods. Briefly, the Pj pods powder was mixed with boric acid as a binder under high pressure (22 tons) to make a pellet. The pellet results were recorded in X-ray fluorescence (XRF) spectrometer S8 Tiger, Bruker AXS, Germany, analysed at SASTRA Deemed University, Thanjavur, India, using a 4-kW rhodium anode X-ray tube44. The acid bleach method was used to isolate and estimate the Pj pods' cellulose. Briefly, 5 g of Pj pods powder was loaded in a Soxhlet apparatus with water and ethanol and reflexed for 8 h to eliminate the wax. The dewaxed Pj pods powder was bleached using 1.5% sodium chlorite for 2 h at 70 °C at pH 3.5. The bleaching procedure was repeated until a consistent white colour was obtained, indicating pure cellulose. The obtained cellulose was filtered, dried and weighed45.Pulping and bleaching are two crucial steps to extracting cellulose effectively from lignocellulosic biomass45.The filtrate from the acid bleaching step was treated with 1 M NaOH at 65 °C for 2 h, neutralized with 6 M HCl at pH 5.5 and precipitated using 46.Acid hydrolysis was used for the isolation of lignin. Briefly, 1 g of Pj pod powder was hydrolyzed with 72% sulfuric acid, autoclaved for 2 h at 121 °C and filtered off. The filtrate was considered acid-soluble lignin, and the residue was acid-insoluble lignin. Further, the acid-soluble lignin was estimated using a UV–Visible spectrophotometer and Eqs.  & 5)46.46.5)46.4UV (abs), average absorbance for sample; ε, absorptivity (specific wavelength); ODW sample, sample weight in mg; path length, the path length of UV–Vis cell in cm.44.Ethanol and benzene extract fat, wax, oil, gums, sugars and other pigmenting components from LCB. In a Soxhlet apparatus, 2.5 g of dried Pj pods powder was extracted with 150 mL of ethanol at 70 °C and reflexed for 8 h. The extracted Pj pods powder was dried in a hot air oven at 105 °C for 2 h. The weight difference between moisture-free pods and extractive complimentary pods powder is represented as extractives47. Briefly, 0.5 g of Pj pods powder was mixed with 10 mL of concentrated sulfuric acid and 5 g of catalyst mixture . The mixture was taken in a digestion tube, loaded into the digester, and heated to 400 °C until the mixture turned light green, indicating the complete digestion process. The digested sample was distilled in a kelplus distylemba distillation system with a hose connected (Model classic dx). One side of the hose was connected with a conical flask and loaded with 20 mL of 4% boric acid and mixed indicator . The other side of the hose was loaded with 1 M NaOH and distilled. The digested sample was heated by passing the vapour constantly, and the released ammonia was absorbed into a conical flask. With the absorption of ammonia, the pinkish colour turned to green colour. The collected distillate was titrated against 0.02 N of sulfuric acid until the sample colour was changed from green to pink. Micro Kjeldahl total nitrogen was estimated from the titer values. Crude protein was calculated using Eq. The anthrone method was used to estimate the starch. Briefly, the 0.1 g Pj pods powder was mixed with 25 mL 80% of hot ethanol and 5 mL of distilled water. The mixture was stirred thoroughly, centrifuged, and decanted off. The residue was again centrifuged with 30 mL of 80% hot ethanol. Two extracts were combined under pressure in a boiling water bath. The alcoholic extract was mixed with 5 mL of distilled water and 6.5 mL of perchloric acid. The mixture was stirred for 30 min at room temperature and centrifuged. The pellet was suspended in 5 mL of distilled water; the perchloric acid extraction was repeated and centrifuged. Two extracts were combined, diluted to 100 mL and filtered.49.Further, 5 mL of the filtrate was diluted to 100 mL with distilled water and used for starch analysis. The starch solution, 1 mL, was diluted with 1 mL of water, and 10 mL of anthrone reagent was added. The mixture was boiled in a boiling water bath for 12 min, cooled, and the amount of glucose was determined by a spectrophotometer. Starch was estimated from the glucose, assuming the 0.9 g starch yields 1 g glucose. Anthrone reagent was prepared as 10 mg of anthrone in 10 mL of concentrated sulfuric acidThe calorific value of Pj pods powder was determined with a Bomb calorimeter . In a bomb, 0.46 g of moisture-free Pj pods powder was taken in a crucible, kept inside the bomb, and filled with 30 bar pressure of oxygen to combust the Pj pods powder.50of model SDT Q-600 to identify the thermal stability of Pj pods, cellulose and hemicellulose. The thermal analysis was carried out under a nitrogen atmosphere of 0.5 lb/in2 with a 100 mL/min flow rate. The different stages of degradation were observed when the components started heating from 37 to 1000 °C.TGA was performed in a Thermal analyzer40 at SASTRA Deemed University. The wavelength was measured in transmission mode from 400 to 4000 cm−1 by the ATR  method51. Methanol was used to clean the ATR crystal before use. Later, the crystal was scooped with samples until they covered the surface area of the crystal and pressed with a mechanical anvil.The functional group of Pj pods, cellulose and hemicellulose were determined by Fourier Transform Infra-red Spectroscopy Spectrum one, Perkin Elmer USA13C spectrum of cellulose and hemicellulose was studied by Bruker Avance HD 500 MHz spectrophotometer from IITM, SAIF, CSAIF, Chennai, India44. The following condition was analyzed: resonance frequency at 125.76 MHZ; proton 90° pulse sequence was 4.30 µs; contact pulse 3300 µs, recycle delay was 5 s; acquisition time 0.02 s; 1024 scan per spectrum were used.CP/MAS 44.The SEM imaging of dried pods, acid-treated pods, cellulose and hemicellulose, was studied by Vega 2 TE-SCAN microscope with a speeding voltage of 10.0 kV. The magnification of the isolated cellulose was focused on up to 500X50 at Bishop Heber College, Trichy, India.The isolated cellulose crystallinity index was analysedusingPANalyticalXpert343.X-ray Fluorescence Spectroscopy analysis was conducted to estimate the chemical elements in the Pj pods powder. The mineral composition was studied by Bruker SPECTRA plus for S8 TIGER X-ray Fluorescence Spectroscopy52. Table 53 and decreases the yield of biomass combustion. Moisture content is represented as the quantity of water present per unit of Pj pods powder, and the low moisture content yields more combustion. High moisture content will decrease the yield of biomass combustion54. Pj pods showed a higher volatile matter content of 87.67 ± 0.002% w/w, which could be a valuable source for pyrolysis. The fuel reactivity has also been influenced by volatile matter55. The high volatile content of biomass has a propensity to easily burn and degrade during the thermochemical reaction, ideally yielding a higher proportion of bio-oil during the pyrolysis56.The proximate and ultimate analyses were used to determine the composition of Pj pods40. The amount of total minerals in biomass indicates the ash content or total ash63. The higher and lower heating values, the amount of solid material to release, dust particles in combustion gases, boiler fouling, and corrosion are all strongly correlated with the ash content64. In addition, a high amount of ash content acts as a heat sink during pyrolysis or combustion that will reduce the fuel energy process65 and biomass with a greater ash content generates more char leftovers during pyrolysis56. Pj pods with a low ash content of 0.21 ± 0.002% w/w, comprising phosphorous, silica, aluminium, sodium, iron, calcium, and potassium, could be better feedstock for fuel energy production. However, ash is one example of a solid fraction that has the potential to be used in agriculture because of its high nitrogen, phosphorous, and potassium content2.Ash is composed of mineral and inorganic matter of biomass and affects the combustion rate of biomass40. The high amount of fixed carbon represents a high energy level. In most gasifiers, the conversion of FC into gases dictates the pace of gasification and its yield, making fixed carbon a significant parameter for gasification study66. Knowing fixed carbon's shape and hardness can aid choose the right combustion equipment since they reveal a fuel's caking qualities67. The high volatile matter will increase biochar yield in thermochemical conversion processes. Pj pods have a fixed carbon content of 4.23 ± 0.002% w/w, producing low char during combustion.Fixed carbon (FC) is the amount of leftover Pj pods powder after releasing volatile matter, moisture, and ash68, low greenhouse emissions42 and thermochemical conversion processes such as pyrolysis, torrefaction and gasification2 and that can produce streams of liquid, solid, and gaseous products. Higher carbon content and a lower oxygen content must result in a higher heating value since the carbon content of biomass is directly correlated with the heating value, and the oxygen content is inversely correlated with the heating value69. The amount of carbon and oxygen are directly correlated with the heating value. Pj pods have a high amount of carbon, meaning that the more significant percentage of carbon, the higher the heating value of the fuel.The elemental composition of Pj pods was determined by ultimate analysis Table . The low64. One of the primary gases emitted during the thermal reactions of biomass is a nitrogen oxide (NOx). Therefore, these emissions need to be controlled as they affect human health. (NOx) consist of seven oxides that are considered the most emissions in an air70. When burning fuel, NOx emissions can be reduced in seven major methods by employing a catalytic combustion system, reduced air preheat, low NOx burner to lower the peak temperature, air staging and natural gas burning. Similarly, the ozone-depleting NO2 can also be treated in a similar way70.Additionally, a more significant oxygen content decreased the heating value of Pj pods, and biomass nitrogen content is crucial for evaluating nitrogen oxide (NOx) emissions, an air pollutant71.As determined by XRF, the micronutrients Zn, Mn, P, Cu, Ca, Mg, Na, K, and Fe were present in the whole Pj pods Table . The mic50. Lignin is the major obstacle to biomass conversion as it inhibits microorganisms' growth72.Chemical analysis data for biomass is crucial information when examining the reaction properties of solid fuels. The term "holocellulose" refers to the total cellulose, which is made up of cellulose and hemicelluloses, and extractives constitute the significant portion of Pj pods powder, 88% with less protein, lignin and starch Table . During 72. Pj pods constitute 57.76 ± 0.14% w/w holocellulose less than the Pj bark, 77%50.Sodium chlorite-based bleaching is an effective method to isolate cellulose from biomass. Alkaline treatment for the isolation of hemicellulose was a widely reported method, as it effectively cleaves the ester bond between lignin and hemicellulose47 and Pj seeds as 4.5%54.The amount of protein in Pj pods was estimated by the micro Kjeldahl method. Pj pods have shown a good amount of protein, 11.63 ± 0.12% w/w, an excellent alternative and low-cost animal feed. Further, the exact protein composition has to explore to assess the nutritional value, net protein utilization and digestibility. The protein content of Pj pods from Ecuador was reported as 10.1% (dry basis)73, and the high amount of extractives needs further evaluation.With the high amount of hollocellulose and less lignin content of 4.71 ± 0.12% w/w, Pj pods could be an effective source for energy conversion through biochemical approaches. The negligible amount of starch 1.23 ± 0.06% w/w was determined, similar to starch obtained from waxy maize starch 1.2 ± 0.9%74. The higher HHV also indicates the Pj pods significance as a bioenergy source. The HHV value was calculated by using Eq. . Therefore, lignin does not exhibit a straightforward weight loss derivate peak−1 corresponding to the axial angular bending of the O–H bond in hydroxyl groups in α cellulose, showing how the hydroxyl group is broadly distributed throughout the cellulose and hemicellulose structures85. Spectral peaks at 1635 cm−1 were assigned carbonyl stretching C–O for the acetyl group in hemicellulose and the aldehyde group in lignin. The C–O–C bond deformation of hemicellulose and cellulose was assigned to 1422 cm−1. The spectral peak at 1056 cm−1 can be assigned to the C–O–C group in Pj pods, and the overlap of stretching vibrations of glycosidic bonds in glucomannan and xylan85. A spectral peak of 606 cm−1 can be assigned to β glycosidic linkages within sugar units , with potentially smaller aspect ratios, giving a large surface area for chemical pretreatment Fig. A. Due to75. Removing amorphous components causes the cellulose index to rise, resulting in the more significant intensity peak associated with bleached fibres99. In Pj pods, cellulose, a well-defined crystalline peak, was observed at 2θ = 10, 22.85 and 38.27 . There were three typical peaks for Pj pods cellulose at 2θ angles of 2θ = 10º, 22.85º and 38.27º regions related to cellulose type I . There is a direct correlation between the degree of crystallinity and the hardness of the samples. The sample becomes more difficult as the crystallinity index risesProsopis juliflora (Pj) pods were characterised using FTIR, SEM, XRD, NMR, HHV and TGA. Lignin, cellulose, and hemicellulose were isolated from Pj pods and evaluated. Pj pods have shown a low high carbon content of 41.77% w/w, a high calorific value of 17.073 MJ/kg, 57.76 ± 0.14% w/w of holocellulose and ash content of 0.21 ± 0.002% w/w. All the parameters were in good agreement with the reported literature. The composition and functional properties of holocellulose, and lignin may differ based on the structure and species of the biomass. However, only marginal differences can be observed in individual biomass based on seasonal and geographical variation. The significant amounts of holocellulose, 57.76 ± 0.14% w/w and the whole carbon content encourage high-value applications of Pj pods in biomass biorefinery. Minor proportions of lignin make it suitable for the fermentative production of biofuels. The present results indicated the significance of the valorization of Prosopis juliflora (Pj) pods. However, further in-depth investigations are required to efficiently utilize the same in environmental remediation, biomass-biorefinery and biofuel technologies. Recently our team has reported the hybrid hydrolysis and fermentation of Prosopis juliflora (Pj) pods for hydrogen production38. The scaleup of hydrogen production is in progress. Further, the combined thermochemical and biochemical routes for refining Prosopis juliflora (Pj) pods for bioenergy and other value-added products could contribute to the circular economy.


Generating polymer–metal structures by means of additive manufacturing offers huge potential for customized, sustainable and lightweight solutions. However, challenges exist, primarily with regard to reliability and reproducibility of the additively generated joints. In this study, the polymers ABS, PETG and PLA, which are common in material extrusion, were joined to grit-blasted aluminum substrates. Temperature dependence of polymer melt rheology, wetting and tensile single-lap-shear strength were examined in order to obtain appropriate thermal processing conditions. Joints with high adhesive strength in the fresh state were aged for up to 100 days in two different moderate environments. For the given conditions, PETG was most suitable for generating structural joints. Contrary to PETG, ABS–aluminum joints in the fresh state as well as PLA–aluminum joints in the aged state did not meet the demands of a structural joint. For the considered polymers and processing conditions, this study implies that the suitability of a polymer and a thermal processing condition to form a polymer–aluminum joint by material extrusion can be evaluated based on the polymer’s rheological properties. Moreover, wetting experiments improved estimation of the resulting tensile single-lap-shear strength. Lightweight systems in particular often require joining multiple components or materials due to size limitations of the production process or locally varying demands concerning functionality and costs [Additive manufacturing (AM) offers huge technical potential, especially with regard to sustainable and customized solutions ,2. One ond costs ,4. Recennd costs  emphasizφ, results from the interfacial tension between polymer and substrate, γPS, polymer and atmosphere, σPA, and substrate and atmosphere, σSA, according to Young’s equation (cf. Equation (1)). The interfacial tension is defined as the mechanical work that needs to be applied to increase the interfacial area by 1 cm2. From adhesive technology, wetting is known to be a crucial factor to buildup adhesive interactions between the substrate surface and the adhesive. Habenicht  distingu2, can be calculated according to the Young–Dupré equation (cf. Equation (2)). Although wetting is crucial to buildup the adhesive interactions, it is generally not possible to predict the strength of an adhesive joint solely based on the wetting conditions (Equation (2)) [The work of adhesion, which is defined as the mechanical work required to separate two phases with a contact area of 1 cmion (2)) .(2)WA=σγPS and σSA), wetting dynamics and thermally induced internal stresses [In general, clean metallic and oxidic surfaces possess a high surface tension, mers—σPA . Howevermers—σPA ,9. As a mers—σPA ). Hence,mers—σPA . Furtherperature . Hence, stresses . While tη, and a high loss factor, tan(δ). On the other hand, geometric accuracy and dimensional stability require high viscosity and a low loss factor. The loss factor, tan(δ) = G″/G′, equals the ratio of loss modulus, G″, and storage modulus, G′, and specifies whether the polymer melt behaves dominant elastic (tan(δ) < 1) or dominant viscous (tan(δ) > 1). Based on the local thermal history and the viscoelastic polymer melt properties, some authors [td. This time corresponds to a long-range relaxation or diffusion, respectively, of a macromolecule and can be calculated according to Equation (3) based on the zero shear viscosity, tro, which corresponds to short-range relaxations at the chain ends and equals the crossover time (cf. Equation (4)) [Das et al.  emphasiz authors ,15,16 delus, GN0 . Anotherion (4)) .(3)td=1Tm, of the hotmelt prior to the joining process. Accordingly, Amancio-Filho and Falck [Te, and the crystallization temperature, Tc, of the polymer processed by ME to optimize polymer–metal bonding. However, in their recent publications regarding the Addjoining® process, Falck et al. [Ts, Falck et al. [Which of the common thermoplastics for ME is most suitable to generate structural polymer–metal joints? Direct comparison between the studies is not feasible, in particular due to different processing and substrate surface conditions.Can structural polymer–metal joints be generated by ME on “simple” practical relevant metal surfaces ? In the relevant studies, either a primer was applied beforehand or complex surface preparation methods were used. Moreover, none of the studies considers joint degradation.For hotmelts, adhesion interface performance is known to be highly sensitive to local temperature management during application. Due to their high thermal conductivity, Habenicht et al.  suggestend Falck  specifiek et al. ,20 applik et al.  generatek et al.  increasek et al.  joined Ak et al. , we showk et al. , Dröder k et al.  and Bechk et al.  observedk et al.  reportedk et al. ). The prThis article focuses on the generation of structural polymer–aluminum joints by means of ME. Structural joints are characterized by high joint strength (shear strength greater than 7 MPa) and significant aging resistance [The hereafter described approach is a continuation of our previous work .2O3), size F150, which was received from Oberflächentechnik Seelmann . Particle size was about 82 µm and laid within standardized range (45–106 µm)—cf. DIN EN 13887-2003. Sandblasting was performed with a ST 800-J  at a pressure of 6 bar, a working distance of 10 cm and an angle of 90° to the surface.The aluminum substrates were received from the water-jet cutting company RS-Evolution . The deburred substrates were prepared to measure 25 mm by 115 mm from 2 mm thick sheet metal of EN AW-6082-T6. This medium-strength aluminum alloy has excellent corrosion resistance and is typically used for structural parts in, e.g., the transportation sector [wPo, of 0.8 mm . Three thermoplastic polymers, common for ME, were considered:ABS), Extrafill™ , yellow-colored filamentAcrylonitrile–butadiene–styrene copolymer (PETG), PolyLite™ , transparent filamentPoly glycol comonomer (PLA), Ingeo™ 3D870 , black-colored filamentPoly(lactic acid) . The extruder was equipped with a water-cooled heatsink, a “volcano”-type hotend and a brass nozzle with an inner diameter, Te, of 240 °C, 220 °C and 200 °C, respectively. Extruder temperature Te was chosen based on polymer melt viscosity. During the deposition of the dPo = 0.3 mm high layers, the extruder moved at a velocity, ve, of 10 mm/s. The polymer-specific substrate temperatures, Ts0, of 100 °C, 80 °C, and 60 °C for ABS, PETG and PLA, respectively, were chosen based on the recommended build-plate temperatures in the datasheets [Ts, was varied. If the substrate temperature at a certain layer i, Tis,, differed from the polymer-specific substrate temperature, Ts0, it is specified in the corresponding section. Slicing was done with Ultimaker Cura 4.0 .ABS, PETG and PLA were extruded at a temperature, tasheets ,27,28. Iω = 2π·10 Hz down to ω = 2π·0.01 Hz. In the first measurement cycle, the set temperature ranged from 200 °C to 130 °C for all polymers. In the second cycle, the maximum temperature was set to 250 °C, 240 °C and 220 °C for ABS, PETG and PLA, respectively, while the minimum temperature was kept unchanged at 130 °C. The third cycle was identical to the first cycle.Rheometry was performed with an Ar2000ex  rheometer using plate–plate configuration. A 1 mm thick polymer disc with a diameter of 25 mm was generated by ME and placed between the plates of the rheometer. After preheating to 200 °C, the disc was carefully compressed by the rheometer plates to prevent the polymer from squeezing out. Using deformation-controlled oscillation rheology, the deformation amplitude was set to 5%. After equalization for 2 min at the set measurement temperature, oscillation sweeps were carried out in the range from Rheometry was used to access the thermo–rheological properties of the polymer melts, which are crucial for wetting, cohesive properties ,30,31 anti, and frequency data, ω, were shifted with the horizontal shift factor Ta (cf. Equation (5)). Based on this time–temperature superposition [G* = G′ + iG″, and viscosity, η* = η′ + iη″, at reference temperature T0 were constructed according to Equation (6). The vertical shift factors, Tb = T0/T, solely depend on measurement temperature, T, and reference temperature, T0, while the horizontal shift factors, Ta, were fit to the Williams–Landel–Ferry equation with the WLF-constants C1 and C2 (cf. Equation (7)).Providing that all relevant relaxation phenomena show the same temperature dependence, relaxation times, position , single Ts,1, the primed extrusion nozzle was placed 1 mm above and 2 mm behind the front edge of the substrate and within the field-of-view of the camera. A polymer melt drop with a volume of about 5 µL (referring to the density at room temperature) was extruded before the extrusion nozzle was moved out of the camera’s field-of-view (standby position). Dynamic wetting was recorded for up to 120 min at several substrate temperatures, Ts,1. The highest considered Ts,1 was equal to the polymer specific extrusion temperature Te. Ts,1 was lowered in increments of 20 °C as long as proper deposition of the polymer melt drop could be achieved.To evaluate wetting behavior, an apparatus for contact angle measurement was added to the ME machine. This apparatus consists of illumination (7W LED spot with diffusor) and a camera , lens: LM50JC ) triggered by the ME machine via specific commands in the G-Code cf. . After pContact angle analysis was done using MATLAB , as shown in Tensile tests were carried out using a Kappa 100 DS  equipped with a 100 kN loadcell at a controlled ambient temperature of 23 °C. In addition to adhesion interface performance, bulk properties of polymer samples prepared by ME were acquired. All tensile tests were driven-displacement controlled, with 1 mm/min crosshead speed.Polymer bulk mechanical properties were obtained according to DIN EN ISO 527-2:2012 (type 1A). The extruded tracks were oriented parallel to the loading direction. The polymer was extruded on polyimide-taped aluminum substrates. Due to local excessive tension in the tapered regions , these rτSLJ = FB/AJ, was calculated based on the breaking load, FB, and the joint area, AJ. Failure patterns are classified as polymer part failure , cohesive failure (CF), adhesive failure (AF) and mixed adhesive and cohesive failure (ACF). The eccentric load path within the joint led to rotation during loading and caused, among other things, additional peel stresses . Deviating from the standard, the joint width of the single-lap joints (SLJ) was increased from 10 mm to 20 mm to improve handling. The clamping length was set to 20 mm for the polymer part and 45 mm for the aluminum part to achieve comparable bending stiffness of both adherents. The dimensions and the ME buildup strategy of the polymer part are shown in f. e.g., ). These Ts,1. For higher layers, the substrate temperature was gradually decreased down to the polymer-specific substrate temperature, Ts0. Ts,1 was equal to the polymer-specific extrusion temperature Te. Ts,1 was lowered in increments of 20 °C until the adhesive strength of the joint was too low for handling.The polymer–aluminum joints were prepared by fixing the aluminum substrates on an aluminum hot plate cf.  while exTs,1, of 200 °C and an extruder temperature, Te, of 220 °C. Sample preparation was carried out on three identical substrates per sequence. In total, 6 specimens were tested for each set of parameters. The fresh SLJ were tested within 5 h after sample production to reduce aging effects.Aging (moist–warm conditions): Darkness, 40 °C and humid air Storing (dry conditions): Darkness, 23 °C and dry air Two different moderate environments, which represent real operating conditions, were considered, and are referred to as aging and storing:Storing was considered as a reference, in order to separate the effect of internal thermal stresses from hygro–thermo–oxidative aging effects.τSLJ, in the fresh state were aged and stored for up to 100 days. Before mechanical testing, the specimens were acclimatized for at least 1 h at the test climate. Dog bone tensile test specimens were also aged for 100 days to achieve the mechanical polymer bulk properties in the aged state as a reference. For each aging time, 6 samples were tested.SLJs with the highest tensile single-lap-shear strength, Tg = 99 °C) showed the highest and PLA (Tg = 58 °C) the lowest glass transition temperature, Tg. The thermal expansion coefficient, α, of the polymers was about an order of magnitude higher than that of aluminum. Hence, the thermal joining process resulted in thermally induced interfacial stresses. Internal stress buildup occurred below the glass transition temperature, Tg, in the amorphous phase and below the crystallization temperature, Tc, in the crystalline phase. Consequently, thermally induced interfacial stresses were particularly relevant for PLA and ABS due to crystallization and high glass transition temperature, Tg, respectively. With regard to the sensitivity to the polymer formulation, the process and buildup parameters, and the strain rate, the observed values for elastic modulus, E, yield strength, σy, and elongation at yield, εy, lay in the data range reported in the literature . Moreove γ˙= Te of 240 °C, 220 °C and 200 °C, respectively, ABS, PETG and PLA showed comparable viscosity at ω = 48 Hz .G′ and G″, or loss factor, tan(δ), revealed pronounced differences, particularly in terms of the loss factor (cf. δ), and thus the viscous character of the polymer melt, was significantly higher for PETG than for PLA and ABS.Comparing the three polymers in terms of modulus, ctor cf. . Especiaη0, could be determined, as there was a continuous increase in viscosity for small frequencies, ω |max, in the available frequency range, ω, was considered. The maximum of the loss factor, tan(δ)|max and the rouse relaxation time, tro, are listed in For ABS as well as for PLA in the second and third run, no zero shear viscosity, s, ω cf. . The plaange cf. . Therefotro, and viscosity, η,  behaved like viscoplastic fluids. These types of fluids do not converge to a zero shear viscosity, d stress . This isThe carboxyl-(end-) groups resulting from hydrolysis of PETG and PLA can form strong physical bonds (H-bonding) to the metal substrate .Due to heat-induced polymer degradation, the first measurement run is most relevant. However, in the thermal joining and wetting process, the polymers were exposed to similar conditions by means of temperature and atmospheric contact. Hence, the observed degradation phenomena may also be decisive in these processes, in particular with regard to the following aspects:φ(t), depended on substrate temperature, Ts,1, and polymer  to insufficient (φeq > 90°) wetting conditions [The temporal change in contact angle, ymer cf. . EquilibTs,1, which is consistent with temperature dependence of surface tension, σPA [η |max, was highest for PETG and lowest for ABS (cf. δ)|max, were adverse for wetting.For all three polymers, wetting improved with increasing substrate temperature, ion, σPA , and visA [η cf. . However ABS cf. . Additio ABS cf. . Since tτSLJ, was investigated, exemplarily for PETG and Ts,1 = 200 °C . Hence, polymer–metal joints with no additional wetting time were considered.Wetting of the aluminum substrates through the polymer melts depended significantly on wetting time and took up to several hours to reach equilibrium. Therefore, first, the influence of wetting time on tensile single-lap-shear strength, 0 °C cf. . IncreasτSLJ, as a function of substrate temperature, Ts,1. In conjunction with the improved wetting  at all substrate temperatures, showed the lowest tensile single-lap-shear strength. Contrary, the well-wetting PETG had the highest τSLJ. With increasing substrate temperature and tensile single-lap-shear strength, the failure pattern changed from adhesive (AF) over than mixed (ACF) and cohesive (CF) to polymer part failure (PF). For PETG, the shear strength reached a plateau for Ts,1 above 180 °C. This is in accordance with the equilibrium contact angle, φeq, which decreased only slightly between 180 and 220 °C  dominated, which means the polymer–metal interface was no longer the weak point. For Ts,1 = 220 °C, there was even a small decrease in τSLJ, accompanied by an increase in polymer part failure (PF). Similar to the effect of the additional wetting time . Consequently, arrangement and orientation of the macromolecules in the wetted area, which is required to form adhesive interactions , proceeded much faster than the macroscopic wetting.For PETG, tensile single-lap-shear strength, utes cf.  and, henutes cf. . HoweverFirst, the effect of aging on the mechanical polymer bulk properties is presented. E, yield strength, σy, and elongation at yield, εy, are given in εf > 10%) in the fresh state, they broke brittle (εf < 10%) in the aged state. Hence, PLA and PETG became brittle due to aging, which made them more sensitive to notches and local stress concentrations. Reasons for the embrittlement could include the extraction of plasticizers, swelling and hydrolysis (ester groups) [Elastic modulus,  groups) . The obs groups) ). One ex groups) . Due to τSLJ in the fresh state, Al–ABS-SLJ was not considered for aging and storing experiments. PETG- and PLA-SLJs, with the highest τSLJ in the fresh state, were aged and stored for up to 100 days  occurred. Hence, the decrease in τSLJ cannot be attributed to interfacial aging effects. Instead, embrittlement of the polymer component due to aging , indicating a weakening of the polymer–metal interface. Contrary to PETG, storing caused an even faster decrease in τSLJ. Consequently, the decrease in τSLJ is attributed to thermally induced internal stresses. These stresses built up below the crystallization temperature, Tc, of PLA. Compared to storing, the mobility of the macromolecules increased during aging due to the higher temperature and the plasticizing effect of water. Moreover, swelling counteracted the thermally induced internal stresses. This explains the faster decrease in τSLJ during storing compared to aging.Tensile single-lap-shear strength, φeq, the term cos(φeq) + 1 was considered, which is proportional to the work of adhesion (cf. Equation (2)). φeq) + 1 as a function of the rouse relaxation time, tro. Temperature dependence of the Rouse relaxation time is given by the horizontal shift factors, Ta. Taking the maximum loss factor, tan(δ)|max, into account revealed a correlation between polymer melt rheology and wetting, which is almost independent of the polymer material |max. This is plausible, as tro describes the time dependence of the polymer dynamic processes and tan(δ)|max the viscous character of the polymer melt.First, wetting was correlated with the polymer melt properties. Instead of the contact equilibrium angle, rial cf. b. AccordτSLJ, as a function of cos(φeq) + 1. There were significant differences between the polymers. For a given equilibrium contact angle, φeq, single lap shear strength, τSLJ, was higher for PETG than for ABS and PLA. Again, by taking the maximum loss factor, tan(δ)|max, into account, the differences between polymers were significantly reduced |max, in By combining the correlations from Which of the common thermoplastics for ME is most suitable to generate structural polymer–metal joints?Can structural polymer–metal joints be generated by ME on “simple” practical relevant metal surfaces ?This study focused on the generation of structural polymer–aluminum joints by means of ME. Based on the relevant literature ,22,23,24These questions were addressed by investigating ME-generated joints between grid-blasted aluminum substrates and the thermoplastics ABS, PETG and PLA as a function of thermal processing (substrate temperature) and aging. For all polymers, tensile single-lap-shear strength increased with increasing substrate temperature. However, there were significant differences between the polymers. For the given conditions and material combinations, PETG was the most suitable to generate structural polymer–metal joints. Appropriate thermal processing conditions for the joining were an extrusion temperature of 220 °C and a substrate temperature of 200 °C. For this case, cohesive failure dominated, and the demands of a structural joint in terms of joint strength and aging resistance were met. Increasing the substrate temperature beyond 200 °C or increasing the time PETG was exposed to the elevated temperatures led to pronounced polymer degradation and reduced joint strength. While storing (dry conditions) had no significant effect on PETG–aluminum joint strength, aging (moist-warm conditions) reduced the tensile single-lap-shear strength due to degradation of the mechanical polymer bulk properties. Hence, this decrease did not result in reduced adhesive strength. Contrary to PETG, ABS–aluminum joints in the fresh state as well as PLA–aluminum joints in the aged state did not meet the demands of a structural joint. PLA–aluminum joint strength decreased faster during storage than aging, which was attributed to internal stresses resulting from the thermal joining process. In particular, crystallization of PLA favored internal stress buildup.τSLJ, equilibrium contact angle, φeq, and the rheologically derived quantities of Rouse relaxation time, tro, and maximum loss factor, tan(δ)|max, were correlated with each other. For the considered polymers and processing conditions, this study implied that the suitability of a polymer and a thermal processing condition to form a polymer–aluminum-joint by ME could be evaluated based on the polymer’s rheological properties  with a


This study aims to reveal the serum tumor marker (STM) levels in lung adenocarcinoma (LUAD) histological subtypes and evaluate their values in predicting the solid and micropapillary components (SMC).We retrospectively analyzed 3100 invasive LUAD patients between January 2017 and December 2020. Associations between preoperative STMs  and LUAD subtypes were evaluated. Multivariate regression analyses were used to determine the independent predictors. Predictive models for SMC were constructed and AUC (area under the curve) was calculated.p <0.001). Tumors with SMC also had higher CEA and CYFRA21‐1 levels than those absence of SMC. Gender, tumor size, CEA, Ki‐67, EGFR mutation (solid components only), and tumor differentiation were significantly independently associated with the containing of SMC. Patients were split into two data sets . The model with gender and tumor size yielded an AUC of 0.723 (training set) and 0.704  for the solid component. Combination of CEA, gender, and tumor size led to a significant increase in the predictive accuracy . The AUC of the model for micropapillary component with only gender and tumor size was 0.699 and 0.711 in the training set and validation set, respectively. Integration of CEA with gender and tumor size significantly improved the predictive performance with an AUC of 0.746  and 0.753 .CEA and CYFRA21‐1 levels differed across the LUAD histological subtypes, with the SPA (solid‐predominant adenocarcinoma) having the highest level and the LPA (lepidic‐predominant adenocarcinoma) harboring the lowest level (Serum CEA and CYFRA21‐1 varied considerably according to LUAD histological subtypes. The combination of serum CEA and other factors showed prominent values in predicting the SMC. Lung adenocarcinoma with solid or micropapillary components had higher levels of serum CEA and CYFRA21‐1 than those absence of solid and micropapillary components.Combination of serum CEA and other clinicopathological factors showed prominent values in predicting the solid and micropapillary components in lung adenocarcinoma. The prognosis of lung cancer is poor and the overall 5‐year survival rate remains less than 30% in most countries.Over the past few years, the radiomics features of LUAD subtypes were depicted and models for predicting the subtype components, especially the SMC, were initially constructed.Traditional serum tumor markers (STMs), such as CEA (carcinoembryonic antigen), CA199 (carbohydrate antigen199), and NSE (neuron‐specific enolase), are widely used for the early diagnosis and classification of lung cancer.To the best of our knowledge, few studies have described the level of STMs in LUAD histological subtypes.In the current study, we systematically evaluated the associations between six STMs  and LUAD histological subtypes, as well as other clinicopathological factors, by retrospectively analyzing the characteristics of 3100 invasive LUAD samples from January 2017 to December 2020 in our department. Further, predictive models for the SMC were constructed based on STMs and other clinicopathological characteristics. This study will provide a deeper insight into the levels of STMs according to histological subtypes of LUAD and an applied tool to predict the SMC in LUAD.22.1We retrospectively screened lung cancer patients who underwent surgical treatment in our department between January 2017 and December 2020. Patients who met the following criteria were initially included in this study: (1) primary lung cancer; (2) histopathological confirmed invasive adenocarcinoma with precise subtype classification: LPA, APA, PPA, MPA, or SPA; (3) single invasive tumor nodule; (4) STMs detection within 1 month before the surgery. Further, patients who (1) received preoperative treatments, including chemotherapy, radiotherapy, target therapy, and immune therapy, and (2) had a history of other malignant tumors in 5 years were excluded.2.2In this study, basic information and clinicopathological characteristics of study subjects were extracted from the medical records in our department. All these data were cleared up by one researcher and reviewed by another investigator. STMs were measured by electrochemiluminescent assay. The normal upper limit for CEA, AFP, CA199, CA724, CYFRA21‐1, and NSE was 4.7, 20.0 ng/ml, 39.0, 6.9 U/ml, 3.3, and 16.3 ng/ml, respectively. Tumors with the micropapillary or solid components <5% of the entire tumor were defined as absent.2.3t test, one‐way ANOVA, or Kruskal test was used for the comparison of continuous variables. The chi‐square χ2 test or Fisher's exact test was adopted for categorical variables. STMs and Ki‐67 levels were shown as the median and interquartile range (IQR). Correlation between STMs and Ki‐67 was evaluated using Pearson correlation. The impacts of various factors on STM levels were estimated by a generalized linear model. Independent factors identified in multivariate regression analyses were reserved to predict the SMCs in LUAD. We depicted the ROC (receiver operating characteristic curve) using the “pROC” package. All the analyses were performed based on R (3.6.0), and p < 0.05 was considered statistically significant.Student's 33.1As summarized in Table 3.2p <0.001). Consistently, 33.8% of SPA patients had an abnormal CEA expression, while the proportion was 33.3% in MPA, 12.7% in APA, 11.9% in PPA, and 5.8% in LPA (p < 0.001). Similarly, the level of CYFRA21‐1 in the SPA subtype was significantly higher than that in other histological subtypes (p < 0.001). Only 9.9% of LPA subjects harbored an abnormal CYFRA21‐1 expression, while the proportion was 24.1% in patients with SPA.As shown in Table p < 0.001). The percentages of patients with aberrant CEA and CYFRA21‐1 levels were 30.7% and 18.4% in patients with solid components, while only 8.4% and 11.8% in those without solid components (p < 0.001). In accordance, patients harboring micropapillary components had higher CEA (p < 0.001) and CYFRA21‐1 (p = 0.008) levels than that absence of micropapillary content. There were 27.9% and 17.4% of patients with micropapillary components had abnormal CEA and CYFRA21‐1 levels, which were much higher than 8.9% (p < 0.001) and 12.0% (p = 0.004) in patients without micropapillary components.To confirm the differential CEA and CYFRA21‐1 levels between histological subtypes in LUAD, we further analyzed the levels of STMs in samples with and without solid or micropapillary components (Table 3.3p < 0.001). Compared with younger patients (<60 years), the older had higher levels of CEA (2.39 [1.58–3.55] vs. 1.67 [1.11–2.56]) and CYFRA21‐1 (2.22 [1.72–2.91] vs. 1.88 [1.40–2.44]) (p < 0.001). The proportion of abnormal CEA and CYFRA21‐1 levels in older patients were 14.8% and 16.6%, which were much higher than that in younger patients , whereas AFP, CA724, and NSE showed no significant differential expression levels in patients with different tumor sizes (p >0.05). The median level of CEA increased from 1.59 ng/ml in tumors ≤10 mm to 1.95 ng/ml (10–20 mm), 2.77 ng/ml (20–30 mm), and 3.39 in tumors >30 mm in diameter . Consistently, abnormal rate of CEA was 3.8%, 7.9%, 22.7%, and 36.2% in tumors ≤10, 10–20, 20–30, and >30 mm, respectively (p < 0.001). Similarly, the expression levels of CEA and CYFRA21‐1 in samples with LN metastasis (N+) were significantly higher than that in LN‐negative (N0) subjects .The levels of STMs according to tumor size, LN metastasis, and tumor differentiation were further estimated. Among these markers, CEA, CA199, and CYFRA21‐1 levels significantly increased with the increase in tumor size (3.4p < 0.05). In brief, older patients, male patients, larger tumor size, solid, or micropapillary components were associated with a higher probability of abnormal CEA level, whereas female patients, EGFR mutation, and high grade of tumor differentiation were associated with a lower risk of aberrant level of CEA. These results suggested that the impact of histological subtypes on CEA levels was not dependent on tumor size, age, and gender.Given the differential levels of CEA and CYFRA21‐1 in histological subtypes and other subgroups, we further evaluated the associations between these clinicopathological factors, CEA and CYFRA21‐1 based on GLM regression models. As presented in Table p >0.05). Older patients and patients with larger tumor sizes had a higher risk of abnormal CYFRA21‐1 levels (p < 0.001). Compared to the LPA subtype, SPA had a higher probability of CYFRA21‐1 abnormal expression  and CYFRA21‐1  in preoperative serum were significantly correlated with the expression of Ki‐67 in LUAD tissues. AFP, CA199, CA724, and NSE levels showed no significant correlation with Ki‐67 expression (p > 0.05).In our previous study, we found that Ki‐67 expression differed across LUAD histological subtypes.3.6The univariate regression analysis showed that age, gender, tumor size, CEA, CYFRA21‐1, Ki‐67, tumor differentiation, and EGFR mutation were significantly associated with the solid components Table . Howeverp = 0.009; validation set: 0.747, p = 0.034). Further, the integration of postoperative factors  and Model 2 greatly improved the predictive accuracy .Given the absence of independent external validation, we split patients into two data sets according to the operative date . Three models were constructed for the solid components: Model 1 (gender + tumor size), Model 2 (Model 1 + CEA), Model 3 (Model 2 + Ki‐67 + EGFR mutation + tumor differentiation). As shown in Figure p = 0.045) and 0.753 . When Ki‐67 and tumor differentiation were further integrated into Model 2, the predictive efficiencies had a great increase with an AUC of 0.837 in the training set and 0.901 in the validation set  to predict whether the micropapillary components existed in LUAD. The AUC of the model with only gender and tumor size was 0.699 and 0.711 in the training set and validation set, respectively Table . As expe4Nowadays, more and more studies focus on the differential prognosis between LUAD histological subtypes. Patients with SPA or MPA might need a more conservative surgical procedure and show different responses to adjuvant therapies. Therefore, the early detection of SMC could have crucial impacts on the choice of surgical procedures and potential follow‐up adjuvant therapies.CEA is a 180‐KDa glycoprotein normally expressed during fetal development but sharply declines before birth. Accordingly, serum CEA is usually less than 2.5 ng/ml in healthy adults but shows aberrantly increased expression in patients with malignant tumors.In addition to CEA, for the first time, we found that the CYFRA21‐1 level varied across histological subtypes of LUAD. The SPA had the highest level of CYFRA21‐1, whereas the LPA had the lowest CYFRA21‐1 level. Consistently, LUAD with solid or micropapillary components had a higher CYFRA21‐1 level than those without solid or micropapillary components. Similar to CEA, CYFRA21‐1 levels in males and older patients were significantly higher than those in females and younger patients, respectively. Besides, patients with larger tumor size, lymph node metastasis, and poor tumor differentiation harbored higher CYFRA21‐1 levels. Notably, the multivariate regression analysis showed that gender and tumor differentiation were not associated with the CYFRA21‐1 level, suggesting that the influence of gender and tumor differentiation on CYFRA21‐1 expression level might result from other underlying confounding factors.Over the past few years, models for predicting SMC have been developed based on radiomics features and histopathological markers.Besides the great values in the early diagnosis and classification of lung cancer, CEA and CYFRA21‐1 correlated with the responses to chemotherapy and immunotherapy. For example, Wang et al. found that NSCLC patients with a higher CYFRA21‐1 level had a lower complete response rate of chemoradiotherapy compared with those with a low level of CYFRA21‐1 (2.9% vs. 20.3%).In summary, this study had three prominent advantages. First, this study had a large sample size with 3100 LUAD patients. Second, we used both the average values and grades of STM expression levels for the comparison. The independent effect of each factor on STM expression levels was further estimated using the regression analyses. Third, we integrated the STMs and other clinicopathological characteristics to predict the SMC in LUAD, preoperatively and postoperatively. However, this study also had some limitations. The underlying reasons for the differential CEA and CYFRA21‐1 levels among LUAD histological subtypes need in‐depth studies. In addition, 91.87% of patients enrolled in the current study were pathological stage Ι, only 8.13% of patients had stage ΙΙ or ΙΙΙ, which could restrict the applicability of our models.5In conclusion, serum CEA and CYFRA21‐1 levels differed across the predominant histological subtypes of LUAD. Serum CEA could be used as a valuable noninvasive predictor for the SMC in LUAD.Conflict of interest relevant to this article was not reported.Zhihua Li: Conceptualization, Writing‐original draft, Methodology, Formal analysis. Weibing Wu: Data curation, Methodology, Formal analysis, Resources, Funding acquisition. Xianglong Pan: Data curation, Writing‐original draft, Investigation. Fang Li: Resources, Data curation, Methodology, Supervision. Quan Zhu: Supervision, Resources, Data curation. Zhicheng He: Writing‐original draft, Data curation, Supervision, Resources. Liang Chen: Conceptualization, Writing‐original draft, Project administration, Resources, Funding acquisition.This study was approved by the Ethical Committee of the First Affiliated Hospital of Nanjing Medical University and individual consent for this retrospective analysis was waived.Figure S1Table S1–S7Click here for additional data file.


Phocaeicola dorei and Lachnoclostridium symbiosum, growing in inulin or xylan, and how they provide a protective effect in cultured cells. P. dorei (previously belonging to the Bacteroides genus) was able to consume inulin and xylan, while L. symposium only used certain inulin fractions to produce butyrate as a major end product. Constrained-based flux simulations of refined genome-scale metabolic models of both microbes predicted high lactate and succinate cross-feeding fluxes between P. dorei and L. symbiosum when growing in each fiber. Bidirectional culture assays in both substrates revealed that L. symbiosum growth increased in the presence of P. dorei. Carbohydrate consumption analyses showed a faster carbohydrate consumption in cocultures compared to monocultures. Lactate and succinate concentrations in bidirectional cocultures were lower than in monocultures, pointing to cross-feeding as initially suggested by the model. Butyrate concentrations were similar across all conditions. Finally, supernatants from both bacteria cultured in xylan in bioreactors significantly reduced tumor necrosis factor-α-induced inflammation in HT-29 cells and exerted a protective effect against the TcdB toxin in Caco-2 epithelial cells. Surprisingly, this effect was not observed in inulin cocultures. Overall, these results highlight the predictive value of metabolic models integrated with microbial culture assays for probing microbial interactions in the gut microbiota. They also provide an example of how metabolic exchange could lead to potential beneficial effects in the host.The gut microbiota is constituted by thousands of microbial interactions, some of which correspond to the exchange of metabolic by-products or cross-feeding. Inulin and xylan are two major dietary polysaccharides that are fermented by members of the human gut microbiota, resulting in different metabolic profiles. Here, we integrated community modeling and bidirectional culturing assays to study the metabolic interactions between two gut microbes, IMPORTANCE Microbial interactions represent the inner connections in the gut microbiome. By integrating mathematical modeling tools and microbial bidirectional culturing, we determined how two gut commensals engage in the exchange of cross-feeding metabolites, lactate and succinate, for increased growth in two fibers. These interactions underpinned butyrate production in cocultures, resulting in a significant reduction in cellular inflammation and protection against microbial toxins when applied to cellular models. The gut microbiota is represented by a complex network of microorganisms occupying different niches in the human gut , 2. Thei3–Bifidobacterium species in in vivo studies  , 34. The37–Phocaeicola dorei (previously assigned to the Bacteroides genus) and Lachnoclostridium symbiosum, are key species in butyrate production within a 15-species synthetic consortium  of 0.70 for L. symbiosum and 0.83 for P. dorei  of mparison  to S6. OP. dorei   and xylan (0.09 h−1) were constrained in the models  balanced growth on the two fibers. For this task, the experimentally observed community growth rates on inulin (0.35 h10.1128/msystems.00646-22.1FIG S1P. dorei and L. symbiosum in bioreactors. (Top) Growth curves (OD600) for inulin and xylan. Experimentally observed community growth rates at midexponential phase growing on inulin and xylan were 0.35 h−1 and 0.09 h−1, respectively (12 h ≤ t ≤ 24 h). (Middle) Relative abundance for L. symbiosum and P. dorei in inulin (left) and xylan (right). In the same period for the community growth calculation, the microbial abundances were stable and were 0.159 on inulin and 0.091 on xylan for P. dorei, and 0.841 on inulin and 0.909 on xylan for L. symbiosum. (Bottom) Concentrations of SCFAs and lactate in bioreactors at the end of each run in inulin (left) or xylan (right). Pd, P. dorei; Ls, L. symbiosum. Download FIG S1, TIF file, 0.1 MB.Cocultures of Copyright © 2022 Hirmas et al.2022Hirmas et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the L. symbiosum was predicted to reach a greater abundance than P. dorei, which was substantially higher when growing on xylan .Under optimal community growth on each fiber, on xylan . Under iymbiosum , whereasscenario . This coymbiosum . This exP. dorei and L. symbiosum ranged, respectively, from 0.113 to 0.558 and 0.442 to 0.887 under inulin growth and from 0.003 to 0.210 and 0.789 to 0.996 under xylan utilization  and a bidirectional cross-feeding of acetate and lactate , while it was significantly lower than zero for L. symbiosum, i.e., it was consumed   correlation  between the exchanges of both microbes  . This waum test) . The casn −0.95, . On the n −0.95, . Lactatemicrobes . Finallyon 0.12, , succinaon 0.12,  under xyP. dorei and L. symbiosum was experimentally assessed in bidirectional assays. In this setup, microorganisms are cultured either in the bottom well or top insert of a Transwell plate, separated by a 0.1-μm membrane filter. The filter allows the exchange of small and medium-sized metabolites that reach chemical equilibrium.The metabolic exchange between P. dorei reached moderate biomass concentration values in inulin and xylan, similar to lactose . On the other hand, L. symbiosum presented a moderate basal growth, which correlates with the ability of clostridia to ferment amino acids . Particularly, L. symbiosum growth using inulin was higher than the basal, suggesting partial fructans utilization (P < 0.05 by t test). lactose . The preno acids . However10.1128/msystems.00646-22.2FIG S2P. dorei and L. symbiosum in bidirectional assays and unidirectional setup under different carbon sources. Cellular growth of P. dorei and L. symbiosum in bidirectional assays (P. dorei [A] and L. symbiosum [C]) and in unidirectional or monocultures (P. dorei [B] and L. symbiosum [D]). In each case, biomass measurements (OD600) were collected at 0, 24, and 48 h. Each condition represents a biological triplicate, and error bars denote one standard deviation. Pd, P. dorei; Ls, L. symbiosum; L, lactose; I, inulin; X, xylan; Z, mZMB culture medium. Download FIG S2, TIF file, 0.6 MB.Cellular growth of Copyright © 2022 Hirmas et al.2022Hirmas et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. dorei in inulin alone or using the spent supernatant from L. symbiosum in inulin . This suggests that P. dorei does not see a major impact on its growth in the presence of L. symbiosum, probably consuming the spent substrates not used by L. symbiosum. Supernatants after 48 h of growth of P. dorei inoculated with inulin or xylan did not stimulate the growth of L. symbiosum in the unidirectional assay (P > 0.05 by t test). This suggests that the growth increase of L. symbiosum in the presence of P. dorei depends on the synergistic growth of both microorganisms.In unidirectional experiments, similar growth values were observed for n inulin . FurtherP. dorei, thin-layer chromatography (TLC) plates and carbohydrate quantification showed a reduction in total inulin concentration after 48 h (P < 0.05 by t test), especially the intermediate and smaller fractions. L. symbiosum also reduced the concentration of inulin in the medium, albeit to a lesser extent . TLC showed that this pair consumed all inulin fractions after 48 h. These results indicate an accelerated consumption of inulin in coculture, reaching almost total consumption at 24 h.To better understand the mechanisms underpinning the higher growth in cocultures on inulin and xylan, we screened the supernatants from bidirectional assays for carbohydrate consumption . For P. r extent  and C. TP. dorei monoculture in xylan resulted only in partial substrate consumption after 48 h, leaving degradation products as shown by TLC  but no visible degradation products (n by TLC  and D. Oproducts . Xylan-dproducts .L. symbiosum monoculture supernatants did not contain lactate in any sample. Single cultures of P. dorei produced 17 mM lactate in inulin and 7 mM lactate in xylan after 48 h. The presence of L. symbiosum reduced lactate amounts in both compartments (P < 0.05 by t test). This corresponds to an approximate reduction of lactate of 15% (in inulin) and 80% (in xylan) with respect to total lactate produced by P. dorei alone. Similarly, only P. dorei produced succinate. Its concentration was higher after growing in xylan than inulin after 48 h (P > 0.05 by t test). A reduction in succinate was also observed in xylan cocultures (P > 0.05 by t test), pointing to the presence of L. symbiosum as possibly being responsible . Xylan utilization resulted in higher acetate concentrations, confirming previous modeling results. Propionate was produced by P. dorei at much lower concentrations in media with no carbon source or xylan, but not inulin . These results indicate that butyrate production is independent of the substrate under these conditions.Finally, concentrations between 20 and 30 mM butyrate were detected in l medium . ButyratP. dorei monoculture in inulin showed the induction of a levanase  were more active in coculture in xylan. This correlates with the higher acetate production reported above. A reduction in butyrate-producing enzymes in L. symbiosum in the xylan coculture was found when comparing the growth in basal medium or L. symbiosum alone. Finally, genes potentially contributing to succinate and propionate production in the P. dorei genome were induced in xylan coculture correlating with concentration data . This concentration was chosen after an optimization assay (−/+ cells (P < 0.05 by t test). This effect was abolished at a higher butyrate concentration (30 mM). In this model, adding supernatants from bacteria cultured in xylan exerted an anti-inflammatory effect, as evidenced by a significant 50% reduction in IL-8 expression (P < 0.05 by t test). This effect was not observed with the inulin supernatant .Supernatants were recovered at the end of each fermentation (26 h for inulin and 48 h for xylan), filtered, and used to evaluate their anti-inflammatory and protective potential in human intestinal cell lines. Confluent HT-29 Glcon assay . In a do10.1128/msystems.00646-22.3FIG S3−/+ cells exposed to increasing concentrations of TNF-α for 6 h. (B) Protective effect of sodium butyrate on HT-29 Glc−/+ cells exposed to TNF-α for 6 h. IL-8 expression estimated by q-PCR. (C) Effect of 30 mM butyrate on cells treated or not with TNF-α. The bars represent the average ± SD of the times of change in IL-8 expression relative to baseline (negative control). (D) Effect of TcdB on Caco-2 cell monolayers for 6 h. Time-dependent alteration of transepithelial electrical resistance (TEER) after apical exposure of 100, 250, and 350 ng/ml of TcdB. (E) Protective effect of different concentrations sodium butyrate on Caco-2 cells monolayers exposed to TcdB for 6 h. The points represent the percentage of the initial TEER value over time (average ± SD). A two-way ANOVA test and Bonferroni post hoc test were performed to assess statistical significance between the groups (n = 3). Download FIG S3, TIF file, 1.4 MB.Evaluation of protective effect of butyrate in model cell lines. (A) Standardization of the intestinal inflammation model in HT-29 GlcCopyright © 2022 Hirmas et al.2022Hirmas et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2) seeded in a Transwell system were apically exposed to different TcdB concentrations for 6 h . In this model, only a lower butyrate concentration (1.65 mM) showed a significant protective effect, preventing the permeability increase induced by 100 ng/ml of TcdB (P > 0.05 by t test). Interestingly, the bacterial supernatants from the xylan fermentation showed a modest but significant protective effect against TcdB action on the epithelial permeability (P < 0.05 by t test). This effect was not observed for the inulin supernatant with P. dorei and L. symbiosum  decreased dextran sulfate sodium-induced damage in mice colonic mucosa  reduced IL-8 expression and NF-κB activation. These results are consistent with our data. Our supernatants were added at 10% vol/vol, so the butyrate concentration administered to the cells ranged between 1 and 3 mM.Similarly, oral treatment with the butyrate-producer c mucosa . It has in CBP22 –75. Thomn CBP22 – observedper se may not be as effective as the restoration of butyrate-producing bacteria or the relevance of dietary fibers to produce this or other fermentation products. It is important to highlight that in this study, in vitro bioreactor samples were taken at the end of the exponential phase. Besides butyrate, supernatants might contain a variety of molecules such as ammonia that might hinder the positive effect of butyrate on epithelial cells. Future studies are needed to elucidate the mechanism of these bacterial supernatants and identify other metabolites or bioactive compounds that exert a protective role in intestinal cells.In a recent study, the addition of 1.5 mM sodium butyrate significantly increased TEER in Caco-2 cell monolayers . This woP. dorei and L. symbiosum, were studied by applying a combined approach of metabolic modeling and experimental cell culture assays. Experimental results were consistent with metabolic modeling predictions, pointing to cross-feeding of lactate and succinate in co-cultures growing on inulin or xylan. Both bacteria showed different characteristics in single culture and in the presence of the other partner, both in inulin and xylan. These changes were observed in terms of their growth, substrate consumption, gene expression, and SCFA production. This study provides an example of the capabilities of metabolic models for predicting microbial interactions and how these predictions could be tested in an experimental setup that evaluates bidirectional interactions. Finally, this work suggests that metabolites differentially produced in xylan provide a protective effect against toxin damage in vitro.The metabolic interactions between two gut commensal microorganisms, P. dorei DSM 17855 and L. symbiosum WAL-14673 and cured manually with literature data  were obtained from AGORA v3.0 (37) for ure data , 76–80.  data 76–, 81. MEMterature , 32, 76.terature . Precisia et al. . Finallyin silico simulations were set to values that ensured they were in excess. The bounds definition for the amino acids uptake rates followed a similar approach. Xylan and inulin are polysaccharides without a fixed chain length; therefore, the formulas described in the Virtual Metabolic Human database were employed for simplicity . For inoculations and initial tests, both microorganisms were cultured in Reinforced Clostridium Media  supplemented with 0.5 g/liter of l-cysteine . All incubations were performed at 37°C for 24 to 48 h in an anaerobic jar  with anaerobic packs . mZMB medium was prepared as in reference g and washed with blank prereduced mZMB media. Each bacterium was inoculated in 1 ml mZMB with 2% inulin  or 2% xylan from birchwood . Substrates were previously filtered using 0.22-μm filters , and microorganisms were inoculated at 5% vol/vol. Samples were transferred to 96-well plates, covered with a mineral oil layer, and cultured in triplicates for 48 h. OD600 was measured in a Synergy H1 spectrophotometer  at 0, 24 and 48 h. Similarly, each microorganism was cultured in 4 ml of mZMB supplemented with 2% xylan or 2% inulin for 48 h and anaerobic conditions. Tubes were centrifuged at 10.000 × g for 5 min, and each supernatant was recovered and filtered as above. 100 μl of each supernatant were added to a well in a 96-well plate that had 100 μl of prereduced mZMB, resulting in a 1:1 mixture. Then, each well with supernatant from one microorganism was inoculated with the other at 5% vol/vol for 24 and 48 h. Absorbance was measured at 600 nm in a Synergy H1 spectrophotometer . A paired Student’s t test assuming equal variances was performed on the maximum OD value for each condition.Single cultures of both microorganisms were carried out in 96-well plates to evaluate the consumption of different metabolites, such as inulin, xylan, succinate, and lactate. As mentioned before, microorganisms were reactivated for 48 h as mentioned before, in 4 ml of RCM-cys at 37°C under anaerobic conditions. They were centrifuged at 5,000 × L. symbiosum was always cultured in the upper insert and P. dorei in the lower well. The bacteria were separated by the permeable membrane of the insert (0.1 μm), which allowed the passage of small carbohydrates and metabolites. Cocultures were performed in 250 μl mZMB in the insert and 1 ml in the bottom well. Nine conditions of mono and coculture were tested in duplicate, using 2% of lactose (Sigma-Aldrich), inulin , or xylan from birchwood  as the sole carbon source. The plate was preincubated in an anaerobic jar under anaerobic conditions at room temperature for 48 h to reduce the medium.Both bacteria were cultured in Tissue Culture Plate Inserts . Later, samples were transferred to Eppendorf tubes and centrifuged at 10,000 × g for 2 min, and the supernatant was separated from pellets. Both were stored at −80°C until use. For the final OD values, the mean of each basal medium without bacteria was subtracted according to the carbon source. A paired Student’s t test assuming equal variances was performed on the maximum OD value for each condition.Bacteria were reactivated in RCM for 48 h, centrifuged at 10,000 × 2SO4 in ethanol as the detecting reagent  was performed in silica gels F-60 , using 1-butanol/ethanol/water 10:8:5 vol/vol as running buffer and 1% orcinol in 10% H reagent , 85. Two reagent , quantift test assuming equal variances was performed on the SCFA data.Acetic, butyric, lactic, propionic, and succinic acids of select supernatants and bioreactor samples from 26 and 48 h were measured in a Lachrom liquid chromatograph (Merck-Hitachi), using an Aminex HPX-87H Ion exclusión column . Thirty microliters of supernatants was injected at a flux of 0.45 ml/min, at 35°C for 35 min. Standard curves were created by measuring nine dilutions of 30 g/liter to 0.155 g/liter of each acid in HPLC grade water. Ten conditions plus a sample of mZMB at hour 48 were tested in biological duplicates. The control of the chromatograph and data analysis was done using Multi-HSM Manager software (Hitachi). A paired Student’s RNA was extracted from the pellets of the samples analyzed by HPLC. Total nucleic acids were extracted using a modified version of the phenol-chloroform/isoamyl alcohol method . Purity RNA was converted into cDNA with the AffinityScript QPCR cDNA Synthesis kit , using the manufacturer’s protocol and random primers and negative control with free nuclease water (Sigma-Aldrich) and an RNase block control for two of each bacterial sample. The final quality of cDNA was assessed by measuring the 260/280 absorbance ratio in a Tecan Infinite M200 Pro plate reader, and absorbance curves from 230 nm to 300 nm were obtained to rule out chemical contamination. All cDNA samples were stored at −20°C until use.L. symbiosum, related to fructan metabolism and butyrate production pathways. Ten genes were selected for P. dorei related to short-chain fatty acids (SCFA) production and inulin and xylan consumption. 16S rRNA genes were used for both microorganisms as the reference gene. Quantitative PCRs (qPCRs) were prepared using the SensiFAST SYBR No-ROX kit  under the manufacturer instructions and 1 μl of each cDNA. Amplification was performed in an AriaMx real-time PCR system (Agilent Technologies), using 96-well optical plates MicroAmp Fast Optical .Primers for calculating changes in gene expression were designed using Primer-BLAST for select genes . Six genes were chosen for TC) values and efficiency of the reactions were calculated using the Agilent Aria 1.7 software. Changes in gene expression were calculated using the efficiency-corrected method , 60°C for 10 s , and 72°C for 10 s (extension). All conditions had two biological replicates with three technical replicates. A standard curve was included by loading 10-fold dilutions of genomic DNA, and a negative control was included for each pair of primers. Threshold cycle (d method . We sepa600) was measured, cultures were centrifuged at 3,000 × g for 5 min, and the supernatant was discarded. The pellet was resuspended in the fixed groups of mZMB without a carbon source so that the final OD600 was equal to 1 for each microorganism before being inoculated to the bioreactor. The bioreactor was autoclaved for 15 min at 121°C, with a 34.2 g/liter of tryptone , 1 g/liter of l-cysteine, and 70 ml of distilled water for each experiment. Xylan (20 g/liter) was also autoclaved in the bioreactor, while inulin (20 g/liter) was sterilized with the remaining components of the medium using 0.22-μm filters and inoculated in the bioreactor after being autoclaved , using xylan or inulin at 20 g/liter as the sole carbon source. Microorganisms were cultured using mZMB with a fixed pH of 5.5, and each fermentation/condition was made in duplicate. Before inoculation, both microbes were grown individually for 48 h at 37°C in an anaerobic jar in mZMB using lactose (20 g/liter) as carbon source. The optical density at 600 nm , supplemented with 10% vol/vol heat-inactivated fetal bovine serum and 1% penicillin/streptomycin . Cells were cultured at 37°C in a humidified incubator with 5% CO2 and were subcultured using 0.05% trypsin at 60% to 90% confluence. To standardize the inflammation model, HT-29 Glc−/+ cells (passages 47 to 50) were seeded at 4 × 105 cells per well in a 24-well polystyrene plate. When cells reached confluence, TNF-α  was added at final concentrations of 0.5, 2.5, 5, 10, 25, and 50 ng/ml. Cultures were incubated for 6 h. Subsequently, cells were washed with phosphate buffer saline , rapidly detached from the plate with 1× trypsin, and centrifuged at 13,000 × g for 2 min at 4°C. Cell pellets were preserved in RNA-Later  at 4°C until use.The anti-inflammatory effect of cell supernatants was evaluated in the human colonic adenocarcinoma cell line HT-29 Glc−/+ . This cu−/+ . Cells wg for 2 min at 4°C, and pellets were preserved in RNA-later until use. As high concentrations of butyrate stimulate IL-8 expression, cells exposed to 30 mM sodium butyrate were used as a positive control, while cells untreated with TNF-α were used as the negative control.To measure the protective effect of sodium butyrate, cells in passages 51 to 54 were treated with 25 ng/ml of TNF-α and sodium butyrate (Sigma-Aldrich) at final concentrations of 3.25, 7.5, 15, and 30 mM for 6 h. After incubation, cells were washed with PBS, trypsinized, centrifuged at 13,000 × 5 cells per well in a 48-well plate. Confluent cells were treated with 25 ng/ml of TNF-α and 30 μl of the supernatant of each bioreactor (10%). After 6 h of incubation, cells were separated and preserved as described before.To evaluate the anti-inflammatory effect of the bioreactor supernatants, cells in passages 55 to 59 were seeded at 3 × 10−/+ cells was extracted using E.Z.N.A. total RNA kit , according to the manufacturer’s protocol and reference TC values were automatically determined, and changes in gene expression were determined by the Relative Standard Curve method   supplemented with 10% vol/vol heat-inactivated fetal bovine serum , 1% penicillin/streptomycin  and 1% (vol/vol) of nonessential amino acids (Corning). Cells were cultured at 37°C in a humidified incubator with 5% CO2 and then subcultured using 0.05% trypsin at 60% to 90% confluence. For standardization, Caco-2 cells (passages 16 to 19) were cultured at a density of 1 × 105 cells per well in polycarbonate Transwell filters of 12-mm diameter and 0.4-μm pore size , previously treated with rat tail collagen (Gibco). Apical and basolateral compartments were filled with 1.5 and 0.5 ml of medium, respectively. The medium was changed three times per week until cells were differentiated (15 to 21 days). When basal TEER reached values higher than 600 Ω·cm2, the apical medium was removed, and TcdB toxin was added to the medium without inactivated fetal bovine serum, at final concentrations of 100, 250, and 350 ng/ml. Monolayers were incubated for 6 h with regular measurement of the TEER.Permeability assays were carried out in Caco-2 cells, considering its similitude to the small intestine epithelium and its high transepithelial electrical resistance (TEER) (>500 to 600 Ohm·cm2) . Cells wTo evaluate the protective effect of sodium butyrate, monolayers of Caco-2 were exposed for 6 h to 100 ng/ml of TcdB and sodium butyrate at final concentrations of 1.65, 3.25, 7.5, 15, and 30 mM. Cells unexposed to TcdB and cells exposed to 30 mM sodium butyrate were used as the negative and positive control, respectively. The TEER was regularly measured at 0, 90, 120, 240, and 360 min during the experiment. The protective effect of the supernatants from inulin and xylan bioreactors was evaluated using monolayers of Caco-2 cells (passages 24 to 28) treated apically with 100 ng/ml of TcdB (10%) of the supernatant of the reactors that showed a good anti-inflammatory response. Cells unexposed to TcdB and cells exposed to 10% of mZMB were used as controls. The TEER was regularly measured, as mentioned below.2. The results of TEER (Ω·cm2) are expressed as percentages of the initial value: TEER = (TEERexp × 100)/TEERbasal.Transepithelial electrical resistance was measured according to reference All raw data, code, tables, calculations, and primer sequences are included in the supplemental material.


Shewanella putrefaciens is a special spoilage bacterium of seafood during cold storage, which is easy to form biofilm and bring serious hazard to the seafood quality. Life cycle of biofilm starts after bacterial adhesion, which is essential for the formation and development of biofilm. As a ubiquitous second messenger in bacteria, c-di-GMP regulates the conversion between bacterial planktonic state and biofilm state. In this study, the adhesion and biofilm formation of S. putrefaciens WS13 under 4°C were compared to those under 30°C. Atom force microscope and scanning electron microscope were used to study the bacterial adhesion. Biofilm was analyzed by Fourier transform infrared spectroscopy, Bradford assay and phenol-sulfuric acid method. High-performance liquid chromatographic-tandem mass spectrometric and quantitative real-time PCR were applied to study c-di-GMP level and genes encoding diguanylate cyclases in cells, respectively. Results showed that the swarming mobility of S. putrefaciens WS13 was weaker under 4°C, however, the adhesive force under 4°C was 4–5 times higher than that under 30°C. Biofilm biomass, extracellular polysaccharides and extracellular proteins were 2.5 times, 3 times, and 1.6 times more than those under 30°C, respectively, but biofilm composition formed under both temperatures were similar. c-di-GMP level in S. putrefaciens WS13 under 30°C was no more than half of that in the corresponding growth stage under 4°C. Quantitative real-time PCR analysis also showed that the expression of genes encoding diguanylate cyclases were significantly enhanced under 4°C than that under 30°C. S. putrefaciens WS13 adapted to the cold stress by enhancing the expression of genes encoding diguanylate cyclases to promote bacterial adhesion and biofilm formation. This study provides a theoretical foundation for the research on the cold adaptation mechanism of specific spoilage bacteria of seafood based on c-di-GMP, and also provides a new idea to control seafood quality from the perspective of microbial molecular biology. Shewanella putrefaciens is a specific Gram-negative bacterium in aquatic products such as Litopenaeus vannamei   and two- in 1987 , and its in 1987  and phos in 1987 , 23. Mos in 1987 , and tem in 1987  to regul in 1987 , 26 have in 1987 . HoweverS. putrefaciens WS13 under cold stress. Our work showed the characteristic differences of adhesive force and biofilm of S. putrefaciens WS13 under 30 and 4°C and indicated the importance of c-di-GMP for bacterial adhesion and biofilm formation. This provides a theoretical basis for the study of c-di-GMP regulating the adhesion and biofilm formation of S. putrefaciens WS13 under cold stress.Based on the above, this study intended to reveal the correlation between c-di-GMP and the adhesion and biofilm formation of S. putrefaciens WS13 strain, isolated from spoiled Litopenaeus vannamei, which was preserved by our group (NCBI No. CP028435.1)  in fresh LB medium under 30°C for 150 rpm, and then, the culture was diluted at 0.1% ratio with LB medium for further use.28435.1)  was applThe diluted culture was cultivated under oscillating conditions (150 rpm) under 4 and 30°C, respectively, and 200 μl of culture was taken out aseptically and added into 96-well polystyrene microtiter plates to measure bacterial growth curves with BioTek Synergy 2  at 600 nm.The swarming mobility of bacteria was measured as described  previousS. putrefaciens WS13 was measured by AFM  as described , respectively. After centrifugation , harvested cells were treated with 0.01 M sterile phosphate-buffered saline  three times and suspended in Milli-Q grade water . The suspension was applied to prepare cell probes  glutaraldehyde precooled under 4°C in advance and kept under 4°C).The diluted culture was cultivated in LB medium under 4 and 30°C to 8–9 log CFU/mL  by electrostatic attraction for 20 min. Bacteria-PS spheres prepared were tenderly cleaned with Milli-Q grade water to divide unattached cells. A biological microscope  at a magnification rate of 900X was used to ensure that cells were fixed on PS spheres. PS spheres with cells were dispersed on the carrier chip and bonded to the MLCT-F tipless cantilever of AFM with AB glue. The experiment which applied the system of Dimension Icon  was operated at room temperature. First, the polystyrene plastic (PS) substrate was probed with a bare probe to set a baseline for comparing the force measurements with the designed probe. The elastic constant of the probe cantilever was 0.1 N/m. Spring constants of the tips were in the range of 0.6 N/m and were measured for each probe. The loading force was set to 1 nN. The adhesive force was measured by the formula F = kAccording to bacterial growth curves, 1 mL of diluted culture was cultivated at 48-well polystyrene microtiter plates containing one piece of PS plastic sheet each under 30°C for 18 h and 4°C for 168 h, respectively. PS plastic sheets were taken out, washed with 0.01 M sterile phosphate-buffered saline  three times, and treated with precooled 2.5 % glutaraldehyde  under 4°C for 4 h. After dehydrated with gradient ethanol  for 10 min each and dried naturally, plastic sheets were treated with gold plating. Scanning electron microscope  at 3 kV was applied to obtain bacterial adhesive state (50 nm objective multiple) and morphology (5 nm objective multiple).Biofilm biomass was measured quantitatively as described  previous–1, and resolutions of4 cm–1.The components of biofilm were analyzed as described , 31 prevCyclic diguanylic acid was analyzed as described  previous600 value = 0.80), respectively, was centrifuged  to extract RNA. The RNA was obtained with the RNA extraction kit , and its concentration was evaluated with an ELISA reader . The prepared RNAs were reverse-transcribed into cDNA following M-MLV 4 First-Strand cDNA Synthesis Kit  which were then kept at – 20°C.The diluted culture, under 4 and 30°C at 8–9 log CFU/mL . The primers used in this experiment for quantitative real-time PCR are shown in Quantitative real-time PCR was carried out with the QuantStudio™ 1 Real-Time PCR System . The reaction system consisted of 10 μl of 2X SYBR Green qPCR Master Mix, 0.4 μl of 10 μM F Primer, 0.4 μl of 10 μM R Primer, 7.2 μl of dd H−ΔΔCT method in triplicate. Statistical analysis was performed by Origin Pro 2021, and error bars represent the SD from the mean of measurements.Quantitative real-time PCR was operated with the following conditions: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s; and 60°C for 1 min. Genes were selected , and thep-value <0.05 were regarded as statistically significant.The experiments were carried out at least three times independently. Analysis of microbiological results was converted to OD and log CFU/mL. Origin Pro 2021  was applied to analyze the content of biofilm, biofilm components, and other data obtained. SigmaStat 3.5 was applied to analyze bacterial adhesive force. Differences at S. putrefaciens WS13 are shown in The growth and swarming mobility of With the prolongation of incubation time, the sizes of colonies cultured under 4°C did not change significantly. However, the colonies cultured under 30°C appeared earlier, and the sizes of colonies increased with the extension of culture time. It showed that cold stress weakened bacterial swarming mobility  and madeS. putrefaciens WS13 under 4°C was stronger than that under 30°C.As shown in S. putrefaciens WS13 under 30 and 4°C are shown in The morphological changes in the mature biofilm of S. putrefaciens WS13 produced more biofilm under cold stress. The results of Fourier transform infrared spectroscopy (FT-IR) characteristics of the mature biofilm formed under 30°C at 18 h and 4°C at 168 h are shown in −1, 1100–1260 cm−1, and 3300–3400 cm−1, respectively. The peak at 1450 cm−1, 1690–1700 cm−1, and 3300–3400 cm−1 indicated the existence of extracellular proteins in biofilm, respectively. Other biomacromolecules such as lipids were verified in the biofilm, which is consistent with the previous studies  of seafood under cold storage and is related to aquatic product disease named shewanellosis  and an optimum growth temperature (30°C) were selected in this work to investigate the difference between those features of S. putrefaciens WS13.nellosis . S. putrd stress , 36. Furfluidity , which wfluidity , 25. HowS. putrefaciens WS13 under 30 and 4°C, were investigated in this work. S. putrefaciens WS13 grew slower under 4°C, but its final density was higher than that under 30°C. The swarming mobility of S. putrefaciens WS13 under 4°C was weaker than that under 30°C. Studies on Listeria monocytogenes and Ralstonia solanacearum strains showed that bacteria under cold stress achieved a greater proliferation rate and a lower growth rate, and bacterial mobility on growing colonies was attenuated  are regulated by many factors, such as c-di-GMP –53. The c-di-GMP . In addi. putida . The inc. putida –53. ThisS. putrefaciens WS13 under cold stress was also investigated. The results showed that the FT-IR characteristics, morphology, and structure of bacterial biofilm formed under 30 and 4°C were similar, which meant the composition of biofilm formed under both temperatures was almost the same. The intracellular c-di-GMP level affects biofilm formation  can be found in the article/supplementary material.RX: data curation, writing—original draft preparation, writing—review and editing, and visualization. JYan: conceptualization, methodology, validation, investigation, resources, data curation, writing—review and editing, and visualization. JM and JYe: visualization. JX: conceptualization, methodology, validation, investigation, resources, data curation, writing—review and editing, visualization, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.This research was funded by the National Natural Science Foundation of China (No. 31972142) and the Science and Technology Commission of Shanghai Municipality (20DZ2292200 and 19DZ1207503).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.