There have been five major mass extinctions and some minor mass extinctions of animals since early animal diversification 540–520 Myr ago. It is said that a sixth mass extinction is already underway. However, the future extinction magnitude has not been quantitatively estimated. Here, I show that the sixth major mass extinction (defined as > 60% species loss) will be avoided, but a minor mass extinction, 20–50% animal species loss (1% now), will occur when humans cause nuclear war and/or fail to stop increasing greenhouse gas (GHG) emissions, pollution, and deforestation until 2060–2080 CE. When humans decrease GHG emissions, pollution, and deforestation in 40 years and prevent nuclear war in the future, 10–15% animal species loss will occur. Humans should stop not only industrial GHG emissions but also deforestation, environmental pollution, and nuclear war to prevent this mass extinction. When humans fail to stop these processes, significant decreases in biodiversity and the human population and a collapse of ecological balance will occur on Earth. Recently, some papers have suggested that a major mass extinction will occur due to human activity in the near future3. Since five major mass extinctions have occurred since animals became common on Earth 540–520 Myr ago6, this expected major mass extinction is called the sixth major mass extinction3. However, analyses of future extinction magnitudes have not yet been reported due to unknown relationships between the causes of future extinctions and the different causes of the Phanerozoic mass extinctions and the current extinction4.The ongoing species extinction rates, which reached the 1% level on land and the 0% level in seas from 1800–1900 to 20107. The ongoing current-future extinction of animals occurred in parallel with rapid global warming, with deforestation and pollution marked by mercury, CO, and black carbon emissions and plastic production3. Only the sunlight reduction category is not ongoing. When a nuclear war occurs, stratospheric soot is formed, thus causing sunlight reductions and global cooling15. Therefore, when nuclear war occurs, all four causes are active.The causes of mass extinctions in the Phanerozoic after the invasion of land by plants and animals are summarized as climate change, pollution, deforestation, and sunlight reduction. Changes in these four categories should have always occurred throughout the major mass extinctions, as evidenced by many geochemical analyses4, no relationships between other causes and extinction magnitudes in the Phanerozoic have been reported. First, I clarify the relevant relationships  the increased rate of mercury concentration in sediments, used herein as a representative proxy for past pollution10 and CO2 emission amounts accompanied by pollutant materials for future pollution11, (iii) occupancy percentages of deforestation areas in pre-event forest areas for the past12 and human population harmonized with deforestation for the future13, and (iv) the amounts of stratospheric soot resulting from the K–Pg asteroid impact for the past14 and estimated nuclear war15 for the future. Only soot introduced by nuclear war is independent of the other causes, whereas the other causes are related both in the past and future. I analyze (i) the former three causes of global warming events on land and shallow seas because the current ongoing crisis is accompanied by global warming and (ii) the global cooling event at the Cretaceous–Paleogene boundary (K–Pg) for the latter cause, soot, because the formation of aerosols causing reduced sunlight is a common cause of major and minor mass extinctions16. The rapid global warming itself may not cause a mass extinction, because the Paleocene–Eocene thermal maximum (PETM) at 56 Myr ago characterized by global warming17 lacks deforestation19 and a mass extinction21. The ongoing biotic crisis without nuclear war is similar to PETM in having global warming, mercury concentration22, and lacking stratospheric aerosols causing global cooling. Whereas, the ongoing crisis with nuclear war will be similar to the past mass extinctions, whereas the ongoing crisis without nuclear war is similar to PETM except for deforestation. Therefore, I calculated animal species extinction magnitude (%) under the PETM case and the mass extinction case.Although the relatively large surface temperature anomalies correlate well with the large extinction magnitudes of marine animals and terrestrial tetrapods in the Phanerozoicips Fig. . Second,ips Fig. . Third, ips Fig. , 4. The 4 , major nuclear war , and the K–Pg impact event occur, respectively  while a magnitude of 23–33% is predicted under the worst case Fig. . When a A minor mass extinction, 20–50% animal species loss, will occur under both the most likely and worst cases when minor or major nuclear wars and/or the worst case for global warming, pollution, and deforestation occurred. Under most likely case without minor and major nuclear wars, a mass extinction will occur, but low, 10–15% animal species loss Fig. . The magThe most likely case corresponds to decreases in GHG emissions, pollution, and deforestation over 40–50 years corresponding to global warming total of 3.0 °C in 2100 CE and 3.8 °C in 2500 CE since 1861–1880 CE, resulting in 8% marine, 16–20% terrestrial animal species extinctions in average, and 12–14% in average of the both without any future nuclear war (the range is due to the PETM case or the mass extinction case), which less than the total extinction magnitude of the end-Guadalupian (25%). However, minor and major nuclear wars under the most likely case will cause 17–19% and 28–31% species extinctions in average, respectively. To avoid a mass extinction , humans must decrease GHG emissions over 20–30 years corresponding to a global warming total of less than 2.5 °C in 2100 CE since 1861–1880 CE, corresponding to less than the mid of Representative Concentration Pathway 2.6 and 4.5 (RCP 2.6 and RCP 4.5); this scenario involves decreasing pollution and deforestation at a similar rate in this century and preventing nuclear war in the future.28 and air/water/soil pollution due to industrial chemical reactions and the combustion of fossil fuels26, resulting in global warming9. At the same time, humans are facing the danger of nuclear war. The magnitudes of future extinctions mainly depend on the feasibility of (i) the CO2 reductions implemented by current world politics, (ii) stopping pollution, (iii) stopping deforestation, and (iv) preventing nuclear war. Humans should continuously monitor GHG emission amounts, temperature anomalies, environmental conditions including air, water, and vegetation conditions, and animal biomasses and species losses on land–near-shore sea areas; in addition, humans should conserve the environment and wildlife and prevent nuclear war to avoid a higher-magnitude crisis and to maintain ecological balance in the current human age, the Anthropocene. These measures will benefit not only animals other than humans but also humans themselves because failure to stop the four causes described above will lead to a significant decrease in biodiversity, the collapse of ecological balance, and a significant decrease in the human population.Humans are inducing deforestationIn global warming events associated with mass extinctions, the current environmental changes are similar to those recorded during the end-Ordovician, end-Guadalupian, and end-Permian mass extinctions. Therefore, I analyzed global surface temperature anomalies, mercury pollution concentrations, and deforestation percentages in these three mass extinctions and in the current crisis. The asteroid impact at the K–Pg boundary and nuclear war cause the formation of stratospheric soot aerosols distributed globally, thus inducing sunlight reductions and global cooling (impact winter and nuclear winter). I also analyzed stratospheric soot aerosols as a possible cause of future extinctions.2 emissions resulting from human conduct, the protection of forests, and the introduction of anti-pollution measures in the future under the Paris Agreement on Climate change and Sustainable Development Goals (SDGs). The worst case corresponds to the scenario in which humans fail to stop increasing global surface temperatures, pollution, and deforestation until 2100–2200 CE.The most likely case corresponds to the reduction of CO8 as the most likely case of GHG emissions, representing the middle of the four potential GHG emissions cases  in Fifth Assessment Report of the IPCC8, approximately corresponding to the middle of SSP2-4.5 and SSP3-7.0 in Sixth Assessment Report of the IPCC9. The timing of decreased global GHG emissions is 2060–2080 CE. Therefore, I use the average GHG emissions and global surface temperature anomalies of the RCP4.5 and RCP6.0 cases as the most likely values and those of the RCP8.5 case as the worst-case scenario, marked by stopping GHG emissions from 2090 to 2100 CE9, as this case corresponds to the highest GHG emissions9.I use the average of the RCP4.5 and RCP6.0 cases in the Intergovernmental Panel on Climate Change (IPCC)4. Changes in industrial GHG emissions and global surface temperature anomalies are sourced from the Fifth and Sixth Assessment Report of the IPCC9.Data on surface temperature anomalies and extinction percentages are from Kaiho8 and in recent sediments deposited in seas and lakes26 because mercury is toxic to plants and animals and because its sources include volcanic eruptions, meteorite impacts, and the combustion of fossil fuels33, which are common sources of pollutants, and because it can be commonly measured from sedimentary rocks recording mass extinctions33. The mercury concentration is related to the CO2 emission amount during global warming because of the common sources of mercury and CO2 . Thus, the future mercury concentrations are estimated based on the CO2 emission amounts estimated by the IPCC9. Since mercury and the other pollutants mainly come from oil, coal, and vegetation33, the amount of mercury released should change in parallel with industrial CO2 emissions because there is a good correlation between mercury and CO2 emissions11.Pollution can be represented by mercury concentrations measured in sedimentary rocks recording mass extinctions28. Thus, future deforestation percentages are estimated based on estimated future population data27 (Supplementary Table 24 and (ii) 2005–2015 CE as a representative of the Anthropocene epoch28 based on the actual forest area relative to the pre-agriculture phase before 4000 BP. Deforestation is related to the human population because agriculture and urbanization have caused deforestation28. I estimate the past and future deforestation percentage using human population data in the past and future21 based on the parallel growth of the human population and deforestation28.Deforestation occurs by the expansion of agricultural areas and urban areas, which are strongly related to human populations34 (Supplementary Table 34.Amount of stratospheric soot was calculated using a method of Kaiho and OshimaI then use those data to estimate the future extinction magnitude based on the assumption that the Earth and contemporary life at the time of each crisis are more or less mutually comparable throughout time and to the present day.15 in the PETM and mass extinction cases, respectively (Supplementary Tables I estimate the magnitude of the species animal extinction crisis between 2000 and 2500 CE using Figs. Supplementary Information.


Soft scalding at 57 °C for 120 s is suitable for broiler chickens for desirable defeathering effects and favorable breast meat and skin color. Hard scalding at 60 °C for 60 s is optimal for layers and the local strain of RF country chickens because of its capacity to ease defeathering and retain acceptable carcass skin color and elasticity.Current soft scalding in broilers is not applicable to various strains of chickens with different market weights and ages. This study aimed to examine the effectiveness of soft (57 °C for 120 s) and hard (60 °C for 60 s) scalding on defeathering and carcass quality of the local strain of Red Feather (RF) country chickens and determine age, breed, and body weight factor in accounting for the defeathering effectiveness using adult layers and juvenile broilers as a reference. Results showed no differences between soft and hard scalding in broilers with 60% and 80% of acceptable defeathering scores, respectively, while a significantly better effect by hard scalding was observed in adult layers, young and old RF chickens with more than 70% of birds exhibiting desirable scores and less than 20% by soft scalding. In contrast to soft scalding, hard scalding had no significant effects on breast meat color but impaired skin color in broilers as assessed by L*, a*, and b* value analysis. Interestingly, hard scalding increased breast skin yellowness and lightness in young RF chickens and lightness in old RF chickens, while there were no significant changes in layers. However, hard scalding decreased skin elasticity in layers and old RF chickens. The effectiveness of defeathering by scalding method was governed by the age to reach a certain threshold in the development of dermal architecture and feather richness, irrelevant to body weight. The alteration of carcass color primarily depends on age, body weight/breed, and their interaction. In conclusion, current soft scalding is suitable for broiler chickens for desirable defeathering effect and carcass color, whereas layers and RF chickens scaled at 60 °C for 60 s showed a better defeathering effect without unfavorable changes of breast meat and skin color. Chickens sent to commercial slaughter facilities are typically rendered unconscious in an electrical water bath, thereafter bled, scalded, plucked for defeathering, and eventually eviscerated. At the scalding step, carcasses are submerged in a hot water tank for a controlled duration and temperature to loosen the skin and facilitate the plucking process by rotating carcasses in the feather-picking machine, which generally can achieve 80% of defeathering . Since aThe scalding process for the defeathering of chickens in a commercial slaughterhouse can be distinguished between hard and soft scalding, depending on the scalding duration and temperature. Hot scalding is typically conducted at 60–66 °C for 45–90 s. By contrast, soft scalding is usually performed with lower temperatures but a longer duration such as either 54–58 °C for 60–120 s , or whenThe soft scalding method is commonly adopted in the broiler industry for desirable defeathering effects and a favorable carcass appearance . In the To exclude the sex effect, only female chickens were selected for the study. Adolescent (12-weeks-old) female Red Feather country chickens (young RF) at market weight were purchased from a commercial farm. A flock of adult Red Feather female breeders  from the Livestock Experimental Farm, National Chung Hsing University  was used and Ross 308 broiler females (Broilers) were raised to juvenile age (7-weeks-old) to achieve BW comparable to young RF chickens. Adult Hyline Brown layer females (Layers) at age of 53 weeks maintained in the Livestock Experimental Farm were used as a reference of comparable age with old RF chickens.Birds were fasted for overnight and shipped to the slaughterhouse 2 h before the experiments. Birds were then slaughtered according to the standard chicken slaughtering process; that is, electrical stunning, bleeding, scalding, defeathering, evisceration, chilling, and refrigeration. The slaughter process was conducted in the poultry slaughterhouse located beside the Livestock Experimental Farm. Totally 160 birds were used in the study; namely 20 birds for each processing group . The soft-scalding process was performed with birds submerged and scalded at 57 °C for 120 s before being mechanically plucked for 60 s, while in the hard-scalding process, birds were submerged and scalded at 60 °C for 60 s before being plucked for 60 s. The processing was conducted in a semi-automatic slaughter line. The sequence of slaughtering was conducted as broilers, layers, young RF, and finally old RF chickens. Chickens were first weighed, and hung upside down in the moving transport line. Subsequently, chickens were moved through an electrical bath one at a time and stunned. The electrical stunning was set as DC (direct current), 50 V, 20 mA, for 12 s. Birds were slaughtered manually by skillful workers through administration of a cut to the throat severing the carotid and jugular veins. Birds were bled for 120 s, released from the shackle, submerged in the tank of scalder, and then manually moved into the feather plucker tank. The scalder featured automatic temperature and time control, a steam circulating heating system, repeated 360° rotation submersion. After scalding, carcasses were transferred into the plucker and then plucked by mechanical rotating for 60 s. The scalder had a 7-chicken capacity for scalding and defeathering, and 4 birds per round were processed simultaneously.Birds of each breed were divided into hard- or soft-scalding groups to assess their responses to scalding temperature and duration in terms of defeathering effectiveness and carcass qualities including breast meat color, skin color, and skin elasticity. Following evisceration of the chickens, a piece of skin 6 × 4 cm in size was removed from both sides of the sternum of each carcass, sealed in a re-sealable bag, and deposited in cold storage (4 °C) for 24 h. The skinned breast meat samples were used for immediate assessment of color change within 2 h after slaughter (defeathering and evisceration).For scalding effectiveness, the parameters developed previously were adopted . After pThe color of breast meat at the site of skinning was measured within 2 h after evisceration. The breast skin samples were removed from cold storage, allowed to return to room temperature for 1 h, and then spread evenly on an opaque white board for color measurement. Breast samples from both sides of each bird were used for color measurement. Measurement was made at the top of eminence site in the anterior part of breast sample and 1 cm below or above the top with 3 replicates in the reading.Color analyses were conducted using a handy colorimeter ; three parameters, L* (lightness), a* (redness), and b* (yellowness) were recorded. Before use, the handy colorimeter was calibrated by measuring the color of a white correcting plate . The L* (lightness), a* (redness), and b* (yellowness) values  of the samples were measured .After skin color measurement, the skin samples were excised into stripes with 3 ± 0.5 cm in length and 0.5 ± 0.1 cm in width and the connective tissue and flesh that remained underneath the skin was removed using a surgical knife. A shear tester  with the Warner-Bratzler shear force up to 25 kg was then employed to determine the magnitude of tensile force . The skit-test or Duncan’s multiple-range test (SAS 9.4) was used for means comparisons. The defeathering scoring results were re-grouped for effective defeathering with the total scores less than 9 for an acceptable defeathering procedure and larger than 10 for ineffectiveness. All factor effects on scalding effectiveness were calculated with the Chi-square likelihood ratio test and analyzed by Nominal Logistic Regression. Data were presented as mean and comparison results with p < 0.05 level were regarded as a significant difference.Data of body weight were analyzed by one-way ANOVA using breed as the variable. A two-way analysis of variance (ANOVA) using scalding condition (hard vs. soft) × age (7-weeks old broilers vs. other breeds of birds at older ages) or scalding condition (hard vs. soft) × BW (layers with 1.8 kg/bird vs. other breeds of birds at 2.6–2.7 kg/bird) was performed to analyze the main effect. If an interaction existed between scalding and age or between scalding and body weight, a Student’s p < 0.05, For comparable variables, namely age and body weight, juvenile broilers, adult layers, and old RF country chickens with optimal ages and body weights were used in the study. Broiler were raised to age of 7 weeks to achieve a similar body weight (2.6 kg in average) comparable to the commercial RF country chickens at market weight (2.7 kg at age of 12 weeks) and a flock of 40-weeks-old RF female breeders (2.6 kg in average) was used as a comparable counterpart with the same breed and body weight but different ages. Layers with significantly lower body weight . These results clearly concluded hard scalding for effective defeathering in layers, in young and old RF chickens. Under soft scalding conditions, juvenile broiler chickens showed a significantly higher defeathering effectiveness than adolescent and adult RF chickens with comparable body weight, as well as than adult layers despite much lighter body weight (p < 0.05), while no differences by hard scalding were found among the four breeds of chickens. Accordingly, the age to reach a certain threshold in the development of dermal architecture and feather richness rather than body weight predominates in determining the effectiveness of the defeathering process.Based on the distribution, the scoring results were re-grouped for net effectiveness with the scoring less than 9 for acceptable defeathering procedure and larger than 10 for ineffectiveness . Resultsp < 0.026) was observed when assessed by scalding condition (hard vs. soft) × BW (layers with 1.8 kg/bird vs. other breeds of birds at 2.6–2.7 kg/bird) ANOVA (p > 0.05).Results by ANOVA using scalding condition (hard vs. soft) × age (7-weeks old broilers vs. other breeds of birds at older ages) as variables suggested no significant differences by the main effects in breast meat color, nor by their interaction . A signid) ANOVA . Howeverp < 0.056), and L* and a* (redness) values by scalding condition (p < 0.083 and 0.054) were found in scalding × age and scalding × BW analysis, respectively. Since young, old RF chickens, and broilers had comparable body weight but different ages, while varied in body weight with adult layers, this susceptibility of breast meat color change to hard scalding  thus is attributed to the age and breed, by which younger birds may have loosened muscle structure and thus are sensitive to protein denaturation, and hemochrome oxidation leading to color change [A very close to significant level in L* values (lightness) by age . Scalding condition had a very close to significant effect on a* and b* values (p < 0.095 and 0.054) in scalding × BW and scalding × age analysis, respectively  with no differences to old RF chickens (p < 0.05) with no differences to old RF chickens  and age (p < 0.001) significantly affected breast skin tensile strength  but no differences with old and young RF chickens (Scalding condition (strength . Under schickens . In contThe present results manifested that the current soft scalding method at 57 °C for 120 s is suitable for broiler chickens for desirable defeathering effects and favorable carcass appearance, while layers and juvenile and adult RF country chickens by hard scalding at 60 °C for 60 s exhibited better defeathering without significant unfavorable changes on breast meat and skin color. The age to reach a certain threshold rather than body weight was concluded to govern the effectiveness of defeathering by scalding process, while the age, BW/breed, and their interaction are the causative factors contributing to the resistance of carcass color alterations. The certain threshold can be attributed to the development of dermal architecture and feather richness ,15.Selection of scalding parameters depends on factors such as the effectiveness of feather removal, age of the birds, chilling method to follow, and desired appearance of processed carcasses and final products . The comHard scalding, namely, at higher temperatures in a shorter duration such as 60 °C for 60 s, is more effective to loosen feathers from follicles and is commonly used for waterfowls, turkeys, and spent layer hens. The higher temperatures of hard scalding cause the outer layer of skin epidermis more loosen and thus is easily removed later during the feather picking operation leading to a white carcass . In the Color is one of the major quality attributes of poultry carcasses and is directly associated with the properties of the meat varying in breeds, ages, feed, and management. Skin color of the carcasses attracts the first sight of consumers and governs their choice of the products . The pigPast studies suggested that increased scalding temperatures can remove the corneum and cuticle in the breast skin and decrease its b* values leading to a pale and less-yellow appearance of the carcass . Soft scBroiler carcasses are typically dissected for sell, and breast meat is among the most popular on the market. Different from broilers, local country chickens with deeper skin color are regarded as portraying meat from healthy and high-quality birds in Asian countries and mostly are sold as a whole carcass. This results in a particular attention for carcass skin color change by scalding procedure. Moreover, the meat of spent layers is commonly used for processed products such as meat floss and meat balls. Accordingly, the parameters of slaughter procedure in commercial electric slaughter houses should be balanced according to chicken age and breeds to ensure the effectiveness of defeathering and carcass quality. The present study demonstrated hard scalding at 60 °C for 60 s for effective defeathering in layers and RF country chickens without unfavorable changes of breast meat and skin color.The current scalding procedure at 57 °C for 120 s in broiler chickens can achieve a desirable defeathering effect and favorable carcass color. Hard scalding at 60 °C for 60 s had effective defeathering for layers and the local strain of RF country chickens with acceptable carcass breast and skin color. The defeathering effectiveness by scalding depends on the age to reach a certain threshold in the development of dermal architecture and feather richness, while carcass color change is governed by age, BW/breed, and their interaction.


Improving the reversibility of lithium metal batteriesis one ofthe challenges in current battery research. This requires better fundamentalunderstanding of the evolution of the lithium deposition morphology,which is very complex due to the various parameters involved in differentsystems. Here, we clarify the fundamental origins of lithium depositioncoverage in achieving highly reversible and compact lithium deposits,providing a comprehensive picture in the relationship between thelithium microstructure and solid electrolyte interphase (SEI) forlithium metal batteries. Systematic variation of the salt concentrationoffers a framework that brings forward the different aspects thatplay a role in cycling reversibility. Higher nucleation densitiesare formed in lower concentration electrolytes, which have the advantageof higher lithium deposition coverage; however, it goes along withthe formation of an organic-rich instable SEI which is unfavorablefor the reversibility during (dis)charging. On the other hand, thegrowth of large deposits benefiting from the formation of an inorganic-richstable SEI is observed in higher concentration electrolytes, but theinitial small nucleation density prevents full coverage of the currentcollector, thus compromising the plated lithium metal density. Takingadvantages of the paradox, a nanostructured substrate is rationallyapplied, which increases the nucleation density realizing a higherdeposition coverage and thus more compact plating at intermediateconcentration (∼1.0 M) electrolytes, leading to extended reversiblecycling of batteries. In this context,Li metal is interesting, having the highest theoretical specific capacity(3860 mA h g–1) and lowest potential (−3.04V vs the standard hydrogen electrode). It is therefore intensivelystudied to break the specific energy bottleneck of current LIBs.4 However, even after decades of intensive research, its poor electrochemicalreversibility and consequentially short cycle life remain the challengesthat prevent commercialization.7Since the appearance of first commerciallithium (Li)-ion batteries(LIBs) in the early 1990s, they have been widely used to power mobileelectronic devices.10 Ideally, Li metal is electrochemically plated asa compact layer, having a small interface area with the electrolyte,where a flexible and stable SEI prevents further electrolyte decomposition.To achieve this, many strategies have been reported, which includeexternal strategies such as applying the pressure14 and increasing the temperature,18 aiming at physically generating a more compact deposition morphology.On the other hand, internal strategies are being investigated, inwhich formulating electrolyte compositions aims at tuning both Limetal morphology and SEI through electrochemical processes.20 Generally, highly concentrated electrolytes [≥4 mol/L (M)]24 and functional additives or alternative salts/solvents28 are employed to induce compact Li metal plating through the formationof a stable SEI with good Li-ion conductivity. However, the largevariety of systems studied make it difficult to establish a coherentperception on how the Li metal microstructural evolution and SEI composition/structureinteract and how these impact the reversibility upon cycling. An opportunityto gain comprehensive understanding is variation of the salt concentration,being one of the very basic parameters that can be used to modifythe electrolyte, determining the Li-ion mass transport through theelectrolyte and through its solvation characteristics, also influencethe SEI composition and structure, both of which play important rolesin the evolution of Li metal microstructure.It is widely accepted thatthe electrochemical reversibility iscorrelated with the evolution of Li metal morphology and solid electrolyteinterphase (SEI) on the Li metal surface. Heterogeneous Li metal platingresults in high-surface-area “mossy” or “whisker-like”morphologies, where the high Fermi energy level of Li metal causesirreversible reactions with the electrolyte that generate the SEI,which leads to the loss of active Li, both as SEI species and as “dead”Li, the latter referring to the formation of electronically disconnectedLi metal particles.In this work, weembark on a systematic study of the relationshipbetween Li metal microstructure and the SEI composition driven fromelectrolytes with varying concentrations. Different concentrationsof lithium bis(fluorosulfonyl)imide (LiFSI) salt were dissolved ina 1,2-dimethoxyethane (DME) solvent, where the high donor number makesDME effective in the dissociation of alkali metal salts, enablingthe study of a wide salt concentration range in the same system. Thisallows us to elucidate the influence of other electrolyte propertieson the microstructure of electrodeposited Li metal to explore theadvantages and disadvantages of lower and higher salt concentrationswith respect to the resulting SEI and Li deposition morphology. Theseresults indicate the importance of achieving a high Li depositioncoverage, which can be achieved at lower salt concentrations, therebymarrying the advantages of low- and high-molarity salts toward higherreversibility for Li metal batteries.–2 for 1.0 mA h cm–2, the averageCoulombic efficiency (CE) increases with the salt concentration (S1), along with a decrease in CE fluctuation. The stabilityand overpotential are also evaluated in Li||Li symmetric cells  where the reduced overpotentialwith increasing molarity appears to be a consequence of the reducedinterfacial charge-transfer resistance after the formation of SEI. The Li-ion transference number. A larger 9The reversibility of the Li metal plating/stripping in LiFSI DMEelectrolytes with different molarities is evaluated in Li||Cu cells.During the first 100 cycles at a current density of 0.5 mA cmntration 1a and S1ctuation1b. A sim–2 to an arealcapacity of 1.0 mA h cm–2 . For the dilute electrolytes (0.05 and 0.2 M), the deposited Limetal is more porous with smaller, whisker-like Li deposits near thecurrent collector, whereas more compact deposition present at thetop results in mushroom-like structures, which explains the largerrelative diameter observed from the top-view SEM images. In contrast,when the molarity increases, larger columnar deposits form, leadingto the larger diameter of the Li deposits. Therefore, a similar diameterin the top-view images of Li metal deposits in dilute and highly concentratedelectrolytes represents a different growth mechanism and morphologyas illustrated in Scanning electron microscopy (SEM) was used tostudy the morphologyof the plated/stripped Li deposits on Cu in Li||Cu cells after platingat a current density of 0.5 mA cmA h cm–2 1c and af cm–2 (+ 1d.From  cm–2 (+ S14. For  cm–2 (+ S14. For  cm–2 (+ S14. For  cm–2 (+ S14. For 7Li solid-state nuclear magnetic resonance (NMR) as anon-invasive method can provide quantitative and temporal informationon Li metal deposition, where the development of operando probes allowsus to monitor the processes of Li plating/stripping during an electrochemicalmeasurement by recording spectra at intervals.35 The chemical shifts in 7Li solid-state NMR can be usedto differentiate the metallic Li and diamagnetic Li species in theelectrolyte and SEI (∼0 ppm), as well as provide insights intothe evolution of the Li metal microstructure during cycling  grows, reflecting the Li metal deposition on the Cu current collector,and as expected, it subsequently shrinks upon Li stripping duringdischarge. The pristine 7Li spectra before charge, at theend of the first charge, and after subsequent discharge extractedfrom the operando dataset are shown for each electrolyte concentrationin Figures S15 and Figure S16). Based on the NMR spectra and the CE, the amount of reversibleLi metal, “dead” Li, and Li in the SEI species can bequantified with the method in Supporting Information Note 1, and the results are shown in Table S2. The percentage of Li in the SEI and the “dead” Liboth decreases with higher molarity, which consequently increasesthe capacity of the reversible Li metal. Only at 5.0 M, the capacityloss is not dominated by the “dead” Li metal, suggestingfavorable SEI properties for highly concentrated electrolytes. cycling 2a. Here,7Li NMR resonanceduring chargeand discharge can also provide insights into the evolution of theLi metal microstructure because the shift of the 7Li metalpeak is sensitive to the orientation and microstructure of the Lideposits due to the bulk magnetic susceptibility.33 Generally,a pure Li metal strip gives rise to a resonance signal at ∼246ppm when placed perpendicular to the fixed magnetic field B0, which shifts to higher ppm values when thestrip is parallel to B0.37 Therefore, mossy or whisker-like microstructures growing perpendicularto Cu, assuming the electrodes to be perpendicular to B0, have been associated with a chemical shift range centeredat around 272 ppm, whereas mossy microstructures encompass a broaderspectral region covering a chemical shift range of approximately 250–290ppm, and more compact Li metal appears to approach ∼246 ppm.33 Also, the vicinityof the deposits to the current collector and the electrode, in combinationwith their bulk susceptibility, impacts the shift. Electrodepositsnear the Li metal substrate result in a shift of ∼260 ppm,whereas whisker-like structures that extend further away from thesurface appear at ∼272 ppm. Due to the small diamagnetism ofCu, the Li metal shift is hardly affected; however, the paramagneticLiFePO4 can induce a +15 ppm shift approximately.36 As shown in Figure S17). These distributions can be deconvoluted in three resonances, wherepeak 1 and peak 3 stand for shoulder peaks on the left (higher ppm)and right (lower ppm) of the peak 2, respectively (S18). In the 0.2 M electrolyte, peak 3 shifts significantlyto lower ppm values along with peak 2 (S14). In the 1.0 M electrolyte, peak 2 and peak3 also shift to smaller ppm values  and thus represents different conditions from the morphologiesshown in the SEM measurements (–2), a thin layerof nanosized Li deposits can be observed for 0.2 M (–2), the number of Li metaldeposits in the 0.2 M electrolyte increases continuously , and its number remains high as summarized in –2), while the average size increasessteadily (–2) until a final diameter of around 4 μm is achieved. The decrease in the numberof Li metal deposits appears to be a result of coalescence of smallerdeposits, further supported by the cross-sectional SEM images shownin Figure S14. Based on the observationabove, the increased coverage observed for the 0.2 M compared to thatfor the 1.0 M electrolyte therefore can be related to the lower shiftsin 0.2 M than the 1.0 M electrolyte as observed in 7LiNMR spectra at the initial stage of plating .During these measurements, there is no applied pressure due to thenature of the in situ AFM setup S19 and t39 Most likely, the amorphous matrix represents organic speciesformed by DME solvent decomposition, whereas the crystalline grainsrepresent the inorganic components of the SEI layer (S22). The latter are attributed to Li2O and LiFfrom selected area electron diffraction (SEAD) measurements . From cryo-scanning TEM annular dark-field(ADF) images combined with electron energy loss spectroscopy (EELS)mapping, it can be concluded that the SEI formed in the 0.2 M electrolyteis mainly organic, being rich in carbon and oxygen . For the 1.0 M LiFSI DME electrolyte, the low-magnificationcryo-TEM image in 40 In the outer layer, the well-defined latticefringes represent large crystalline grains (∼10 nm), whereasthe inner layer is largely amorphous (S22). Theinorganic components in the outer layer are Li2O and LiF,as determined by SEAD . Inthe ADF and EELS mapping, a strong oxygen signal is present , further confirmed by X-ray photoelectronspectroscopy (XPS) measurements . Thelarge fraction of C-containing species detected in the SEI formedin the 0.2 M electrolyte indicates that solvent decomposition dominatesthe SEI formation (S32), which is consistentwith the large redox peak observed at ∼0.5 V using cyclic voltammetry. The higher salt concentrationin the 1.0 M electrolyte results in more SOx and Li2O, suggesting that LiFSI decomposition dominatesthe SEI formation process .Since the organic components of the SEI are mainlydecompositionproducts of the DME solvent, the carbon content in the SEI is expectedto be higher. This appears to be true for the organic-rich SEI (moreorganic components such as C–O and C–C/C–H speciesby solvent decomposition) formed in the 0.2 M electrolyte based onthe XPS measurements in ompounds S27–S31. tammetryS33. The 41The higher nucleation coverage and longer nucleation periods inlow-concentration electrolytes, driven by the more severe Li-ion depletion,can be considered as a favorable starting point for dense Li metalplating and thus for more reversible cycling. However, the same iondepletion is responsible for a more organic-rich SEI through DME solventdecomposition, which promotes inhomogeneous Li plating/stripping andstalls the growth of large and dense Li metal deposits. The largersurface area of the smaller deposits in dilute electrolytes causesmore SEI growth, which leads to more irreversible capacity loss andelectrolyte consumption during cycling. The benefit of a higher saltconcentration is the thin, well-defined multi-layer SEI that is richin inorganic species that guarantee a higher stability as well asa higher and more homogeneous Li-ion conductivity. This seems to beresponsible for the continuous growth of large and dense Li metaldeposits, which in turn suppresses the formation of “dead”Li metal, and the smaller surface area of these large deposits leadsto a much smaller amount of SEI species, both these factors promotingthe reversibility. The disadvantages are however that the lower nucleationdensity at higher electrolyte concentrations leaves parts of the Cuuncovered, leading to a lower Li deposition coverage and limitingthe Li metal density. This in addition to the known disadvantagesof higher salt concentrations, that is, the increase in viscosity(lowering conductivity), reduction in wettability, and increase incosts, has so far limited its practical application.44 However, the pulsed charging/discharging induces continuous consumptionof both solvent and Li from the cathode side, which leads to morerapid degradation of the battery.45 Instead,we propose to the commercially available current collector coveredby nanosized Cu particles to replace the regular Cu current collector and study the deposition in combination with a 1.0 M LiFSI DMEelectrolyte.Therefore, the challenge is to combine the favorableproperties of both high and low salt concentrations. With respectto the SEI composition, a stable SEI requires at least an intermediate,around 1.0 M, salt concentration (excluding the possibility of improvingthe SEI with additives). Therefore, a rational strategy is to aimfor increasing the density in nucleation sites at 1.0 M to achievedenser plating while keeping the favorable SEI morphology and composition.Several studies have demonstrated that initial nucleation pulses canincrease the density of Li metal nucleation on the electrode surface.ollector5a that aS36). Operando 7Li NMR was performed to further verify the Li metal microstructuralevolution on the nanostructured Cu. Compared with using regular Cu,the Li metal peak on nanostructured Cu results in a chemical shiftat lower ppm values in the initial stages (S37), resemblingthe chemical shift observed in dilute electrolytes, indicating anincreased Li deposition coverage. The nucleation on this nanostructuredCu was further studied by in situ electrochemical AFM, where a largernucleation size was achieved as expected for the 1.0 M electrolyte. In order to evaluate the potential application,Cu||LiFePO4 full cells were assembled to study the cyclingstability of the nanostructured and the regular Cu. The results showstable cycling over 130 cycles for the nanostructured Cu, and thecell using regular Cu fails after around 60 cycles (S39), extending the cycling life more than two times.Introducing the nanostructured Cu in a Li||Cu celldemonstratesa marked improvement in the average CE and in cycling stability ascompared to the regular Cu 5c. The Sctrolyte3, but in6 dissolved in ethylene carbonateand dimethyl carbonate (EC/DMC 1:1 in weight), including a 5% FECadditive which is known to improve the cycling stability of Li metal.The electrochemical cycling of the Li||Cu cells demonstrates thatalso in this case, the nanostructured Cu results in a higher CE (91.6vs 90.5%) and better cycling stability (7Li NMR was carried out to gain insights into the Li metalmicrostructure evolution during plating and stripping in the esterelectrolyte using the regular or nanostructured Cu in Cu||LiFePO4 cells. Comparing the nanostructured and regular Cu shownin 7Li chemical shifts that can be associated with more compact platingand larger deposition coverage. In contrast, the deposition on regularCu results in a higher shift for the 7Li chemical shifts,indicating more whisker-like growth and less deposition coverage (7Li NMR spectra after discharge, there islittle difference in the amount of residual lithium metal betweenthe two copper current collectors, which may be related to a similarSEI composition, also suggested by the similar CE. Even though theimprovement in the CE upon cycling is small, the more compact platingon the nanostructured Cu enhances the cycling stability, extendingthe cycle life of the Li||Cu cell.Finally, the impact of increasing the Li depositioncoverage and density via the nanostructure of the Cu substrate isinvestigated using a typical ester electrolyte. The standard carbonateelectrolyte with 1.0 M LiPFtability 6a, as wetability 6c, as cotability 6b. The Ltability 6d than ttability 6h. Aftertability 6i, compatability 6e. Operacoverage 6g,k. IntBased on the above observations, the complex dependence of the depositionmorphology on the concentration due to the different nucleation conditionsas well as the different SEI growth conditions can be clarified. Toachieve dense plating, it not only requires large Li deposition diameters,as achieved in higher concentrated electrolytes, but also requiresa high deposition coverage. This is demonstrated by Li plating in5.0 M LiFSI DME, where large Li deposit diameters are realized, butthe low deposition coverage can be held responsible for leaving gapsbetween the deposits 7a, whichIn summary, the Li microstructure was systematicallyinvestigatedas a function of electrolyte concentration using a combination ofoperando, in situ and ex situ experimental techniques that probe theLi metal morphology and SEI on all length scales, formulating a comprehensivepicture of the relationship between the Li deposition coverage andmicrostructure in lithium metal batteries. The higher deposition coveragecan be formed in the dilute electrolytes, which provide a favorablestarting point for dense Li metal deposition. However, the formationof the organic-rich mosaic SEI, also a consequence of salt depletionat the Li metal surface, prevents the growth of large Li depositsand dense Li metal deposition. In contrast, higher concentrated electrolytesinduce a thin and stable SEI, which induces the growth of large Lideposits. In this case, however, the relatively small deposition coveragelimits the final density of the Li metal deposition. These resultsimply the importance of deposition coverage in the microstructureof Li metal. Furthermore, the deposition coverage can be improvedthrough the substrate surface structure, making it possible to combinethe favorable aspects of low-concentration electrolytes with thoseof highly concentrated electrolytes. For intermediate concentrationelectrolytes, the combination of the high deposition coverage withstable SEI driven from the functional additives or alternative salts/solventsprovides a promising research direction for practical applications,which has also been demonstrated by the commercial carbonate electrolytes.


By adding successive amounts of post-flotation sludge to the high-quality lamellar mass, typical parameters such as firing shrinkage, water absorbability and bending strength after firing were assessed. The structure of the obtained ceramic materials, using scanning electron microscopy (SEM with EDS), was also determined. Obtaining positive results would allow not only to significantly reduce the production costs of ceramic tiles, because the sludge is finely divided and no grinding is necessary, but, above all, will allow to eliminate the environmental risk. The present study has shown that it is possible to introduce up to 20% post-flotation sludge for gres porcellanato tile production and up to 50% post-flotation slugde for faience tile production. Both types of ceramic materials with an appropriate proportion of sludge, meet the requirements of tile standards in terms of mechanical strength and water absorption.The development of the mining industry has resulted in the accumulation of large amounts of waste, which effectively degrades the environment. The aim of this study is to check whether the addition of post-flotation sludge from copper production to the typical ceramic mass of the “ Generally, ceramic materials, due to their water absorbability, can be divided into: porous and sintered . The basDescribing the raw material compositions of these materials by means of the mentioned components, the areas of rational compositions of typical masses of ceramic materials are shown in the Although the raw materials from different deposits vary in their mineral and chemical compositions, the so-called rational composition is very useful in production practice as it allows to obtain a material with desired parameters.3 and MgCO3 in the form of dolomite. These types of faience are fired at a temperature of 1000–1180 °C. In particular, calcium faience has a light body with a low apparent density and water absorbability. The porosity and brittleness of the body increases with increasing calcium content.Among the mentioned, there is also a ceramic material containing limestone, called lime- or feldspar-faience . FeldspaThe average water absorbability of lime faience is 20% by mass, although for wall tiles, it does not exceed 18%. Since the faience body is permeable to water and gases, it absorbs liquids and gets dirty quickly, which is why it is usually covered with glaze.vitrified tiles, utility properties intermediate between stoneware and porcelain can be obtained. Its body is non-translucent, sintered and characterized by water absorbability below 6% and high mechanical strength [In the case of the production of semi-porcelain ceramic tiles, also called as strength . Semi-pogres porcellanato. In this case, the water absorption rate must not exceed 0.5%wt and the mechanical flexural strength be less than 35 MPa [wt fieldspar, 25%wt quatrz and 45%wt clay minerals  production mass was used as a base for the study.A special type of ceramic tile is the so-called n 35 MPa ,5. Generrals see . Since tGenerally, post-production wastes are threatening to all components of the environment, earth’s surface, the hydrosphere, the biosphere and the atmosphere. These threats basically occur at all stages of waste management, i.e., during their production and collection, transport, utilization and disposal, as well as during their storage .mining concretes, i.e., mixing with ashes from coal combustion, and as a sealing medium for the production of self-solidifying mineral powder, as well as in terms of neutralization of sulfuric acid. Overall, about 1% of flotation wastes is utilized, the remainder is the so-called anthropogenic deposit–resource reserve of trace elements [Flotation tailings are the dominant type of waste generated in the copper ore enrichment process and account for about 94% of the mass of produced wastes. It is a mineral waste in the form of a finely graded gangue, which is essentially neutral to the environment and harmless to health. In the case of Poland, this waste is produced in the amount of about 24 million Mg/year and is landfilled. Until now, over 800 million Mg of this waste has been deposited in the copper industry’s landfills and, in principle, it is utilized in a minimal extent . AttemptThe particle size composition of waste produced from sandstone ore is dominated by the grain fraction below 0.06 mm, whose share ranges from 62 to 66%, while the amount of the coarser grain fraction above 0.2 mm is approx. 8%. The waste mainly consists of inert silica, aluminosilicates and carbonate minerals .2, 13% CaO and 5% MgO, while fine-grained carbonate waste contains about 24% CaO, 20% SiO2 and more than 5% MgO [The chemical and mineralogical composition of post-flotation waste is variable and depends on the type of processed ore. In the waste generated as a result of the sandstone ore flotation process, the predominating part takes quartz, approximately 40%, and carbonate minerals in face of dolomite—30%, and calcite,10%; those are the most common wastes. Calculated as oxides, those are approximately 50% SiOn 5% MgO . This aln 5% MgO .Carbonate waste is finer-grained, and the share of the mentioned grain fractions are 90% and approx. 3%, respectively. Taking into account the good fineness of the waste, it is hereby proposed to use them also for the production of ceramic tiles.For sintered ceramic tiles, the temperature and width of the sintering interval play an important role, especially contain lithium silicate  in compogress porcelanato lamellar mass   . All masFiring of formed beams in a gradient furnace in the temperature range 800–1250 °C (KENT);Determination of the mechanical bending strength after firing (Wick Roell Z150);Determination of post-burn shrinkage;Testing water absorbability;Examination of the microstructure on a scanning microscope SEM (JEOL–5400) with additional Energy-dispersive X-ray spectroscopy EDS;Particle size distribution; Mastersizer 2000 ;The following scope of research was assumed:gres porcelanato NG and sludge is shown in The grain distribution of gres porcelanato NG slurry relative to sludge is about 12 μm larger. The successive introduction of the typical gres porcellanato mass into the flotation sludge increases the amount of clay minerals by approx. 11%, quartz by approx. 14%, feldspar by approx. 12% and decreases the proportion of carbonate minerals by approx. 38%. (Na + K)-feldspar act as low–temperature fluxes [The shapes of the curves represent similar unimodal distributions. The most common grain fraction in e fluxes . Such a 3 were used for the tests. Seven samples were made from each set. They were dried and fired in a gradient furnace in the temperature range of 900–1250 °C in two rounds.Samples in the form of compressed beams dimensions 80 × 9 × 10 mmgres porcellanato shrinks successively during sintering and stabilizes at the level of 8.5% in the temperature range 1170–1240 °C.The results of the firing shrinkage tests as an average value of 7 samples are shown in The mechanical bending strength after firing the masses P1–P8 is shown in First of all, it should be noted that the highest mechanical strength is achieved by samples fired at a temperature of approx. 1170 °C. The strength decreases rapidly with increasing sludge content in the bulk.The highest mechanical bending strength (>30 MPa) was obtained for the P5, P6, P7, P8 compositions fired at a temperature of about 1170 °C. The lowest mechanical strength is demonstrated by samples contained more than 40% of sludge. In this case, the mechanical bending strength does not exceed 28 MPa.The results of water absorbability measurements for individual samples after firing are presented in The tests showed that the water absorbability of the tested materials after firing depends on both the firing temperature and the sludge content. As the firing temperature is lowered, there is a marked increase in absorbability for compositions containing less than 70% sludge. From a temperature of about 1170 °C for P6–P8 sets, the absorbability value is close to zero. P5–P8 masses show the best sintering tendency. With an increased share of sludge at temperatures above 1170 °C, the water absorption initially decreases and then increases (dashed line in chart), which is the result of melting and expansion of the material. Lightweight expanded clay aggregate (LECA) is formed.Samples fired at a temperature of 1170 °C, i.e., the ones with the highest mechanical bending strength, were selected for microscopic examinations and EDS. Depending on the sludge content, the structure of ceramic materials differs significantly .gres porcellanato mass increases. The liquid phase is already visible in samples P4–P8.All the tested samples, except P8 and P7, are characterized by a significant presence of pores. At the temperature of 1170 °C, the P1–P3 compositions contain weakly bound crystals which firstly grows and then diminishes as the content of Averaged EDS analysis for samples P7 and P8 showed the presence of Al probably related to the presence of mullite, although no needles of secondary mullite were noticed. On the other hand, in the P1–P3 masses, a low quartz content and an exceptionally high presence of Ca can be observed. Scanning microscope images of the samples show that the masses containing increased amounts of sludge have more pores, which may be caused by thermal dissociation of mainly calcium carbonate during firing.gress porcelanato mass should be summarized in two areas: possibilities of using the sludge for the production of absorbent faience-type tiles and maximum amount of sludge additive to the sintered masses in order to reduce the firing temperature.The preliminary tests carried out on eight masses containing various amounts of flotation sludge and 3), the higher the water absorbability values and the narrower the sintering process is, especially at temperatures above 1150 °C.Research shows that shrinkage increases as the firing temperature increases, which indicates sintering of the masses. The sludge increases the sintering ability of masses and narrows the sintering interval. This fact is confirmed by water absorption measurements. The more sludge by weight added (>40% CaCOMasses with low sludge content (<30%) start sintering at lower temperatures, i.e., from 1150 °C. At a temperature of about 1170 °C, P1–P4 compositions have zero water absorption.The analysis of the results of mechanical bending strength shows that the masses fired at a temperature of approx. 1170 °C have the highest strength values and it decreases with increasing sludge content.gres porcellanato tiles (B Ia)  are allowed for production. This applies to faience wall tiles (B III GL) and non-frost-resistant tiles (B IIa) as well as frost-resistant s (B Ia) .gres porcellanato tiles, a maximum of 40% of sludge can be included. However, taking into account the width of the sintering interval, the addition of the sludge should not exceed 20% , only porous material can be produced, e.g., glazed faience tiles or stoneware tiles. This fact is confirmed by textural (microscopic) tests. A further increase in the amount of sludge results in more pores what is mainly related to the decomposition of calcium carbonate.EDS tests confirmed the presence of calcium, silicon, aluminum and magnesium atoms. At temperatures above 1050 °C, it suggests the formation of solid solutions, probably calcium (and magnesium) aluminosilicates.A thorough analysis in this area requires detailed X-ray examinations, especially since in these systems with the participation of alkali, many silicate and aluminosilicate compounds may be formed .Much more restrictive parameters of ceramic tiles concern water absorption .gres porcellanato) should contain less than 18% of sludge. Group B IIa frost-resistant floor tiles may contain 29–53% of sludge. And group B III wall tiles may contain 65–77% of sludge [In this case, frost-resistant tiles of group B Ia  allow for the production of three types of ceramic tile materials at a firing temperature of approx. 1170 °C;The compositions of the typical Flotation sludge generally reduces the mechanical strength of the material and increases its water absorbability;gres porcellanato porcelain is obtained, with a mechanical bending strength of approx. 35 MPa and a water absorption capacity of less than 0.5%;With a sludge content of up to 18% in the tile base mass, a frost-resistant sintered With a sludge content of 28–53%, a non-frost-resistant (stoneware) plate material is obtained with mechanical bending strength of 21–35 MPa and water absorption within 3–6% by weight;With a sludge content of 65–77%, a faience-type plate material with mechanical bending strength of 18–25 MPa and water absorption within 10–15% by weight where obtained.The post-flotation sludge contains large amounts of calcium carbonate and/or dolomite, which significantly affects the sintering interval and burning temperature of the material as well as other operational and technological parameters.Based on the current study, the following conclusions can be drawn:The tailings sludge from copper production can contain varying amounts of water. For this reason, it can be difficult to incorporate it into the ceramic mass. Further research should take this aspect into consideration. Due to the significant changes in the parameters for the tested sets fired at temperatures above 1100 °C, especially those containing larger amounts of tailings sludge, follow-up measurements should also be made with special attention to measurement errors.Also of interest may be mixtures of raw materials of various mining wastes together.


Water and sanitationare important factors in the emergence ofantimicrobial resistance in low- and middle-income countries. Drugresidues, metals, and various wastes foster the spread of antibioticresistance genes (ARGs) with the help of mobile genetic elements (MGEs),and therefore, rivers receiving contaminants and effluents from multiplesources are of special interest. We followed both the microbiome andresistome of the Code River in Indonesia from its pristine originat the Merapi volcano through rural and then city areas to the coastof the Indian Ocean. We used a SmartChip quantitative PCR with 382primer pairs for profiling the resistome and MGEs and 16S rRNA geneamplicon sequencing to analyze the bacterial communities. The communitystructure explained the resistome composition in rural areas, whilethe city sampling sites had lower bacterial diversity and more ARGs,which correlated with MGEs, suggesting increased mobility potentialin response to pressures from human activities. Importantly, the vastmajority of ARGs and MGEs were no longer detectable in marine watersat the ocean entrance. Our work provides information on the impactof different influents on river health as well as sheds light on howland use contributes to the river resistome and microbiome. Genes related to antimicrobial resistanceand transfer wereanalyzed together with bacterial communities along the Code Riverin Indonesia. Genes were disseminated by fecal bacteria, and pollutionincreased their mobility potential. Low- and middle-income countries carry most of the burdenof infectious diseases and thus are strongly affected by the increasedprevalence of antibiotic-resistant bacterial pathogens.13 It has been suggested that surveillance of watersreceiving effluents from households could be utilized to gather informationon the resistance burden within the population;14 however, this type of surveillance would require priorknowledge of the sources of resistant bacteria as well as their genesand how different human activities contribute to these.Indonesia, a home of 261.4 million people (2017),is the fourthmost populous country in the world. Despite its population size beinglarge enough to have a considerable role in the global antibioticresistance crisis, the dynamics between the environmental resistomeand pollution caused by different anthropogenic factors has not beeninvestigated. Overall, fragmented antibiotic resistance monitoringin low- and middle-income countries is considered as one of the keyproblems in tackling the spread of antibiotic resistance.7 and the impact of human activities on ARGs found in surfacewaters has been previously investigated.20 While this work has been important for instance to understand theinfluences of wastewater treatment plant effluents on the water resistome,those studies commonly consider only one contributing factor at atime. In this study, we investigated the impact of multiple sourcesof antibiotic resistance contamination as well as the dynamics betweenthe microbiome and resistome along the Code River starting from itspristine source, a spring at the Merapi volcano, to the estuary atthe coast of the Indian Ocean. The investigated samples included surfacewater from the river in rural areas as well as the city of Yogyakarta.For profiling the river resistome and microbiome, we used a high-throughputSmartChip quantitative PCR (qPCR) array23 with 382 primers26 targeting ARGs or MGEs together with 16S rRNA gene amplicon sequencingto characterize the bacterial community compositions. Our work describesthe contribution of rural and urban areas as well as the intrinsicresistome to the river resistance burden, provides information onthe impact of different human activities on river health, as wellas gives suggestions how the resistance load could be reduced.Contaminantsand the role of the environment in the evolution ofantibiotic resistance are under increasing research,27 We choseCode River for this study, since it is heavily impacted by both agriculturein the rural area as well as a large city, which makes it a suitablemodel for studying the impacts of different human activities on microbiomesand resistomes in river water. For instance, wastewater managementin the settlement along the riverside of Code is deficient. Thereare only a few communal wastewater treatment plants in the Yogyakartaarea, and residents of the riverside tend to flush their domesticwaste directly to the river without any treatment. Due to this, manywaterborne and water-deprived diseases are endemic in the area.28 The river belongs to the tropical rain beltand has high rainfall, with precipitation >100 mm in the wet seasonand <60 mm in dry season. The samples were taken in 3 days duringthe dry season in May 2017. All non-Indonesian researchers that participatedin sampling have received the Foreign Research Permit approved byThe Ministry of Research, Technology and Higher Education, Republicof Indonesia.Sampling was donein the Code River in Java Island, Indonesia. The Code River is approximately63 km long, starting from the spring at the Merapi volcano to theestuary at the Indian Ocean; 30% of the drainage basin of the riveris used for agriculture, 16% of the area is forest with residents,and 52% of the area is considered urban. Besides the city of Yogyakartawith a population of 422 732 (2017), the urban area also hashospitals, laboratories, and industry. Two percent of the drainagebasin is classified as other.3/s, and the range of river flow rates variedfrom 1 m3/s in November to 9 m3/s in February.We took samples at eight locations along the river:(1) Spring Water, a pristine site at the spring where the river originates(7°32′39.5″S 110°26′45.8″E);(2) Rural + Cattle Farm, a site that was impacted by untreated wastewaterfrom a local dairy farm (cattle consisting of less than 20 head) (7°35′32.6″S110°26′31.0″E); (3) Chicken Slaughterhouse, a sitethat was impacted by a small chicken slaughterhouse (processing approximately100 chickens daily) (7°44′52.3″S 110°22′32.0″E);(4) Hospital, a site that was impacted by effluent from a nearby hospitalwastewater treatment plant (7°46′07.3″S 110°22′24.6″E);(5) City, a site that was impacted by untreated wastewater from riversidehouseholds (7°48′05.4″S 110°22′16.5″E);(6) City Downstream, a site after several kilometers below the city(7°50′17.0″S 110°22′37.1″E);(7) Estuary-Freshwater, a site at the estuary where the water salinitywas under 0.5 ‰ (7°53′34.7″S 110°23′08.3″E);and (8) Estuary-Seawater, an estuary area with brackish water withtidal influence (8°00′50.3″S 110°17′34.2″E)1. DuringThree biological replicate samples were collected at each site.The volume of collected surface water was 1 L per each replicate,except for the Spring Water site where 3 L of water was collected.The samples were filtered on site using a sterile disposable filterunit with a 0.2 μm pore size PES membrane . The filter membranes were transported ondry ice to the laboratory at Universitas Gadjah Mada, where they wereplaced in a −20 °C freezer within 3 h of sample collectionand stored until DNA extraction.The DNA was extracted from filtermembranes using DNeasy PowerWaterkits (QIAGEN), and the concentration was measured with NanoDrop One(Thermo Fisher Scientific). The transfer of the extracted DNA samplesfrom Indonesia to Finland, which was approved by the Ministry of Health,Republic of Indonesia, was done based on the Nagoya Protocol on Accessto Genetic Resources and the Fair and Equitable Sharing of BenefitsArising from their Utilization to the Convention on Biological Diversity,Material Transfer of Agreement (MTA) between Universitas Gadjah Madain Indonesia and the University of Helsinki in Finland.Table S1), which were validated inearlier studies30 as well as by the analysis provider before the runs with our DNAsamples, and false-positive amplification was not observed. The targetedgenes (Table S1) included 16S rRNA genes,four other reference genes in case 16S rRNA primers would not haveamplified, antibiotic resistance genes (ARGs), mobile genetic elements(MGEs), integrons, and other genes associated with antibacterial compoundsdescribed in previous reports.26 Briefly, 5184 parallel 100 nL reactions were dispensedinto the SmartChip using Multisample Nanodispenser (Takara Bio). PCRreactions, cycle conditions, and initial data processing were conductedas previously reported.30DNA (1 μg) from each replicate sample wasanalyzed using the SmartChip Real-Time PCR (Takara Bio) with 384 primersets 38 was used for dataexploration,39 graphics, and analysis togetherwith the ggplot2 package.40 Analyses ofdifferential abundances of ARGs and MGEs were carried out using gammadistributed GLMs. p-values were obtained with Tukey’sposthoc test and were adjusted with false discovery rate control41 using glht function in themultcomp package.42 The community and resistomecompositions were analyzed using the vegan package.43 Nonmetric multidimensional scalings (NMDSs) . Spearman’s rank correlationsbetween ARGs and MGEs within sampling areas and their p-values were obtained with package psych46 using false discovery rate control.41 Only ARG-MGE-pairs that were detected at least in half of the samplesin a sampling area with a strong positive correlation  were included.In the SmartChip data analysis, the cyclethreshold (Ct) value of 27 was used as the cutoff for detection . The ΔCt values,ΔΔCt values, and relative gene abundances were calculatedfrom the Ct values as previously described. (NMDSs) 4 were coSupporting Information (Table S2) as well as at https://github.com/sjmuurine/CodeRiverIn, together with raw SmartChip qPCR array results and all data setsused in the statistical analyses, including the R code. The materialtransfer agreement (MTA) is available from the corresponding authorupon reasonable request.The Illumina sequencing data of the16S rRNA genes have been deposited in the NCBI Sequence Read Archive(SRA) database under project no. PRJEB51169. The SmartChip qPCR arrayresults are in available in the Burkholderiales and Rhizobiales were themost abundant orders in the Spring Water sampling site. Members ofthese orders include known pathogens but also common environmentalheterotrophs, bacteria capable of extracting nutrients from mineralsand species able to fix atmospheric nitrogen,48 indicating oligotrophic or mesotrophic conditions at the SpringWater site. Clostridiales, Bacteroidales, Bacillales, Rhodobacterales,and Campylobacterales became abundant at the secondsampling site   were found in all sampling areas 2A, imply estuary 2A. Membeng areas2B. All fng areas2B, so evS1). Fromthe Rural + Cattle Farm site, the number and relative abundance ofdetected ARGs and MGEs mostly increased along the river until theestuary, where only a few genes remained abundant , 159 assays were positive  (Table S3). Many of these genes have been previously found in agroecosystemsunder restricted antimicrobial use and in environments that were notheavily human impacted,25 like the environmentof the Rural + Cattle farm site. The ISEfm1 elementtogether with intrinsic penicillin resistance gene pbp were enriched in the rural sampling sites compared to city samplingsites  , suggesting that runoff from fieldsfertilized with manure from production animals could be the sourceof these genes.Animal agriculture is aknown disseminator of ARGs to the environment,ngsites 3, indicap-value <0.05, gamma distribution GLMs) (Table S3). It is plausible that discharges of various wastes 60 caused the elevation of these genes in the cityarea.64 However, even though the abundanceof MGEs and related ARGs increased in the city sampling sites, theincrease can be caused by a shift in the taxa from rural agroecosystem-adaptedbacteria to bacteria that are better adapted to humans and built environments.65 It is common that human-associated bacteria carry MGEs that arepacked with multiple features, including various resistance genes.66 There were more ARGs co-occurringwith MGEs in the city area than in the rural area sites  . There were also more ARGs that correlatedwith integrons in the city area ; thus, the city area resistome had increased mobility potentialcompared to the rural area resistome. Many of the MGE-ARG pairs thatco-occurred in the city area  have been previously found to co-occur in production animal manuresin Finland and in China,29 indicating that increasedfecal contamination could explain the elevated abundance of ARGs andMGEs and that ARGs persist in globally emerging antimicrobial resistanceunits.67The abundance of most ARGs and MGEs increased significantly fromrural sampling sites to the city sampling sites (Table S3). This was partly due to a change inthe river water conditions from fresh water to seawater and consequentlya shift in the bacteria community composition. The few genes thatwere abundant in estuary samples can be linked to certain widely disseminatedmobile elements.54 Therefore, althoughmany of the genes related to resistance and transfer were abundantin the river water, especially in the city area, most of these genesdid not seem to spread to the marine ecosystems.The vast majority of ARGsand MGEs were significantly more abundantin the city area sites and in the rural area sites than in the estuary  5A. The re <0.01) 5A. Howeve >0.05)5A, and oe >0.05)2D.Genere >0.05)5A,C, indctively)5A. Interctively)5B–D.Thep < 0.05), which suggests that the taxonomic composition did notexplain the resistome composition very well when all samples wereincluded in the analysis. Interestingly, when rural, city, and estuarysamples were analyzed individually, the correlation coefficient betweenOTU and ARG and MGE distance matrices was high in the rural area  and low in the city and estuaryareas . This supports thehypothesis that the ARGs and MGEs were brought into the river by bacteriain runoff from agricultural settings in the rural area. Also, theARGs in the city area could be maintained at elevated levels by bacteriacarrying MGEs, in which these ARGs were embedded.Mantel’s tests with Spearman’s rankcorrelation wereused to analyze if the bacterial community structure determines theresistome structure. The correlation coefficient between OTU and ARGand MGE distance matrices was moderate . Comparisonrevealed that rural and city area belonged to a group of sample typeswith detection frequencies above average, whereas the estuary areabelonged to a group with detection frequencies below average  than upstream  or downstream of the city (City Downstream). The increasedmetal and detergent concentrations60 couldpartly explain the elevation of MGEs and related ARGs we observedin the city area, since MGEs commonly carry metal and quaternary ammoniumcompound resistance genes.62 Concentrations of PO43– and total suspendedsolids were higher in the rural area  than in the city area, possibly due to soil erosion.60 Although making statistical connections betweenpreviously analyzed water quality parameters68 and the data of this study was not possible due to different numbersof samples and sampling times, the river quality parameters revealedthat different sources contributed to the river contaminant load throughdifferent mechanisms. In the rural area, due to higher concentrationsof PO43– and total suspended solids,60 it can be postulated that ARG contaminationarises from soil erosion and agricultural runoffs containing fecesof the production animals, whereas in the city area, the ARG contaminationis most likely increased due to direct flushing of wastes to the river68 with elevated NO2–, NO3–, and detergent concentrations.60 Thus, decreasing the resistance load of the river couldbe possible with measures reducing soil erosion in the rural areaand by improving wastewater treatment in the city area.Water quality parameters of the Code River were analyzed in previousstudies, and it was found that both rural and city areas contributeto the bacterial load of the river.69 most of the environmentalbacteria are not associated with the genes our assays targeted, andonly a small proportion of all bacterial species are responsible forthe majority of clinically emerging resistant infections.70 To discover the sources of possible ARG traffickersas well as potential human, animal, or plant pathogens, genera belongingto families of Enterobacteriaceae, Streptococcaceae, Staphylococcaceae, Campylobacteraceae, Moraxellaceae, Enterococcaceae, Pseudomonadaceae, Clostridiaceae, and Aeromonadaceae were filtered from the OTUdata and analyzed separately. The changes in the relative abundanceof these genera in rural, city, and estuary sampling areas were comparedagainst the pristine Spring Water sampling site. However, in mostcases, the differences were not statistically significant using theWilcoxon rank-sum test, likely due to low number of observations andhigh variability in abundances within the sampling areas . Other genera thatwere more abundant in rural area samples than in the Spring Watersite were Acinetobacter, Aeromonas, Arcobacter, Eschericia-Shigella, Klebsiella, Staphylococcus, and Tolumonas , Eschericia-Shigella, Streptococcus, and Tolumonas . Dickeya, Kosakonia, Pectobacterium, Plesiomonas, and Samsonia were more abundant in the SpringWater site than in rural or city areas  could have caused the decreased abundance Dickeya and Pectobacterium. Although antimicrobialsare commonly recommended for protecting food crops from these bacteriain southeast Asian countries,81 accordingto our inquiries, antimicrobials were not used for food crops in theCode River drainage basin.In two sites of the rural sampling area, the abundance of nificant7 (Wilcoxolumonas 7. Many oolumonas 7. Arcobay area77 3 and S2.ty areas 7. Except16 building up infrastructure forwastewater treatment could decrease the potential risks caused bythe combination of fecal bacteria, ARGs, and MGEs observed in thecity area. Despite that ARGs, MGEs, and bacteria trafficking themwere abundant in the river, only a few of them remained detectablein the marine environment, most likely due to drastic changes in thewater conditions at the estuary, although dilution due to tidal cyclescould also contribute to their status. In summary, our work showsthat rivers with drainage basins covering both rural and urban areascan be utilized in surveillance for gaining information on the sourcesof contaminants with potential for public health threats.The Code River resistomes and bacterialcommunities reflected differenthuman activities in the drainage basin. In rural area sampling sites,the microbiome and resistome profiles supported our hypothesis thatrunoff including fecal contamination brought bacteria, ARGs, and MGEsinto the river. Controlling soil erosion could perhaps limit the spreadof agricultural ARGs and MGEs to the river. Interestingly, our resultsshow that the potential role of MGEs in keeping ARGs elevated becamemore important in the city area, indicating selection toward bacteriacommonly found in human impacted environments and a higher ARG mobilitypotential. Since wastewater treatment plants can be efficient in reducingARG pollution,


The VMO/VL ratio improved significantly on the BTA side only. The VMO/VL ratios on the BTA side were higher than those on the untreated side (p < 0.05). Knee pain decreased significantly after the BTA injection. The EMG ratios were negatively correlated with the NRS and WOMAC scores. Conclusion: BTA injection effectively reduces knee pain and restores the EMG ratio between the VMO and VL.Objective: To determine the pain and electromyographic (EMG) amplitude ratio of the vastus medialis oblique (VMO) to the vastus lateralis (VL) after botulinum toxin type A (BTA) was injected in the bilateral osteoarthritic knee of patients with patellar malalignment for analysis. Material and methods: A total of fifteen patients were recruited; the more symptomatic knee of each patient received a BTA injection (BTA side). The other set of patients were left untreated. In all, fifteen healthy participants comprised the control group. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and numeric rating scale (NRS) for pain were assessed. The EMG amplitude of VL and VMO activity was recorded using an isokinetic dynamometer and synchronized using the BIOPAC MP100. The data were collected before and at 4, 8, and 12 weeks post–BTA injection. Results: The EMG ratios of the patient group were lower than those of the control group at all testing velocities ( Among older adults, the most prevalent type of arthritis and a leading cause of disability is knee osteoarthritis (OA) . Knee OAThe conservative treatment was the main initial management for PFPS, while surgical treatment was reserved for patients who not only failed the conservative treatment for >6 months but also with documented malalignment, a tight lateral retinaculum, or an articular cartilage lesion . AlthougPLS mainly results from abnormal bony structures and lower extremity muscle imbalance. Abnormal bony structures are more likely associated with the femur bone having a flattened trochlear groove, pronation, or forward tilting ,18,19,20The present study used surface EMG to conduct a comparison of EMG ratios between the VMO and VL before and after BTA injection to thoroughly investigate the effects of BTA in treating PLS-related AKP.This research was approved by the institutional review board of a tertiary medical center (IRB number: 101-5028A3). All the patients signed informed consent forms before participation. Additionally, fifteen patients with symptomatic osteoarthritis of the knee were recruited from an outpatient clinic between 2013 and 2016. Further, anteroposterior radiographic assessment was employed to assign each case of knee OA an Ahlbäck classification grade of 1 or 2. Merchant’s view was used to identify patellar malalignment. In the experimental group, the knee with the worst pain was selected for BTA injection (BTA side). The other knee remained untreated (untreated side).The exclusion criteria were as follows: (1) an inability for independent walking; (2) no Merchant’s view–identifiable PLS or tilting; (3) neurological comorbidities, including multiple sclerosis, Alzheimer disease, or Parkinson disease; (4) rheumatoid arthritis or other inflammatory diseases of the joints; (5) a history of previous lower extremity joint  injury; (6) severe medial tibia degeneration, as indicated by a Kellgren–Lawrence or Ahlbäck’s grade higher than II; (7) previous surgery related to the lower extremities, including amputation, replacement, or ligament repair; (8) a BTA allergy, plan to attempt a pregnancy within 2 years, or current pregnancy; and (9) an inability to comprehend or complete the experiment.p > 0.05; The fifteen healthy participants without any knee pain symptoms and normal alignment of the patella, according to the merchant’s view, were recruited as a healthy control group. The experimental and control groups exhibited no significant differences in age, height, or body mass index  to a concentration of 10 U/0.1 mL. The VL was located using a 27-gauge open-lumen needle that was Teflon coated. The repetitive square wave pulses were applied once per second for a duration of 0.25 ms to the targeted muscle at a location 3 to 5 cm above the patella and lateral to the midline at an oblique angle . A notabThe consent of each participant was sorted and a profile survey and clinical questionnaire were completed. The profile survey was used to obtain each participant’s name, sex, age, height, and weight. The participants’ knee stiffness, pain, and functional status were determined using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). On the WOMAC, of the 96 possible points, 68 points represented functional status, 20 points represented pain severity, and 8 points represented stiffness level. Numerous studies have reported on the responsiveness and sensitivity of the WOMAC, and the WOMAC’s function and pain subscales have been reported to demonstrate a higher responsiveness to change than the function and pain subscales of the SF-36 ,39. An 1A Cybex Norm  dynamometer was used to complete an isokinetic concentric assessment of the extensor muscles of the knee. To test the flexors and extensors, the participants were asked to assume the seated position on the Cybex dynamometer. The backs of the participants were supported, and their hips were flexed to an angle of 85° . AdditioThe EMG signals were obtained using a BIOPAC MP100 system with six channels . A total of four channels were used to record direction (flexion/extension), angular velocity, position (degree of knee extension), and torque signals, while two channels were applied to record the muscle activity amplitude. The skin of the participants was prepared using isopropyl alcohol, and bipolar gold-plated surface electrodes were placed over the center of the VMO and distal VL muscle bellies at a 3-cm interelectrode distance, as specified in Basmajian and Blumenstein . The gro2) analysis. The BTA and untreated sides at four time points  and their interactions with respect to the VMO/VL ratios were compared by using a two-way repeated measures analysis of variance. After the main effects were detected to have significant differences, Bonferroni-corrected paired t tests were applied as post hoc tests. Furthermore, a Friedman test was performed in order to analyze the effect of time points on WOMAC scores. If the Friedman test result was significant, Wilcoxon’s signed-rank test was performed between both time points and between the BTA side and the untreated side. The Spearman correlation test was conducted to determine the association between two variables measured on an ordinal scale. Significance was considered to have occurred when p < 0.05.The statistical analysis was conducted using SPSS 21.0 for Windows . The demographic data and the VMO/VL ratios were compared using a Student’s t test, while the sex ratios of the patients and normal participants were analyzed using a chi-squared . Notably, no significant difference was identified between the knees of the normal control group and the BTA side at angular velocities of 60, 120, and 180 rad/s at 4 weeks after the BTA treatment. The VMO/VL ratio was significantly larger for the BTA side than for the untreated side at angular velocities of 60, 120, and 180 rad/s at 4, 8, and 12 weeks after BTA treatment (p < 0.05).p < 0.05).p < 0.01) and a negative correlation between the VMO/VL ratios and NRS scores  when they completed a timed stair climbing task (p < 0.002) [The safety issue and excessive muscle weakness have been major concerns in BTA injection. Singer et al. reported that BTA injections improved participants’ ability to produce extensor isometric force at 30° flexion (< 0.002) . In our < 0.002) .In conclusion, our study demonstrated that BTA injection was safe and effective in restoring VMO and VL muscle balance and treating clinical symptoms of PFPS; further work should be targeted at managing existing OA change by regenerative technique or intraarticular BTA injection. However, there were still several limitations to this study, including the relatively small sample size and the lack of radiographic image comparison before and after BTA injection.