Orange pomace (OP) is a solid waste produced in bulk as a byproduct of the orange juice industry and accounts for approximately 50% of the quantity of the fruits processed into juice. In numerous literature references there is information about diverse uses of orange pomace for the production of high-added-value products including production of natural antioxidant and antimicrobial extracts rich in polyphenols and flavonoids which can substitute the hazardous chemical antioxidants/antimicrobials used in agro-food and cosmetics sectors. In this work and for the first time, according to our knowledge, the eco-friendly aqueous vacuum microwave assisted extraction of orange pomace was investigated and optimized at real industrial scale in order to produce aqueous antioxidant/antimicrobial extracts. A Response Surface Optimization methodology with a multipoint historical data experimental design was employed to obtain the optimal values of the process parameters in order to achieve the maximum rates of extraction of OP total polyphenols and/or total flavonoids for economically optimum production at industrial scale. The three factors used for the optimization were: (a) microwave power (b) water to raw pomace ratio and (c) extraction time. Moreover, the effectiveness and statistical soundness of the derived cubic polynomial predictive models were verified by ANOVA. Citrus sinensis and to the family Rutacae and it originates from China. It is designated as sweet orange in order to be discriminated from the bitter orange which is known as Citrus aurantium [Orange fruit belongs to the species urantium  and it iurantium , 33% of urantium .w/w, crude fibers: 14.17 ± 0.36% w/w, protein: 12.43 ± 0.20% w/w, ash: 7.8 ± 0.01% w/w. carbohydrates: 52.90 ± 0.43% w/w. In addition, concerning the phenolic content of orange pomace, it contains polyphenols belonging mainly to the class of flavonoids and in particular flavones, flavonols, flavonons, isoflavons, anthocynidines and flavanols. The main flavonoids of orange fruit are hesperidin, narirutin, eriocitrin, naringin, neohesperidin, neoeriocitrin and among them hesperidin and narirutin are dominant [In an investigation carried out by Sharma et al. , it was dominant . The che−1 of dry orange pomace. Min et al. [Acinetobacter and Listeria. Faber et al. [The extraction of high-added-value phenolics and flavonoids from orange pomace is one of the first steps of any scheme of biorefinery targeting a total discharge of this solid waste. Thus, a variety of methods, with some of them very innovative and cutting-edge, have been used for the effective extraction of them. Luengo et al.  successfn et al.  & Kim etn et al.  presenten et al. ,21 studir et al.  & Memduhr et al.  carried r et al. ,25,26.−1 h−1 for MAE, UAE, ASE and CSE, respectively); (iv) in the case of MAE, microwaves are selectively absorbed by the residual water present in C. sinensis peels; and (v) better customer acceptance of the byproducts (peels) made through this MAE ‘‘cleaner, greener’’ extraction technology. In addition, Nguyen et al. [Citrus maxima). Their research had shown that continuous microwave-assisted extraction was more effective than intermittent extraction. Alcohol content, extraction time, microwave power, material-to-solvent ratio, that significantly influence the extraction process, were the parameters to be investigated. Ethanol concentration of 60%, microwave power of 300 W, microwave-assisted time of 2 min, and material-to-solvent ratio of 1:30 were significant parameters obtained in the research. Hayat et al. [According to Nayak et al. , microwan et al.  studied t et al. , compareinvestigate and optimize, for the first time at industrial scale, and with both recovery and economic criteria, the microwave extraction of raw orange pomace and obtain the optimum conditions in terms of microwave power, water to raw orange pomace ratio, extraction time, and with the aim to get maximum yield or productivity of polyphenols or/and flavonoids. This information can then be used in order to produce, at industrial scale and in a financially favorable way, high-added-value bioactive aqueous orange pomace extracts and thus to exploit a mass-produced solid agro-food waste by the orange juice production industry. The optimized aqueous orange pomace extracts can be used either as natural antioxidants or/and as natural antimicrobials (extracts with maximum polyphenols and/or flavonoids content) targeting food, nutraceutical, cosmetic and agro-protection applications and leading to the improvement of the carbon footprint of the orange juice production industry.The target of the present work is to The results of the total polyphenol content of the orange extracts are presented in 2 value = 0.8417 is satisfactory and simultaneously there is a high degree of proximity between the calculated values of Adjusted R2 = 0.8133 and Predicted R2 = 0.7705 as the difference between them is, by far, less than the limit of 0.2 set by the method. Therefore, the derived RSM model can be used for effective prediction of the extracted amount of OP polyphenols within the design space as well as for optimization.Furthermore, the ANOVA statistics of the RSM model, derived to correlate the extracted OP total polyphenols with the experimental factors, are presented in Microwave power: 5999.997 W,Water to orange pomace ratio = 26.09Extraction Time: 120.00 minThe robustness of the derived model is further supported by the data presented in Whereas, the corresponding maximum amount of extracted OP total polyphenols, at the above-mentioned conditions, was found to be 13,559.802 mg per 2 Kg of extracted raw orange pomace.Type of polynomial Model: Reduced Cubic modelResponse: AMOUNT OF EXTRACTED OP TOTAL POLYPHENOLS (mg/2 kg raw OP)Transform: Inverse SqrtConstant: 0Fit StatisticsStd. Dev.0.0003R²0.8417Mean0.0101Adjusted R²0.8133C.V. %2.97Predicted R²0.7705Adeq Precision27.7094The results of the extracted amount of OP total flavonoids are summarized in 2 value (0.8552) is high, which reveals a satisfactory correlation of the extracted amount of OP total flavonoids with the three experimental factors. In addition, the value of the Adjusted R2 (0.8293) is very close to the Predicted R2 (0.8105) value, which means that there is satisfactory prediction by the developed model equation. The effectiveness of derived model is additionally supported by the data presented in Microwave power: 2000 WattWater to solid orange pomace ratio: 24.12Extraction time: 53.45 minMoreover, in And, the maximum amount of extracted OP total flavonoids, at the above mentioned optimum conditions, was found to be 1909.27 mg QE/2 kg of extracted raw OP.Type of polynomial model: reduced cubic modelResponse: AMOUNT OF EXTRACTED OP TOTAL FLAVONOIDS (mg QE per 2 kg raw OP)Transform: Inverse SqrtConstant: 0Fit StatisticsStd. Dev.6.27R-Squared0.8552Mean0.046Adj R-Squared0.8293C.V. %13.72Pred R-Squared0.8105PRESS3.45Adeq Precision23.712In addition, the simultaneous maximization of the amount of extracted OP total polyphenol and total flavonoids was carried out. The methodology to obtain the optimum amount of the extracted OP total polyphenols and flavonoids simultaneously, was based on the function of Design Expert software that can calculate the values of the extraction parameters which lead to a common convergence of the extracted amounts of OP total polyphenols and total flavonoids to values as close as possible to their independent optima.Microwave Power: 2000.00 WWater to solid orange pomace ratio: 24.95Extraction Time: 63.96 minBy this methodology, the following set of optimum extraction conditions that simultaneously maximize the extracted amounts of OP total polyphenols and OP total flavonoids was obtained:At the above-mentioned optimum extraction conditions for simultaneous optimization of the amount of OP total polyphenols and total flavonoids in the orange pomace extracts obtained by VMAE, the corresponding optimum values found to be 9977.48 mg GAE for total polyphenol, which represents the 73.58% of the independent polyphenols optimum, and 1847.42 mg of QE for total flavonoids, which represents the 96.76% of the independent flavonoids optimum respectively.The predicted optimum extraction conditions and the corresponding optimum values of responses (extracted amounts of OP polyphenols and flavonoids) independently or in combination of two are summarized in 2 = 0.9823 and also to a high degree of proximity among the adjusted R2 = 0.9788 and predicted R2 = 0.9737 (difference by far less than the proposed maximum acceptable limit of 0.2) which implies that the derived model can be successfully used for prediction within the selected design space as well as for effective industrial scale economic optimization.By using the derived Equation (7) in paragraph 4.6, the extraction rate of orange pomace total polyphenols was calculated for each one of the 80 experimental points and recorded in −1 min−1)Response: TOTAL OP POLYPHENOLS PRODUCTIVITY )Microwave power: 4484.777 WWater to solid orange pomace ratio: 19.619Extraction Time: 15.00 min−1 min−1.The optimum rate of OP total polyphenols extraction (maximum productivity) 154.074 mg GAE KgFurthermore, the optimum extraction conditions and the corresponding maximum value of the raw OP total polyphenols extraction rate (productivity) were found to be the following:2 = 0.8784 and also a high degree of proximity of the Adjusted R2 = 0.8627 and Predicted R2 = 0.8500, which implies that the derived model can be successfully used for prediction purposes within the selected design space as well as for effective optimization at industrial scale.By using the Equation (8), which was derived in paragraph 4.6., the rate of extraction of orange pomace total flavonoids was calculated for each one of the 80 experimental points and the calculated values are listed in −1 min−1)Response: OP TOTAL FLAVONOIDS PRODUCTIVITY :Microwave power: 2000.00 WWater to solid orange pomace ratio: 24.35Extraction time: 15.00 minFinally, the optimum extraction conditions and the corresponding maximum rate of extraction of raw OP total flavonoids were found to be the following:−1 min−1.Optimum (maximum) rate of OP total flavonoids extraction  equaled 26.08 mg QE KgFor the validation of the precision of the four developed predictive mathematical models, the absolute average % error between the measured and predicted values were calculated and they are presented in the following From the values of the % mean error of prediction for the four RSM models presented in the Lycium barbarum and the explanation that was given by them is the following: (a) the increase in microwave power results in an increase in the extracted amount of total polyphenols which may be explained by the easier penetration of the solvent into the plant structure as a result of increasing power that facilitates cell rupture and thus enhances the extraction; (b) higher phenolic yield can be obtained at the longer extraction time and higher solvent-to-sample ratio and the extraction yield increases with the increase in solvent-to-sample ratio, but, when the ratio exceeded a certain limit, the amount of extracted total polyphenols began to decrease. This decrease that is observed after a limit of water-to-solid ratio is reached can be attributed to the excessive increase of the total volume exposed to the microwaves which reduces the wave effect per Kg of total mass. Moreover, the significant terms of the derived predictive model for the extracted amount of OP total polyphenols are: A, C, AB, AC, B2, C2, A2B and AB2 which, according to the p-data listed in In the case of the extraction of OP total polyphenols, the factor A (Microwave power) has a significant and very clear increasing effect on the total amount of the extracted OP polyphenols, while the increase of factor B (Water to orange pomace ratio) has initially a positive effect on the extracted amount of total OP polyphenols up to a maximum value is reached and after that the extracted amount total OP polyphenols declines. Finally, as far as the extraction factor C (Extraction time) is concerned, this has a clear increasing effect on the amount of extracted total OP polyphenols. Similar trend concerning the effect of extraction parameters on the MAE extraction yield, reported by Özbek et al.  and Mend2, B2, C2, A2B, AB2 and B2C which, according to the ANOVA statistics summarized in In addition, in the case of extraction of OP total flavonoids factor A (microwave power) has a clear and substantial decreasing effect on the total amount of the extracted OP flavonoids, which can be attributed to an inherent sensitivity of OP flavonoids to high-intension MW treatment. Moreover, regarding factor B, initially, by increasing it, a substantial positive effect on the extracted amount of OP total flavonoids is observed up to a maximum value and afterwards the extracted amount of OP total flavonoids starts to decline by further increase of B factor. Finally, with regards to the extraction parameter C (extraction time) initially has a clear increasing effect on the amount of extracted total OP flavonoids until this reaches a maximum value. Then any further increase of extraction time causes a rapid reduction of the extracted amount of total OP flavonoids probably due to the sensitivity of the OP flavonoids to long exposure to the MW power and high temperatures. This finding is fully in line with the views expressed by  who have2, C2, A2B, A2C, AB2 and C3 which, according to the ANOVA statistics listed in In the case of the rate of the VMAE extraction (productivity) of OP total polyphenols, by increasing the factor A (Microwave power), the rate of extraction of OP total polyphenols increases until it reaches a maximum value and after that it is getting reduced. Similarly, by increasing the factor B , initially a positive effect on the rate of the extracted total OP polyphenols is observed up to a pick value and subsequently the rate of extraction of OP total polyphenols decreases. Conversely, as far as the extraction factor C (Extraction time) is concerned, as this increases it causes a remarkable very steep decrease of the rate of extraction of total OP polyphenols. This suggests that the maximized extraction rate of OP total polyphenols which provide the most economic operation  is obtained at a very low extraction time (just 15 min). Moreover, the significant terms of the OP total polyphenols rate of extraction predictive model are A, C, AB, AC, BC, B2, B2, C2, A2B and AB2, which, according to the data listed in In addition, in the case of the rate of extraction (productivity) of OP total flavonoids the factor A (Microwave power) has a clear and substantial decreasing effect on the extraction rate of the OP flavonoids which can probably be attributed to an inherent sensitivity of OP flavonoids to high intension MW treatment. Moreover, concerning factor B (water to OP ratio), initially, by increasing B, a substantial positive effect is introduced to the rate of extraction of the OP total flavonoids and, then, as a certain maximum value is reached, afterwards the rate of extraction of OP total flavonoids starts to decline as factor B further increases. Finally, regarding parameter C (extraction time), by increasing this, a very steep decrease on the rate of extraction of total OP flavonoids is observed. This means that the maximum productivity of OP total flavonoids is obtained after a short interval (just 15 min) and low MW power. Finally, the significant terms of the extraction rate predictive model for OP total flavonoids are A, C, AB, A−1 raw orange pomace. Furthermore, as the dry matter of raw OP was determined to be 18% w/w, the maximum extracted OP total polyphenols expressed on a dry basis is calculated to be 6780 × 100/18 = 37667 mg GAE Kg−1 of OP dry matter. Similarly the maximum amount of the extracted OP flavonoids expressed on a dry basis is 1909.27 mg QE/2 × 100/18 = 5304 mg QE Kg−1 of dry OP.According to the results of the present study the maximum amount of the polyphenols extracted from raw OP by vacuum-microwave-assisted extraction (VMAE) is 13,559.802 mg per 2 Kg of raw orange pomace or, equivalently, 6780 mg GAE Kg−1 of dry peel while the corresponding maximum of flavonoids falls in the range of 2000–30,000 mg QE Kg−1 of dry peel. Further, Dahmoune et al. [−1 obtained by using as extraction solvent water/ethyl alcohol solutions. In addition, Londono-Londono et al. [−1 of dry orange waste and, for total flavonoids, a value of 40,250 mg QE kg−1 but this was obtained by using lab-scale equipment and OP raw material in dry powder form.According to Putnik et al. , the maxe et al.  & Mhiri e et al.  reportedo et al.  cited th−1 of dry OP depending on the experimental conditions. In addition, Anagnostopoulou et al. [−1 DM and Casquete et al. [−1 DM. Finally, Escobedo-Avellaneda et al. [−1 of DM. From the above mentioned literature references, it is concluded that, in general, the yield of the extracted total polyphenols from the orange juice industry solid waste lies in the wide range of 36.3–66,360 mg GAE Kg−1, whereas the yield of total flavonoids was reported to be in the range of 2000–40,250 mg QE Kg−1 of dry solid waste. It is also worth noting that, according to Barbosa et al. [Furthermore, Faber et al.  & Memduhu et al. , reportee et al.  2840 mg a et al. , by usin−1 of dry OP compared to the highest value of 66,360 mg GAE Kg−1 reported in the literature) whereas the optimum value of the yield of OP total flavonoids of 5304 mg QE Kg−1 of dry OP obtained by VMAE extraction in the present work is within the range 2000–42,250 mg QE Kg−1 of dry OP reported in the literature. This can be attributed first to the quality of the raw OP used in the present work, which is substantially different to the dry and finely comminuted orange peel used in almost all cases by other researchers and also to the mechanical removal of the orange essential oil before the mashing of the fruits to obtain the juice by our supplier of the OP. This operational mode removes the major part of the natural antioxidants that originally are contained in the orange peel in the form of essential oil and therefore reduces the concentration of the phenolics in the orange pomace. In addition, the orange cultivar and the extractor dynamics can be considered as parameters that could cause this difference. Finally, in the present research, for the first time, according to our knowledge, optimization of the aqueous VMAE for raw orange pomace at industrial scale was carried out. For this reason, the results obtained in the context of the present work cannot be directly comparable to those of previous research works because all of them were conducted at lab scale. However, our results are particularly interesting and readily usable by the industry as no scale up is required for them.Comparing the literature values with the findings of the present work, it is concluded that our optimum value for OP total polyphenols yield obtained by VMAE extraction is slightly above the middle of the range reported in the literature  [In the literature, there are two types of mathematical models dedicated to microwave extraction: iffusion ,40,41,42gy (RSM) ,43,44,45gy (RSM) ,47.From the above-mentioned models, the theoretical ones are not very useful in the case we need to obtain the optimum conditions for industrial-scale optimization of the VMAE extraction of bioactive phytochemicals. This is because they involve only the dependence of the extracted amount of the targeted substance vs. time and not the combined effect of the three significant extraction parameters . On the contrary, the empirical statistical models connect the extraction yield to all extraction parameters and they can give the overall optimum of the response. Furthermore, the empirical models incorporate the effect of the disintegration of a part of the total polyphenols and flavonoids due to shear or thermal stress during the process which is not taken into account in all of the above-mentioned theoretical models.For the above reason, in our case, a novel empirical model was selected based on the RSM methodology but with significant differences compared to the typical Box and Behnken experimental design used by previous researchers. In particular, with the target to increase the accuracy of the optimization, a denser experimental plan designated as “historical data design” was adopted with a significantly larger number of experimental points compared to the well-known Box & Behnken design used by other researchers. On top of this, by using the Cox & Box plot provided by the statistical software (Design Expert), a proper transformation of the model response was selected in order to improve the model accuracy and, in addition, a cubic instead of quadratic polynomial model was used to fit the experimental data. The increase of the degree of the polynomial model from 2 to 3 is suggested by the Design Expert software in cases where there is a strong nonlinearity in the dependence of the response  on the extraction parameters. This high nonlinearity can be easily concluded if we observe the form of theoretical model equations given in the literature above.However, the novel approach of predictive modeling and optimization given in this paper can be tested further with more applications in order to be validated. Then it can be used to increase the effectiveness of the derived empirical statistical models dedicated to microwave extraction, by using a larger number of experimental points which can eliminate the negative effect of a single erroneous measurement and by proper transformation of the response. Finally, despite the fact the experimental effort is heavier by this approach because of the higher number of experiments and the industrial size, the much better precision and the avoidance of scale-up can pay back for the extra effort.Concerning the novelty of the present research, this is based on the following points: (a) predictive modeling and optimization of OP extraction by aqueous “green” vacuum-microwave-assisted extraction for the first time at industrial scale and involving both maximum recovery as well as economic criteria; (b) use of raw orange pomace against dried used in previous research works and therefore avoidance of the costly and quality-deteriorating pomace drying and milling pretreatments; (c) use of statistical criteria for the correct selection of the optimum degree of the RSM polynomials and the optimum transformation of the dependent variables; and (d) use a comprehensive and multipoint “historical data” experimental design involving 80 experimental points in total in order to improve the accuracy of the derived predictive models and the precision of the determination of the optimum values. The orange pomace was kindly supplied by the Greek orange juice producer, ALBERTA S.A. which is established in Argos Peloponnese-Greece. The orange variety from which the obtained pomace was coming from was the well-known orange fruit variety “Navel”. The obtained pomace was passed through a commercial meat mincer  with a 3 mm hole diameter screen in order to become comminuted and then it was kept in a properly sealed plastic vacuum bag (2 Kg per bag) at −25 °C until used for extraction. Drying was not applied to the orange pomace in order to avoid oxidative degradation of the bioactive compounds.The extraction of the orange pomace samples was conducted by using the industrial-scale vacuum-microwave extractor model MAC-75 –Italy) which is established in the premises of PELLAS NATURE Co  and is illustrated in The extraction trials of the orange pomace samples were conducted following the procedure described below. The frozen orange samples were first thawed at ambient temperature and 2 Kg of each sample were then collected and used as the extraction sample. The 2 Kg orange pomace sample was first put in a plastic basket which was then adjusted to the extraction cavity of MAC-75 vacuum-microwave extractor. Consequently, the machine door was closed and filled with the appropriate quantity of distilled water. The quantity of the water used in each trial was according to the water/solid ratio suggested by the experimental plan shown in . In addi2 value was 0.9982. Calculation of the total polyphenols of extracts of OP was carried out following the same procedure and using the following equation of the standard curve:For the determination of the total polyphenols as GAE  of the obtained orange extracts, a slightly modified version of the method of Singleton et al.  and WateEach measurement concerning total polyphenols was carried out in triplicate and the result was the average of the three obtained values.3, as described by [3 reacts with the hydroxyls of the flavonoids and produces a colored complex which has maximum absorbance at 420 nm. The total flavonoids content was expressed as quercetin equivalents (QE) per L of extract. The determination method for the total flavonoids was carried out as follows: 1.0 mL of the orange pomace extract or standard solution  was added in a glass test tube to which 3 mL methanol, 200 μL of aqueous solution of 10% w/v AlCl3, 200 μL 1Μ potassium acetate solution and 5.6 mL distilled water were added. The tube was then agitated by vortex and incubated for 30 min at ambient temperature for the completion of the chemical reaction. The absorbance of each sample was measured at 420 nm against a blind solution which contained all the reagents except for the orange pomace extract which was replaced by distilled water. The total flavonoids content expressed as mg of quercetin equivalents (QE)/L of the obtained orange pomace extracts was determined by using the colorimetric method of AlClribed by . The met2 value of the obtained linear correlation was 0.9834.For the construction of the calibration curve, a quercetin stock solution of 1000 ppm was prepared as well as a series of standard solutions of 50, 100, 200, 500 and 1000 ppm by serial dilutions of the stock. The absorbance of standard solutions was measured and plotted against their concentration and the linear equation obtained by Excel was used for the determination of the concentration of the total flavonoids of the OP extracts. The RCalculation of the total flavonoids of OP extracts was carried out following the same procedure and using the following equation of the standard curve:Each measurement concerning total flavonoids was carried out in triplicate and the result was the average of the three obtained values.All the chemicals used for the above mentioned antioxidant tests were selected from the standard catalog of the Sigma Aldrich company and they were supplied by the Greek representative Life Sciences Chemilab, 33 Amarantou str, 56431 Thessaloniki, Greece.Modeling and optimization of total orange pomace polyphenol extraction;Modeling and optimization of total orange pomace flavonoids extraction;Simultaneous optimization of total orange pomace polyphenols, total flavonoids;Modeling and optimization (maximization) of the rate of extraction of orange pomace polyphenols to achieve the maximum productivity (economic optimum);Modeling and optimization (maximization) of the rate of extraction of orange pomace flavonoids to achieve the maximum productivity (economic optimum).The methodology used for modeling and optimization had the following aims:A multipoint historical data experimental design employing 80 experimental points evenly spread in the design space was used along with response surface methodology (RSM) to derive the relevant polynomial mathematical models for the prediction of total polyphenols and total flavonoids of the orange pomace extracts and obtain the corresponding optimum values. Three factors were used as optimization factors: (a) the microwave power in the range of 2000 to 6000 W; (b) the ratio of extraction water to orange pomace in the range from 10 to 30; and (c) the extraction time in the range of 15 min to 120 min. There were five optimization responses: (a) total polyphenol content; (b) total flavonoids content; (c) total polyphenols and total flavonoids content simultaneously; (d) the rate of the extraction of the total orange pomace polyphenols; and (e) the rate of extraction of the total orange pomace flavonoids. The Design Expert 12.0.0 statistical software was used to preform predictive modeling and optimization and derive the mathematical models. The selection of the appropriate order for the polynomial models to fit the experimental data was based to the statistical evaluation tools of the Design Expert software and concerning the optimum response transformation in order to obtain satisfactory fitting this was selected in all cases according to the suggestion of the Cox & Box Diagram. The reliability of the obtained models was validated by statistical analysis (ANOVA) and in all cases the statistical significance of the derived models as well as the desirable nonsignificance of lack of fit were confirmed. The validity of the derived models was also tested by conduction of measurements at experimental points within the design space different than the ones used to develop the models and, consequently, by comparison of the obtained predicted and measured values and determination of the magnitude of the mean prediction error.The most interesting target of optimization for the industry, on economic grounds, is the maximization of the rate of extraction of polyphenols or flavonoids from raw orange pomace. -The amount of the extracted total orange pomace polyphenols as well as the corresponding extraction time (t) are given in -The mass of the extracted raw OP was in all cases equal to 2 Kg;-delay) between successive extraction cycles was 15 min.Furthermore, the delay time  between successive extraction cycles was 15 min.As above, the delay time  Microwave power = 5999.997 W, Water/OP ratio = 26.09 and Extraction time = 120.00 min for total polyphenols, and (b) Microwave power = 2000.00 W, Water/OP ratio = 24.12 and Extraction time = 53.45 min for total flavonoids. It is also worth noting that a set of extraction parameters corresponding to Microwave power = 2000.00 W, Water/OP ratio = 24.95 and Extraction time = 63.96 min could be an effective compromise in order to simultaneously optimize the total polyphenols and the total flavonoids of the orange pomace extract. Yet another important observation is that, in order to achieve the optimum (maximum) yield of polyphenols, operation at high microwave power and long time intervals, is required, while the optimum flavonoids yield is obtained at low microwave power and moderate time probably due to the sensitivity of their chemical structure.Moreover, as industrial reality demands high productivity in order to achieve cost effective production, in the course of the present work the productivities of the VMAE extraction of raw OP total polyphenols and raw OP total flavonoids were optimized and the optimum conditions for total OP polyphenols and total OP flavonoids respectively were found to be (a) Microwave power = 4484.777 W, Water/OP ratio = 19.619 and Extraction time = 15 min for total polyphenols, and (b) Microwave power = 2000 W, Water/OP ratio = 24.35 and Extraction time = 15 min for total flavonoids. The optimization of the productivities can be used as explained for financial reasons while the maximization of the amounts of the extracted OP total polyphenols and flavonoids can alternatively be useful in case the emphasis is on the minimum content of phenolics in the postextraction waste. Something like that would be very important in the case that this waste is to undergo further processing (biorefinery principle) by fermentation in order to avoid undesirable retard of the used starter cultures by the remaining phenolics postextraction.


The proposed bottom-up approach, reasonably expensive in terms of computational resources, has been validated by finite-temperature molecular dynamics simulations of clusters composed of up to fifty molecules.The C, N and O 1s XPS spectra of uracil clusters in the gas phase have been measured. A new bottom-up approach, which relies on computational simulations starting from the crystallographic structure of uracil, has been adopted to interpret the measured spectra. This approach sheds light on the different molecular interactions (H-bond, π However, the study of the weak interactions in gas-phase clusters of increasing size can give information on structures and mechanisms at work in both the liquid and condensed phases. For instance, gas-phase nucleobase pairs may also follow the Watson–Crick pairing mechanisms2. X-ray photoemission spectroscopy (XPS) provides detailed information on the environment of an emitting atom in a sample3. In a molecular cluster, the weak, non-covalent interactions modulate the position of nominally equivalent XPS peaks. The measurement and interpretation of such fine variations have provided a better understanding of the chemical equilibrium in ionized aggregates, as well as of their stability and reactivity5. In the present work the C, N and O 1s XPS spectra of uracil clusters have been measured with synchrotron radiation. The spectra have been interpreted using a simplified bottom-up procedure to describe the interaction patterns within the cluster and a systematic calculation of the ionization energy of all atoms based on ab initio simulations. Different studies on core spectroscopy and fragmentation processes of the isolated uracil molecule have been previously reported7, and their results provide the reference for the present work.H-bonds and van der Waals interactions are ubiquitous in nature and influence the structure, stability, dynamics, and function of molecules and materials throughout chemistry, biology, physics, and material science. Molecular clusters are weakly bonded systems◦C, are reported in Fig. 6. The uracil ring is formed by two -aza- nitrogen atoms in positions 1 and 3 and four carbon atoms. The C2 and C4 atoms form carbonyl groups with O8 and O7 atoms, respectively. In the isolated molecule  have been cut out from the uracil crystal structure9 in order to sample all kinds of local connectivity present in the periodic structure, and fully optimized in gas phase, with the BE of each atom calculated, as reported in Table “dimer1” in Table “dimer2”) and a stacked dimer . The formation of large clusters is driven by the anisotropic distribution of H-bond donor and acceptor sites, which can be also modulated by weaker dispersion forces between the π-conjugate charge distributions. Calculated average values of the C, N and O1s BEs are shown in Fig. To understand how the different interactions in the cluster affect the observed shift a simplified bottom-up theoretical approach has been developed. This approach is motivated by the un-sustainable computational cost of the calculation of XPS lines in large (>10 molecules) clusters, performed on several configurations sampled along equilibrated molecular dynamics trajectories. −⋯H–O+–C, we have also checked if it did not lead to an increase of charge on N3 already in the neutral dimer. The results of such investigation, described in detail in the Supplementary Information The two different dimer configurations having the same formation energy per molecule, have been simulated first. In both dimers and for all atoms, the BEs shift towards lower values, with a maximum shift of about 1.1 eV for N3 (“dimer1”). This immediately remarks a characteristic and expected property of NH groups H-bonded to electron-rich C=O groups, where the core-hole in the N atom is very efficiently screened by the carbonyl group. This is not the case for N1, which experiences instead a slight increase with respect to the monomer. As for "dimer3" the absence of H-bonds leads to BE shifts ≤ 50 meV  with respect to the stacked configuration (0.37 eV). Such a stable planar motif, likely ubiquitous in realistic aggregates, will be discussed below for the interpretation of the intermolecular screening. Finally, the additional shift in the dodecamer with respect to the hexamer varies between 400 meV (C6) and 70 meV (O7), with an average of 220 meV. All calculated C1s BEs are in excellent agreement with the experiments. The shifts in this case depend on the size of the cluster, but not on the connectivity between molecules, because C 1s atoms are not involved in the formation of H-bonds. We note that our “crystallographic structure” model likely tends to overestimate the energy separation between N3  and N1  values and to underestimate the one between O7 and O8. On the basis of the overall satisfactory agreement between calculated and measured BEs, and considering that all the intermolecular and side interactions among the three stacks of uracil molecules . The two carbonyl groups of molecule A (red arrows labeled 2) are directly involved in the accumulation of the charge density to screen more effectively the core hole. The overall result is a red shift of about 1 eV with respect to the isolated molecule. The similar, but slightly more complex evaluation of the intermolecular screening in the case of molecules B, C and D and the extension to the dodecamer are reported in Supplementary Information As an example the ionization of the C2 atom in each one of the four molecules (A to D) of the tetramer in planar configuration has been chosen, due to the abundance of the different intermolecular configurations and a relatively simple graphical representation of this cluster. Two kinds of map have been calculated: in the first case  used for the crystallographic structure. The comparison of the connectivity in the crystallographic and randomly oriented clusters ⋯O) intermolecular distances, whose first maximum around 2.8 Å Fig.  describe method BLYP used In summary a theoretical modeling of uracil clusters with a bottom-up approach based on the crystallographic structure has been proposed. Molecular dynamics simulations at finite temperature have confirmed that the model used is substantially able to reproduce connections and structures found in realistic gas phase clusters. Hence this approach can be proposed as a computationally sustainable method to study the chemical physical properties of weakly bonded clusters, provided their crystal structure is known. It can be applied to a large variety of systems from the bases of nucleic acids up to large proteins and therefore find a broad use.14.The modeling of the XPS spectra has allowed to disentangle the contribution of the different intermolecular interactions for increasing cluster size and shown the relevance of the H-bonds in the different positions of the cluster to determine the screening of the original core hole. The ability to display the contribution of intra- and inter-molecular variation of the charge density following inner shell ionization provides valuable information on the stability of hydrogen-bonded biomolecules, like for example DNA base pairs in interaction with a damaging radiation. It is indeed known that charge transfer via hydrogen bond, like for example the transport of the protons, can lead to induced and spontaneous mutations16. The details of the experimental procedure are provided in Supplementary Information The XPS measurements have been performed at the PLEIADES beamline of Soleil synchrotron. A gas aggregation source was used to produce neutral clusters of uracil molecules with a log-normal size distribution centered about 30–50 molecules, depending on the source parameters17 in a plane-wave/pseudopotential framework20. In addition, molecular dynamics simulations to determine the structural properties of clusters at finite temperature have been performed using both ab initio10 and tight-binding11 methods. As for the metadynamics (MTD), the computational protocol described by Grimme12 in his introductory article assessing the functionalities of the xTB program, based on the GFN2-xTB Hamiltonian11, for the exploration of potential energy surfaces has been followed. A complete account of all the employed computational protocols is provided in Supplementary Information Ab initio simulations of the properties of uracil clusters, including the calculations of core-ionization energies, have been performed by using the Quantum ESPRESSO suite of programsSupplementary Information


The composited cementitious materials usually have superior performance; for example, using limestone powder (LP) and fly ash (FA) as the admixtures of cement in concrete/mortar is a popular way of improving the properties of concrete/mortar structures. In this work, we performed experimental tests to study the hydration process and pore distribution in mortar containing different ratios of LP and FA. Based on the results of mercury intrusion porosimetry (MIP), a numerical mortar model with random pore is built. The model can reflect the synergistic hydration interaction and filling effect caused by the admixtures of LP and FA. After analyzing the hydration process, the coupled chemical–thermal–mechanical method was used to simulate the characteristics of mortar containing LP and FA. The coupling model can simulate the “hump-type” hydration acceleration stage of the mortar at early age, which is specifically caused by the LP, proved in the experimental test. Additionally, the special, “hump-type” stage is important to enhance the early strength of the mortar. At different levels of admixture content, the random pore model and coupled method can predict the evolution process of the mechanical properties well, at early age and for long-term strength. Both experimental and numerical results suggest that the mortar containing admixtures of the proper ratio of LP to FA have good mechanical properties, which can be applied to engineering structures. Portland cement has been widely used in hydraulic engineering, transportation, industrial, and civil construction for many years . To imprThe concrete containing limestone powder (LP) and fly ash (FA) has been widely tested and analyzed in previous studies. The effects on strength, stability, and durability were gradually discovered. Kirk Vance et al.  and V. BThe strength evolution of concrete is closely related to the hydration of cement-based materials. For an experimental test, the mechanical properties of concrete can be easily obtained through a destructive test, but it is hard to predict the strength via an undamaged test. Therefore, numerical simulation is ideal for predicting strength evolution, especially when considering the hydration process of the cement paste ,15. To sConcrete is regarded as a typical porous material, whose strength depends much on the hole due to the weakness of the pores. For the concrete containing LP and FA, LP has a filling effect that changes the porosity, which also directly affects the mechanical property. To simulate the evolution of the mechanical property accurately, the pore structure should not be neglected in meso-modeling . Pores aThis paper aims to investigate concrete containing high amounts of LP and FA, including their hydration characteristics, macro-mechanical properties, micro-hydration products, and pore size distribution. In addition, a multi-field coupling method that can reflect the hydration characteristics of composite cementitious materials is developed, which can consider the effect of different ratios of LP and FA in the mesoscale mortar. The model can also simulate the evolution of strength and fracture characteristics under uniaxial compression at different hydration ages of the mortar.2 produced by decomposition, which proves that the limestone powder contains a high purity of 95% CaCO3 content.The cement-based materials in the mortar were ordinary Portland cement (OPC), fly ash (FA), and limestone powder (LP). The strength grade of OPC is 42.5 MPa, the fly ash conformed to the stair FA property of the Chinese National Standard DL/T5055-2007, and the limestone powder was from carboniferous limestone, which was ground for 50 min by a vertical grinder. The chemical compositions of these three components determined by X-ray fluorescence (XRF) are listed in To study the characteristics of mortar containing a high content of admixtures, the mortar consisted of composite cement and was designed with different ratios of LP and FA. The total admixture content was kept at 50% with the water–binder ratio setting at the same value of 0.5. The mortar mixture proportions are shown in To obtain the hydration heat characteristics of composite cement-based materials containing different ratios of LP and FA, the heat flow of the mortar samples was monitored using a TAM Air isothermal calorimeter at 25 °C during the initial 72 h of the hydration process. The TAM Air had eight parallel twin-chamber measuring channels maintained at a constant temperature: one chamber containing the sample, and the other containing the reference, always water. The temperature fluctuation was less than ±0.02 °C, and the precision could reach ±20 mµW. The water-to-binder ratios (W/B) were 0.5 and 5 g for each sample used. First, 5 g of powder and 2.5 g of water were weighed for each specimen as the testing reference, and 2.90 g of water were used as the control reference. The testing reference was mixed, and the testing and control references were placed into certain tunnels at the same time. Then, marking the data till the equipment became stable.The compressive strength and flexural strength of the mortar was tested following a method in the Chinese National Standard GB/T 17671-1999 (Method of Testing Cement-Determination of Strength) after curing the specimens at a temperature of 20 ± 1 °C and a relative humidity of ≥90%, until the stipulated age of 7, 28, 90, 180, and 365 days. The strength was tested by a YAW-300C, a battery solution type flexural and compressive testing machine. The flexural strength was calculated from the average value of the three prism specimens of 40 × 40 × 160 mm. The compressive strength was calculated from the average value of six cubic specimens testing results.After the mechanical test of the mortar was performed at each stipulated curing age, the inner crushed cement slurry powder of the mortar was collected and soaked in anhydrous ethanol to terminate the hydration, and the microscopic characteristics test was performed.Mercury intrusion porosimetry (MIP) was used to test the pore distribution of the hydrated composite cement matrix with three different ratios (LF-0/LF-30/LF-50). Mung-bean-like crushed powder was prepared for this experiment. The prepared samples were immersed in anhydrous ethanol and dried in a 60 °C desiccators for 24 h. After that, each sample (about 1.5 g) was put into the glass tube of a dilatometer for a low pressure test, and the glass measuring tube filled with mercury was then placed into a high-pressure measuring tank for a high pressure test. The mercury injection apparatus was the Auto Pore IV (9500). The pressure ranged from 0.20 to 60000.00 psi (about 1.38 kPa to 413.76 MPa), and calculation of the measuring pore size range was from about 30 Å to 0.9 mm.The rest of the sample inner pieces were ground in the agate mortar and dried at 60 °C for 2 h to reduce carbonization for X-ray diffraction (XRD). Here, the XRD test was used to identify the hydration products of the cement paste during the hydration process, in order to find out the effect of the limestone powder on the microstructure in the composite cementitious materials.The compressive and flexural strength of the mortar is shown in In order to study the role of limestone powder in the hydration process of cement slurry, the heat of hydration and the hydration rate of the blend paste were analyzed by the isothermal calorimeter. The tested samples include LF-0, LF-30, and LF-50. 2. In 3 was mainly caused by carbonation. A synergistic interaction of LP and FA was observed when studying the additional reaction of aluminates. The interaction product was calcium carboaluminate hydrate [To understand the synergistic hydration interaction of LP and FA, we compared hydration products with pure cement and those with composite cement containing LP and FA. The hydrated cement paste powder of the OPC and LF-30 were tested by XRD at hydration ages of 28 and 180 days. The XRD results are shown in Because of its convenience and repeatability, MIP test is widely used in comparing the pore structure characteristics of different proportions of cement-based materials ,43. Althore size , whereasore size . Pore clIn the engineering structure, although the mechanical properties of concrete/mortar can be obtained through physical experiments, it can take as many as several months or even a year. It is essential to use an appropriate method to predict the properties of concrete/mortar through a numerical test. Because of the variation in hydration properties of complex cement paste, the process of the strength evolution of the concrete/mortar cannot be neglected. Here, an effective method called the chemical–thermal–mechanical method and the random pore model is used to model the mechanical evolution process of mortar, according to the information of aggregate grade, cement hydration, and porosity.In concrete/mortar, the exothermic reaction of the cement paste is due to the water combination, which can release heat known as the heat of hydration. The degree of hydration is defined as the heat released during hydration to the ultimate released heat when the hydration is complete. It can be formulated as follows ,49:(1)ξ is the superposition of Equations (3) and (4), in which Equation (3) can demonstrate the hydration of the OPC, and Equation (4) can demonstrate the hydration of the FA and LP:The transient heat transfer process of the mortar can be described as follows:In the process of iteration, the rate of hydration degree was calculated based on the Equations (2) and (6), according to the hydration degree and temperature at previous moment. The hydration degree The damage and failure process of mortar can be simulated by a damage model, which can be described by the damage factor Here, Poisson’s ratio is assumed to be a constant, and the relationship between the elastic modulus and the degree of hydration is as follows :(11)E(ξ)nt ratio , and it research . The parSimilar to Young’s modulus, the compressive strength can also be defined as a function of the degree of hydration :(13)fc(ξThe damage factor and (15)  and the The pore system in cement-based materials consists of four types of pores, namely macro-pores due to inadequate compaction, macro-pores due to deliberately entrained air, capillary pores, and gel pores. Pores can also be divided into four grades: very harmful pores  , which hThe heat capacity of the matrix can be estimated using the law of mixtures when giving the volume fractions and heat capacity values of the composite cement. Equation (25) shows the law:’s study , these vThe thermal conductivity of the mortar can be calculated from the well-known Hashin–Shtrikman (H-S) bounds  because e phases  for λ1 ≥The effective thermal conductivity of the composite By using the approach above, the thermal conductivity of the mortar components are derived. Nine specimens with a size of 40 × 40 mm are constructed. The numerical models of LF-0, LF-30, and LF-50 are made of a mortar matrix, a fine aggregate, ITZ, and pores. For each kind of sample, three specimens that are different in aggregate and pore distribution are generated. One of the three specimens (Sample 1) is shown in In this section, the cement pastes of samples LF-0, LF-30, and LF-50 are established to simulate the TAM test. The parameters of the cement paste in the hydration Equation (6) are calibrated, as shown in To obtain the relationship between the elastic modulus and compressive strength with the hydration degree in the numerical model, the elastic modulus of different ages is calculated by the method of the Chinese National Standard GB 50010-2010). Of course, other method mentioned in CEMHYD3D, HYMOSTRUC, etc., can also be used. The calculated elastic modulus is then matched with the degree hydration calculated by the chemical–thermal coupling method, and so is the compressive strength. The relationship above is developed by Equations (11) and (13). As described in In order to predict the strength of the mortar using the method of coupled chemical–thermal–mechanical method, it is advisable to use the same ternary Portland cement, so that the parameters in The damage factors can reflect the crack state. For the porous mortar specimen, the cracks first appear in the porous zone of ITZ, then develop along the porous zone of the matrix, and then gradually form complete cracks, which can be seen in (1)In the OPC-FA system, limestone powder provides the nucleation sites that can accelerate the hydration effect at an early age, and 10%~20% of limestone powder is appropriate while excessive replacement may have a negative influence. Combined with fly ash, limestone powder has a filling effect, also there exitsthe aluminate phase reaction, which can both reduce harmful pores in the mortar and optimize the pore structure.(2)In terms of cement-based materials, the coupled chemical–thermal–mechanical method can simulate the “hump-type” hydration stage of the composite mortar well, which is specifically caused by limestone powder. For the microstructure, the random pore model considers the pore differences between the ITZ and the mortar matrix, which can simulate the effect of harmful pores in the mortar.(3)Combined with the coupled chemical–thermal–mechanical method, the random pore model can simulate and predict the hydration characteristics and mechanical properties of the mortar at different hydration ages, according to the information of aggregate grade, hydration rate, and porosity. The prediction property is in good agreement with the experimental results. The method can also be applied in other cement-based materials, while the fitting parameters should be recalibrated.This paper investigated the composite cement-based material with high amounts of fly ash and limestone powder by experimental tests and numerical simulation. The following conclusions have been drawn:


Under the terms of the Montreal Protocol, its production for dispersive uses was banned from 2010. In this work we show that, despite the controls on production being introduced, CCl4 emissions from the eastern part of China did not decline between 2009 and 2016. This finding is in contrast to a recent bottom-up estimate, which predicted a significant decrease in emissions after the introduction of production controls. We find eastern Asian emissions of CCl4 to be 16 (9–24) Gg/year on average between 2009 and 2016, with the primary source regions being in eastern China. The spatial distribution of emissions that we derive suggests that the source distribution of CCl4 in China changed during the 8-year study period, indicating a new source or sources of emissions from China’s Shandong province after 2012.Carbon tetrachloride (CCl Carbon tetrachloride is one of several man-made gases that contribute to the depletion of the ozone layer high in the atmosphere. Because of this, restrictions were introduced on the use of this ozone-depleting substance, with the expectation that production should by now be close to 0. However, the slower than expected rate of decline of carbon tetrachloride in the atmosphere shows this is not the case, and a large portion of global emissions are unaccounted for. In this study we use atmospheric measurements of carbon tetrachloride from a site in East Asia to identify the magnitude and location of emissions from this region between 2009 and 2016. We find that there are significant ongoing emissions from eastern China and that these account for a large part of the missing emissions from global estimates. The presence of continued sources of this important ozone-depleting substance indicates that more could be done to speed up the recovery of the ozone layer. Emissions from the United States  and HCFC-22 (CHClF2) to infer emissions of CCl4 . However, the emissions estimates from the ISC methods may be prone to error in this assumption, and the two most recent studies estimate very different emission magnitudes of under 5 Gg/year  or 13 (7–19) Gg/year for the NAME and FLEXPART estimates, respectively, over this 8-year period. Our estimates for China are significantly higher than the recently published bottom-up estimates of 4 Gg/year  flight campaign in May–June 2016. The KORUS-AQ data show some of the largest and most frequent mole fraction enhancements over the Yellow Sea between China and the Korean Peninsula  in this region, in addition to another 10 measurements above 100 ppt that were at least 25% greater than baseline  and 0.3 (0.1–0.5) Gg/year from NAME and FLEXPART, respectively, over the 8-year inversion period. This average is larger than the prior estimate of emissions of 0.2 Gg/year, although not statistically different. These relatively small emissions were concentrated in the southeast of the country . The KORbaseline . Back trbaseline . While tWith the exception of 2011, we find emissions from Japan to be relatively small, with an 8-year mean of 0.6 (0.2–1.3) and 0.3 (0.0–1.0) Gg/year from NAME and FLEXPART, respectively, consistent with the bottom-up industrial estimate of emissions used as the prior . In 20114.Based on the output of both NAME and FLEXPART inversions, our results show that eastern Asian emissions averaged 16 (9–24) Gg/year over the period 2009–2016, 24–58% of our estimated mean global emissions. Coupled to the regional estimates for the United States, Western Europe, and Australia from previous studies , this co4 emissions from China that are larger than those estimated from bottom-up industrial estimates , and in the assumption that emissions of CCl4 and HCFC-22 are colocated. Approximately 50 Gg/year HCFC-22 emissions have been estimated to be from the room air-conditioning sector in China in 2014 (4 are thought to be primarily from industrial sources (The results of this work contrast with two separate studies using atmospheric data based on ISC to HCFC-22 . The ISC in 2014 , account sources , and as  sources .4 is used in the production of CFC-11, and the timing of the change in source distribution after 2012 is consistent with an increased CFC-11 source around this time found by 4 from China since the 2010 ban on production for dispersive uses came into force. Renewed efforts to control and remove emission sources of CCl4 around the world could help to enhance the decline of total tropospheric chlorine and speed up the subsequent recovery of ozone in the stratosphere.The discovery of a new source of emissions in Shandong province may potentially have wider implications linked to the recent discovery of a new source of CFC-11 into the atmosphere , given tSupplementary Material


The properties and the behaviour of plaster mortars designed with Polyurethane Foam Waste (PFW) are studied in this investigation. A characterization of the mixtures is completed, in accordance with the technical specifications of European Norms. The incorporation of polyurethane waste foam can yield porous and lighter mortars, with better resistance to water-vapour permeability, although with weaker mechanical strength and higher levels of absorbency. Nevertheless, suitable mechanical strengths were achieved, resulting in a new material that is compliant with the requirements of the construction industry. The use of PFW in the the manufacture of gypsum mortars for construction reduces the consumption of natural resources and, at the same time, recovers an industrial waste that is otherwise difficult to recycle. The degree of development of our society has brought with it important levels of wellbeing for the population, although social inequalities unbefitting a civilized world still persist. Development has also provoked considerable negative effects, both for the environment and for the quality of life of many people. One of the most significant is industrial waste, with a particular impact on the physical deterioration of the environment and the global warming of our planet ,2,3,4.Concerns are growing over the efficient management of the waste generated in industrial processes ,6,7,8,9.Polyurethane is a basic material that is used in numerous industrial applications, such as the manufacture of foam for the automobile industry, the manufacture of packaging, the furniture industry, and as insulative materials. These activities generate significant volumes of polyurethane waste with other materials that, in most cases, are only managed through dumping in landfill sites. This sort of waste may be characterized by its low biodegradability and because its molecular structures persist over time, and are difficult to transform into simpler and more stable shapes ,12,13. IPUReSmart project is investigating the use of both rigid and flexible PFW for the design and manufacture of new polymetric materials [Life Repolyuse project is investigating the use of rigid polyurethane foam in the design of new construction materials for the automobile industry and refrigeration industry in [One of the direct uses of Polyurethane Foam Waste (PFW) is its recovery for the production of energy through its incineration in industrial furnaces. It is also used to obtain compound foams from panel-cutting waste, which, once shredded, are added to chemical binders such as diphenylmethane diisocyanate (MDI) or polymethylene polyphenyl isocyanate (PAPI) . Neverthaterials . In the ustry in .REPUR project is investigating chemical and mechanical recycling processes for their recovery in the manufacture of detergents, adhesives and new polymeric foams [Foam2Foam project is developing technologies for the chemical recycling of generic PFW, to produce green polyols as a raw material in the manufacturing of new polyurethane foams [RECALZA project has developed investigations to close the life cycle of the polyurethane foams used in the manufacture of footware through chemical recycling by glycolysis [In Spain, there are various Research Projects with investigations into development to recover PFW. The ic foams . The Foane foams . Finallyycolysis .Numerous investigations have likewise been carried out for the design of new construction materials based on polyurethane waste. Its properties mean that polyurethane may be incorporated in new materials with good thermal and acoustic performance. Some examples are the manufacture of concrete, and cement mortars with rigid polyurethane foams from the refrigeration chamber-panel-making industry, and for the automobile industry ,27,28,29Life Repolyuse project proposed, to good effect, the manufacture of gypsum panels with polyurethane waste, for their use in the manufacture of technical roofs [There are investigations oriented towards the design of improved gypsum mortars through the inclusion of rigid polyurethane foam waste ,34,35,36al roofs .In this work, the design of gypsum mortars fabricated with rigid PFW is reported for their use either as conglomerate materials or as raw materials for the manufacture of prefabricated construction products.The objective of this investigation is the design and characterization of gypsum mortars for the manufacture of Interior Thermal Insulation Systems (ITIC) panels, with the objective of improving the energetic behaviour of buildings.Various mixtures of gypsum were designed with the added value of PFW, characterizing their properties in both the fresh and the hardened state.3. The binder consisted of calcium sulphate in its different phases of hydration  and anhydrite (CaSO4)), obtained from partial dehydration of the calcium sulphate dihydrate (CaSO4·2H2O) phase;Gypsum binder Type A1, as per EN 13279-1 , with a 3. The binder consisted of gypsum with a minimum of 50% calcium sulphate as the principal active component and a lime content 2)) below 5%;Gypsum binder Type B1, according to EN 13279-1 , with a The gypsum mortars were designed with two types of binders, together with variable quantities of recovered PFW. Subsequently, standardized sample specimens were cast in moulds, in preparation for the characterization tests and to study the effects of those additions on the physical–mechanical properties of the resulting mixtures, in accordance whit standard EN 13279-2 . The folThe two gypsum binders were manufactured and supplied by the firm Yesos ALBI (Grupo CALCINOR), from its factory at Villalómez . Their technical characteristics are shown in 3, was supplied by the firm Kingspan Prax, S.A. . The waste product is from a factory cutting process, in which large-sized blocks of polyurethane are sawn to the sizes required by the manufacturer. This polyurethane dust is recovered directly from the sawing mill by a vacuum coupled to a sleeve filter where the waste accumulates. Distribution by grain sizes, represented in Polyurethane Foam Waste (PFW), with a density of 90,687 kg/mThe chemical composition of the polyurethane foam was determined by means of elemental analysis, a LECO CHNS-932 Elementary Chemical Analyzer. The results are shown in Water: from the urban mains supply, acceptable for use in the manufacture of mortars.The three raw materials employed for the manufacture of the mortars was analyzed with a model JEOL JSM-6460-LV Scanning Electron Microscope (SEM) , equipped with an elemental INCA analysis system, with an acceleration voltage of 0.20–30 KV. The samples had previously been coated by low-vacuum cathode sputtering (gold) with an EMITECH k550X sputter coater, at a current of 30 mA over 2 min in an argon atmosphere. The SEM images obtained are shown in Gypsum mortars have been designed from two types of binders: A1 and B1, together with different amounts of PFW, by volume.In flow table method. The w/b (water/binder) ratio of the reference mortar, A1, was also determined by the same method.Except for binder Type A1, the other gypsums were considered pre-mixed gypsums, as per the specifications in standard EN 13279-1 , for whiIn accordance with these criteria, the necessary amount of water was added to the mixtures, to yield a diameter on the flow table of 160 ± 5 mm, as per the specifications of standard EN 13279-2 . The gypVicat Cone test procedure was followed to determine the Time to Onset of Setting [Mortar density in the fresh state was calculated as per standard UNE 102042 , by diff Setting . A rubbe3 were prepared for each dosage, to perform the flexural and compressive tests. The specimens were demoulded on the following day and remained in the laboratory for seven days, at a temperature of 23 ± 5 °C and at a relative humidity of 50% ± 5%.Three standardized specimens 40 × 40 × 160 mmPrior to the completion of the tests, the specimens had been dried in an oven at a temperature of 40 °C, until they reached a constant mass. On the following day, the specimens were removed from the oven and introduced in a drier, until their temperature fell to the same level as the temperature in the laboratory, and the dry weight of the three specimens was determined, to obtain the dry bulk density.A Shore durometer with a sensitivity of 1 Shore unit was employed, in order to measure the surface harness or Shore hardness of the specimens . Five me2 surface, applying a constant load and with no accelerations until its breakage, in accordance with the specifications of standard EN 13279-2 [Having determined the mortar density and its surface strength, the specimens were then broken in a Supercar MEM-101/SDC hydraulic press of 200 kN. A load centred mid-span on the specimens of hardened mortar was applied up until failure, in order to determine their flexural strength. The separation between supports was 100 mm . The two 13279-2 .A 10 mm layer of gypsum was applied to a ceramic brick, previously submerged in water for 1 min, to avoid loss of mix-water from the mixtures. Before starting the setting process, the circular surfaces of a diameter of 50 mm were drawn on the gypsum layer. The samples remained for 7 d in the laboratory at a temperature of 23 ± 5 °C and at a relative humidity of 50% ± 5%. Following 7 d of curing and hardening, metallic pads were adhered to the samples with a bi-component glue to apply traction force. A traction test apparatus applied a perpendicular load to the test areas through the metallic pads. The load was applied without shaking and at a uniform speed .3), with the objective of maintaining a constant internal water-vapour pressure, different from the exterior atmosphere. Subsequently, the sides were sealed with a waterproof tape and kept in an environment at 20 ºC and at a relative humidity of 50%. Then, the recipients were weighed at appropriate intervals of time, to determine the flow of water vapour through the gypsum mortar.The procedure from standard EN 1015-19  was used3 were moulded and the absorption of water by capillarity was determined by following the procedure from standard EN 1015-18 [As the properties of gypsum mortar material are affected by humidity, specimens with dimensions of 40 × 40 × 160 mm 1015-18 . To do s3 specimens in a recipient until their saturation, to obtain their saturated weights.Finally, total water absorption of the mortars was determined by submerging the 40 × 40 × 160 mmThe results of the in-fresh mortar characterization tests designed in the fresh state are shown in As may be observed, both in the mortars containing gypsum Type A1 and in the mortars with gypsum Type B1, the higher the amount of added polymeric waste, the higher the amount of water, because of the high water-absorption capacity of the PFW . This beLikewise, their in-fresh density was higher in the B1 mortars when compared with the A1 mortars. This behaviour may be explained by the mineral aggregates that accompany the Type B1 binder, as specified in standard EN 13279-1 . A behavThe reduction in mortar density is related to the quantity of water added to the mixtures, as may be observed from In relation to the onset of setting time test, the mortars dosed with binder Type A1 and PFW, as may be seen from When the analysis was completed as a function of the binder that was employed, it was observed that the times to onset of setting of the mortars manufactured with binder Type B1 were lower with respect to their counterparts dosed with binder Type A1. This behaviour can be explained by the presence in binder Type B1 of mineral aggregates and dihydrates that function as a nucleus of crystallization. These impurities contribute to more rapid crystal formation in the hydration process than in the case of binder Type A1, because the presence of Anhydrites I and II are frequent, on account of their greater purity, which slows the reaction process of water with the material .The mechanical characterization test results are shown in As may be observed, the incorporation of PFW in the mixtures led to a progressive reduction in density in the hardened state, which was higher as larger quantities of foam were added, showing a similar behaviour to mortars in the fresh state. The reduction in the density of the hardened mortars as larger amounts of PFW were added likewise reduced the mechanical strengths , as may be observed in According to the data in When the loss of mechanical strength, due to the incorporation of maximum amounts of PWF, was analysed, the flexural strength of mortar A1:PFW was reduced with regard to its reference mortar, A1, by 54.5% and its compressive strength was reduced by 56.2%. In the case of mortar B1:PFW with regard to B1, the flexural strength was reduced by 65.8%, and its compressive strength by 67.8%.In accordance with this criterion, when the amount of PWF added to the mixtures was 0.6, the reduction in their mechanical strength was approximately 40%, so that amount is considered the optimal option for the quantity of waste/mechanical strength ratio. In this way, mortars A1:0.6PFW and B1:0.6PFW were selected for detailed study in a second stage of the investigation.Nevertheless, the strengths of all the design mortars surpassed the minimum references established in standard EN 13279-2 , regardlOn the one hand, in the case of adherence strength, greater strengths were likewise obtained in the mortars dosed with binder type A1, while the mortars dosed with binder type B1 recorded lower values in all cases. On the other hand, as can be seen from With regard to Shore C hardness, the values were quite uniform in all cases (>90), except for the mortars in which the amount of PFW was greater (A1:PFW and B1:PFW). Gypsum materials are sensitive to humidity. As observed, the incorporation of PFW in the mixtures increased the amount of mix water and, in consequence, their porosity. Capillary Water Absorption, Total Water Absorption, and Water-Vapour Permeability were determined, to establish the behaviour of the mortar designs against the action of water. The test results are shown in Taking the Coefficient of Water Vapour Diffusion (µ) as a reference, the lower the value, the lower the resistance of the mortar to the passage of water vapour through its internal pores. All the mortars under study were shown to have very low coefficients (µ ≤ 7), and so they all presented a minimal risk of interstitial condensation within their interior.It is worth pointing out that the mortars with larger amounts of PWF, A1:PFW and B1:PFW, presented the lowest values: µ = 3. With regard to capillarity water absorption, the mortars presented high coefficients, corresponding to gypsum mortars, and, in consequence, the water penetration height within the mortar was very high. It was observed that the water penetration height in the mortars was greater when binder type A1 was employed. This result implied that the consolidation of their interstitial capillary network was also greater, due to the purity of the material.When the study was completed it was observed that, as a function of the amount of PFW added to the mixtures, the water reached higher levels as larger amounts of PWF were added.network of open pores was greater in mixtures A1, so they presented lower µ values and were more permeable to the water vapour passing through their pores. In addition, the test results of the suction test, to determine the height reached by the water, led us to assume that a fine capillary network with many interconnected pores formed, for which reason the water rose easily, due to the rising forces of capillarity [Likewise, in the Total Water Absorption Test, the mixtures made with binder type A1 had higher levels than those dosed with B1. This result implies that the illarity .The characterization tests of the gypsum mortars dosed with PFW have clarified how the dosing of PFW influences the mortar properties. -The incorporation of PFW significantly reduced the density of the gypsum mortars, both in the fresh and in the hardened state, increasing the water/gypsum ratio in the mixtures, due to the hygroscopic nature of PFW;-The gypsum mortars reduced their mechanical strength under both flexural and compressive as larger quantities of PFW were incorporated. Nevertheless, in all cases, the gypsum mortar designs with PFW showed good mechanical properties for use as a construction material;-With regard to their adherence, the incorporation of PFW in the mixtures also reduced adherence to the ceramic substrate, but not significatively, yielding acceptable values for a rendering material;-Despite the fact that PFW is a soft and a flexible material, the Shore C surface hardness was maintained at acceptable values in all the mixtures, in comparison with the reference mortars, A1 and B1, where it was lower whenever larger dosages of PFW were incorporated;-The mortars dosed with PFW presented higher water vapor diffusion levels. In addition, the greater the amount of added foam waste, the greater the water absorption by capillarity.The first general conclusion from the experimental process leads us to affirm that the use of PFW in the manufacture of gypsum mortars determines an important change in the properties with regard to the reference mortars:


Thus, the effect of MgO on reducing the coking hardness is stronger than that of the other four metal oxides, reducing the hardness of carbonized products by approximately 81%. Furthermore, the adding charge can reduce the hardness index by at least 60%. When the carbonization temperature is higher than 800°C, the hardness index of the carbonized product decreases by approximately 5% each 50°C of increase in temperature. This study shows that the addition of metal oxides can effectively reduce the hardness of coking during the treatment of high-salt organic wastewater by carbonization and oxidation and provide theoretical support for the subsequent treatment of high-salt organic wastewater by carbonization and oxidation.Slag is difficult to treat quantitatively due to the formation of a molten mixture in the carbonization process of high-salinity organic wastewater. Thus, aiming at solving this difficulty, the effects of metal oxide additives, additive ratio, furnace burden ratio, and carbonization temperature on the carbonization and coking of high-salinity organic wastewater are systematically analyzed. The analysis is performed using scanning electron microscopy, X-ray diffraction, and Vickers hardness tests. The results show that all five metal oxide additives can reduce the hardness of carbonized products. The relative effect of reducing the coked hardness is as follows: MgO > CaO > kaolin > Fe Domestic sewage, industrial wastewater, and other useless water, such as the inflow of the first rain path into the drainage pipes canals, are called wastewater. The direct discharge of wastewater can substantially damage the environment due to its complex composition. According to data from the China Environmental Statistics Yearbook, although the discharge of industrial wastewater in China showed a downward trend in the past decade, by 2018, the industrial wastewater discharge in China was still high, 18.16 billion tons, accounting for 23.55% of the national wastewater discharge. Industrial wastewater is characterized by having a wide range of sources, high salt concentrations, and complex components  of recovA large number of salts and other impurities are often present in hypersaline organic wastewater. When this water is treated in a high-temperature process such as incineration , 6, pyroFurthermore, biological methods have advantages such as low cost and wide application scope. In this regard, halophilic bacteria or salt-tolerant bacteria in sewage treatment processes could increase the salt content in wastewater or reduce the organic matter in wastewater. Some studies have shown that when the salt concentration is 10 mg/L, the COD removal rate becomes the highest . HoweverIn this study, the carbonization-oxidation method was used to treat organic wastewater with high salinity. According to the difference between the carbonization point of the organic substances in wastewater and the melting point of salt, the organic substances were carbonized. Subsequently, the carbonized product was dissolved to separate the carbon and salt. Unlike the molten salt oxidation method, the carbonization oxidation method can extract organic matter from wastewater and reuse it as an industrial raw material. Similar to fused salt oxidation, inorganic salts easily agglomerate with raw materials in the wastewater during carbonization and oxidation, resulting in partial loss of salt and a lower conversion rate of salt.2O3, Al2O3·2SiO2·H2O, and Al2O3) and shale ash (solid waste) were added to the furnace charge. The coking structures were analyzed under different conditions using X-ray diffraction (SEM) and scanning electron microscopy (XRD). The effects of CaO, MgO, Fe2O3, kaolin (Al2O3·2SiO2·H2O), and Al2O3 on the hardness index of the carbonized products and the effect of reducing the hardness of coke were also studied. The adequate additive and proportion, as well as the temperature, could be determined through the experiment. Therefore, this study can provide theoretical support for the subsequent carbonization-oxidation method to deal with high-salinity organic wastewater.In conclusion, few studies have considered adding metal oxides to reduce the coking of carbonization products formed during the carbonization of high-salt organic waste liquid. In this study, five varieties of metal oxides  and dehydrated in a blast oven. Sodium chloride, furnace charge, and additives were weighed with a precise balance with an accuracy of 0.1 mg for accurate development of the experiment. An ultrasonic vibrator was used to thoroughly mix the sodium chloride, furnace charge, and additives. The hardness index (SII) of the carbonized products was measured using a Vickers hardness tester, and the coke after heating was tested by electron X-ray diffraction (XRD) and scanning electron microscopy (SEM) using a TD-3500 X-ray diffractometer and an electron microscope. The detailed experimental steps are shown in F is the test force (N),and d is the arithmetic mean of the diagonal lengths d1 and d2 of the two indentations (mm).The hardness of the carbonized product was evaluated using the hardness index SII. After the experiment, the hardness of the carbonized products was calculated using a Vickers hardness tester to develop a quantitative comparison. During the experiment, under the action of a particular load, an indentation with a square cone shape was created on the surface of the sample. The diagonal length of the indentation was measured to calculate the surface area of the indentation. The value of the load divided by the surface area represents the hardness of the sample. The calculation equation presented in  is writtIn each experiment, equal volumes of the developed hypersaline organic wastewater were added to different beakers. The quantity of the metal oxide additive was added to the beaker. The additive was added using a 1 : 1 molar mass ratio of the cation in the metal oxide to Na+ in the high-salt organic wastewater. The amount of the furnace charge was added to the beaker. The amount was added using a 1 : 1 mass ratio of the furnace charge to NaCl in the organic wastewater. After drying, the samples were heated in a corundum crucible, away from the door of the muffle furnace. After setting the temperature of the muffle furnace at 900°C for 30 min, the samples were carbonized after drying treatment. 2O3, kaolin, or Al2O3, the hardness index of the high-salinity organic wastewater is reduced by at least 25%. Among them, the addition of MgO and CaO significantly reduced the hardness index of the product by 63.6% and 43.2%, respectively. The relative effects of the five metal oxides on the hardness index of carbonized products were Al2O3 < kaolin < Fe2O3 < CaO < MgO.The hardness index directly reflects the coking effect of carbonized products. SEM and XRD enabled to observe the microstructure of the carbonized products to understand how metal oxide additives affect carbonized products of organic wastewater with high salinity. The specific analysis is discussed in the following paragraphs.2O3, the surface of the carbonized product becomes rough, but there is still obvious coking. After adding kaolin and Al2O3, the microscopic morphology of the carbonized products is similar. The surface of the charged particles was smooth, and the edges and corners were clear. Large gaps between the particles with no flocculent filler were observed. Moreover, less NaCl was present on the surface of the carbonized product in the molten state, which was because Al3+ and Si4+ are having similar ionic interaction forces with molten Na+, resulting in a similar degree of sintering on the particle surface [ surface .The XRD analysis chart of the high-salinity organic-wastewater carbonized products is shown in 2O3, kaolinite, or Al2O3, indicating that the three kinds of metal oxides had similar mechanisms to inhibit the coking of the carbonization products.However, the chemical reaction between sodium chloride and silicon oxide did not occur because the sample was dried in a blast dryer before heating, and no additional water was added to the chemical reaction. This shows that only the physical reaction of NaCl occurred during the heating process. After adding CaO, the XRD analysis diagram of the carbonized products showed that only two crystal phases were present, CaO and NaCl, in the carbonized products, and no material with a high melting point was generated by the reaction between sodium silicate and CaO . This agThe results of SEM and XRD show that the metal oxide mechanism reduces the coking of the carbonized product, as shown in 2O3, kaolin, and Al2O3, the microstructures of the carbonized products were similar. Moreover, a slightly molten state of NaCl remains on the surface of the carbonized products. This may be because the Fe3+ and Al3+ are positive trivalent cations, having similar interaction force with molten Na+, resulting in a similar sintering degree on the surface of the particles [2O3, kaolin, and Al2O3 were added, the surfaces of the burden particles were smooth with clear edges and corners, with larger gaps between the particles. This indicates that the three additives may play a role in the framework for melting the ash melt, effectively raising the melting point of the eutectic to inhibit coking [In this experiment, because the CaO wraps the low-melting-point eutectic, a layer of refractory-like material was formed on the surface of the residual slag sample, and the material was not cohesive, thus, reducing the bonding between the molten NaCl and the furnace charge . After aarticles . After Ft coking .From the above results, owing to the protective effect of the furnace charge due to the additive, the NaCl adhered to the surface of the carbonized product was reduced, and more NaCl was separated; thus, the conversion rate of waste salt increased.In the following experiment, the quartz sand considered in the previous section was replaced by shale ash with the same quality, and the remaining conditions remained unchanged. The hardness indexes of the carbonized products were measured after the experiment. 2O3 and Al2O3 have no significant effect on hardness reduction because Na+ in quartz sand interacts with Fe3+(Fe2O3) and Al3+ (Al2O3) [2O3 > Al2O3. In conclusion, MgO is an additive that can significantly reduce the coking caused by the cohesion of molten NaCl and furnace charge in processing high-salinity organic wastewater by carbonization-oxidation. (Al2O3) . ConcernUnder the same conditions, different molar mass ratios of metal oxide additives  were added to the organic wastewater with high salinity. The hardness indexes of the carbonized products were measured after being dried and carbonized. The relationship between the measured hardness index and the ratio of the additives is shown in As shown in Three groups were considered based on adding different mass ratios of furnace charge  to high-salinity organic wastewater. In the first group, no additional additive was added, and the furnace charge was shale ash. In the second group of experiments, the burden was shale ash without any additives. The third group was based on the first group of experiments, adding MgO, whose molar mass ratio is 1 : 1. The fourth group was based on the second group of experiments; however, the same proportion of MgO was added, in this case. After drying, the samples were placed in a muffle furnace, and the heating conditions were consistent with the above experiments. The relationship between the hardness index and the charge and its ratio is shown in The first group of samples used quartz sand as the furnace charge, while the second group of samples used shale ash as the furnace charge, without adding additional additives. In the third group of samples, quartz sand was used as the furnace charge, and MgO with a molar mass ratio of 1 : 1 was added. After drying the above three groups of samples, the hardness indexes of each sample at 600°C, 700°C, 800°C, and 900°C were measured, as shown in 2O3 > Al2O3. Among them, MgO can reduce the hardness index by approximately 80%.The relationship between the metal oxide additives and the hardness index of carbonized products showed that an increase in the molar mass ratio of the additives decreases the hardness index of the carbonized products significantly. The relative effect of the five metal oxide additives on reducing hardness was MgO > CaO > Kaolin > FeBurden and temperature also affect the hardness index of carbonation products. The hardness index of the carbonization products decreases with an increase in the added burden ratio. When quartz sand and shale ash were used as the furnace charge, the hardness index decreased by approximately 60% and 66%, respectively. For temperatures above 800°C, the hardness index decreased by approximately 10% for every 50°C incrementsCarbonization and oxidation technology can recycle the inorganic salts and organic matter in high-salt organic wastewater largely. This research can effectively solve the problem of coking in carbonization and oxidation treatment of wastewater. Therefore, adding metal oxide additives can increase the recovery rate of inorganic salts and organics in organic wastewater in actual industrial production. However, this study uses a laboratory to configure a mixed liquid to simulate high-salt organic wastewater and does not consider the impact of other particles in the actual wastewater. In future research, a wider range of metal oxide species should be selected, and the impact of other particles in actual industrial wastewater should be consideredThe hardness index was introduced as an index to evaluate the hardness of carbonized products. The structure of the carbonized products after adding metal oxide additives was analyzed through SEM, XRD, and Vickers hardness test. The effects of the type and proportion of metal oxides, burden, and temperature on the hardness index of carbonized products were studied. The conclusions can be drawn as follows:


Mustela nivalis contains total 16,502 bp in length and consists of a control region and a conserved set of 37 genes. The phylogenetic tree of the family Mustelidae constructed using 18 mitogenome sequences from 16 mustelid species of 6 genera shows that the mustelid species are separated into two main groups. All the members of the genus Martes form a monophyletic group, which has the genus Melogale as sister clade, and the clade of the two genera is sister to that of the genera Enhydra and Lutra. Among species of the genus Mustela, with the exclusion of M. frenata with Neovison vison as sister species, all the other species of the genus Mustela are well grouped. All members of M. nivalis are well placed within the species M. nivalis clade with the clade of M. itatsi and M. sibirica as sister group.The complete mitogenome (MF459691) of  Mustela nivalis is the smallest carnivorous predator of the genus Mustela and is widely distributed throughout Holarctic region, taking in much of Europe, northern Asian and northern North America . The voucher specimen (MUMUNI-1) was deposited in the Wildlife and Fish Conservation Center of the Institute of Forest Science, Kangwon National University. Genomic DNA extraction, PCR and genome annotation were conducted according to the previous studies  was used as a reference for gene annotation and primer design.We characterized the complete mitogenome of a M. nivalis contains total 16,502 bp long, which consists of a control region and a conserved set of 37 genes including 13 protein-coding genes (PCGs), 22 tRNA genes, and two ribosomal RNA genes (12S rRNA and 16S rRNA). The majority of 13 PCGs (10 of 13 PCGs) use ATG as start codon, whereas Nd3and Nd5 initiate with ATT and Nd2 starts with ATC. Of the 13 PCGs, the incomplete stop codons (T– or TA–) are used for termination of Nd1, Nd2, Cox3, and Nd4 (T–) and Nd3 (TA–). ATT and AGA are used as stop codons in Nd6 and Cytb, respectively, and the other six genes end with TAA. The 22 tRNA genes range from 62 bp (Ser(AGY)tRNA) to 75 bp (Leu(UUR)tRNA) in length. Lengths of the two rRNA genes and control region are 1009 bp (12S rRNA), 1572 bp (16S rRNA), and 1016 bp (control regions), respectively. The replication origin RO, which is 35 bp long, is located between AsntRNA and CystRNA within the WANCY tRNA cluster as seen in most vertebrates (Kim and Park The complete mitogenome (MF459691) of Martes form a monophyletic group, which has the genus Melogale as sister clade, and the clade of the two genera is sister to that of the genera Enhydra and Lutra. In Group 2, with the exclusion of M. frenata with Neovison vison as sister species, all the other species of the genus Mustela are well grouped. All members of M. nivalis are well placed within the M. nivalis clade with the clade of M. itatsi and M. sibirica as sister group.According to the phylogenetic analysis of the family Mustelidae , the mus