Here, we report a first case of ampullary cancer with solitary metastasis of the spleen, which was successfully treated with pancreatoduodenectomy and splenectomy and was discharged 7 days after the operation with outpatient chemotherapy. In such cases, physicians should consider splenectomy as an effective treatment option. Ampullary cancer accounts for about only 0.2% of malignant tumors of the intestine. Most ampullary cancers are carcinomas, such as adenocarcinoma 65%), carcinoma 8.1%), and other rarer types such as papillary adenocarcinoma (5.6%), mucinous adenocarcinoma 4.7%), and signet‐ring cell carcinoma (2%) %, and ot.7%, and %, carcinIn this report, we describe a case of solitary splenic tumor metastasized from ampullary carcinoma that was successfully treated with pancreatoduodenectomy and splenectomy.A 65‐year‐old woman, of an emaciated appearance, presented at our hospital with jaundice and complaining of epigastric pain. She had no history of abdominal surgery, but 6 years prior to presentation had been diagnosed with hypertension and pulmonary tuberculosis. Clinical examination found left upper quadrant pain on palpation and blood testing showed an increase in plasma bilirubin (5.09 mg/dL) in which direct bilirubin was dominant (4.71 mg/dL), aspartate aminotransferase (AST) was 75 U/L, alanine aminotransferase (ALT) was 84 U/L, cancer antigen 19‐9 (CA 19‐9) was 71.06 IU/mL, and carcinoembryonic antigen (CEA) was 1325 ng/mL.An ampullary tumor Fig. A, the diPancreatoduodenectomy and splenectomy were performed. An ampullary tumor (2 × 2 cm) and a hard splenic tumor (3 × 3 cm) were surgically resected. Again, there was no sign of any invasion to the superior mesenteric vein and portal vein or any peritoneal metastasis. The operation took 7 h with a 700 mL perioperative blood transfusion for 200 mL of blood loss. On the first day after the operation, the patient could sit up with a little wound pain, and the nasogastric tube and Foley catheter were withdrawn. On the second day, the patient was encouraged to take some food and drink because she had flatus, and the pancreatic duct drainage released approximately 100 mL of fluid.Histopathology confirmed ampullary carcinoma metastasizing to the spleen, maturation medium in histodifferentiation, invading to the duodenum and pancreas, and metastasizing to the hilus hepatis and superior mesenteric arteric lymphatic nodes, but no malignant cells were detected at border of the dissection Fig. A,B. The The patient was discharged from the hospital 7 days after the operation without any complication and received a course of outpatient chemotherapy.Clinical decision‐making is difficult in cases of this rarity where the metastatic mechanism is not fully elucidated. Hellman and colleagues In our patient with a solitary splenic tumor, performing a splenectomy alongside the pancreatoduodenectomy helped to decrease the total number of cancer cells and avoid the spread of cancer cells in the blood stream or along the peritoneum. Splenectomy, indicated by the oligometastatic state of the solitary metastasis, was, therefore, a very appropriate treatment option.Here, we have described the first case to be reported of a patient with ampullary cancer with solitary metastasis of the spleen, which was successfully treated with pancreatoduodenectomy and splenectomy and was discharged 7 days after the operation with outpatient chemotherapy. Despite its rarity, ampullary carcinoma should be added to the list of possible primary tumor locations for secondary splenic metastasis. In such cases, physicians should consider splenectomy as an effective treatment option.Our Institute's  representative was fully aware of this submission, and this scientific activity including writing manuscript was approved by the Ethic Committee of Cho Ray hospital, where the patient was operated.None declared.TMP: patient management, surgical treatment, wrote, and submitted the manuscript.


Wetland restoration and creation efforts have been widely attempted as a way to compensate for wetland losses and to recover wetland functions; however, to date, there has been no comprehensive evaluation of the efficacy of soil carbon (C) and nitrogen (N) content recovery at a regional scale. This meta-analysis synthesizes 48 articles to identify the general patterns of soil C and N change after wetland restoration and creation in the United States. Our results indicate that, after 11–20 years, soil C and N in restored and created wetlands are still significantly lower by 51.7% and 50.3%, respectively, than those in natural wetlands. The soil C and N in restored wetlands recovered faster than in created wetlands. Furthermore, the soil C in restored organic flat and created depressional wetlands recovered more rapidly than in restored and created hydrologically open wetlands , respectively. Mean annual temperature and soil texture were recognized as two crucial abiotic factors affecting soil C and N recovery. Linear regression analysis revealed a positive relationship between the restoration and creation effect sizes on soil C and N, indicating that wetlands may alleviate N limitations intrinsically during C recovery processes. The primary causes of wetland loss include draining and filling for agriculture as well as human settlements. Aerobic conditions in degraded wetlands accelerate microbial respiration, result in the oxidation of accumulated organic C and lead to a net release of C into the atmosphere5, which has strongly influenced atmospheric CO2 levels and global C balances.Natural wetlands store a large amount of carbon (C), with 202–377 Gt C stored in the top 100 cm of soil8 ha of wetlands at 2178 sites around the world have been designated Wetlands of International Importance. Following the convention, many countries have implemented wetland conservation and restoration projects. In North America alone, over $70 billion (U.S. dollars) have been spent attempting to restore more than 3 × 106 ha of wetlands in the last 20 years7.The Ramsar Convention, signed in 1971, is an intergovernmental treaty that provides the framework for national action and international cooperation in the conservation and sustainable use of wetlands and their resources. To date, more than 2 × 108. In terms of soil C stocks, when wetland hydrology regimes are restored, plant litter and root biomass and external riverine/tidal inputs of organic particles provide the primary inputs of organic C into soils10, and anaerobic wetland soils enable organic C to accumulate slowly; however, it is still unclear whether or when these restored and created wetlands attain the C levels of natural wetland soils. Some studies have found that restored and created wetlands can develop soil C and soil organic matter (SOM) conditions comparable to those in natural wetlands within7–20 years following restoration or creation13. However, many studies have demonstrated that restoration and creation do not necessarily recover the soil organic C or SOM of the degraded wetlands to pre-disturbance levels within a certain period17. Some studies of individual sites have indicated that the restored and created wetlands may never achieve soil C levels equivalent to natural wetlands19. Assuming there is a trajectory of C accumulation, several studies have estimated that it would take 15–300 years for soil C pools to reach the levels observed in natural wetlands when based on several different models22. Moreno-Mateos et al.6 conducted a meta-analysis to examine the temporal change in soil C storage in restored and created wetlands throughout the world, and found that the level had not recovered to natural levels after 20 years.The restoration of degraded wetlands and the creation of new ones has been undertaken with the objective of recovering their physical, chemical and biological processes and properties23. Aerobic conditions in artificially drained wetlands also perturb N storage and cycling, allowing for the mineralization of organic N3. Inorganic N is rapidly processed by microorganisms and plants, leaving the original pool of N in the soil depleted or unavailable. Although wetland restoration and creation have been reported to accrue soil N to some extent, soil N does not necessarily reach the original levels after a set number of years25. Some studies at single sites have predicted that it will take 2–40 years for restored and created wetlands to develop soil N levels equivalent to natural wetlands26.As one of the limiting nutrients in terrestrial ecosystems, nitrogen (N) controls primary production in the biosphere and plays a crucial role in the C cycle27. Wetland recovery might be affected by the physical characteristics of wetlands, land-use history, restoration activities, topography, and environmental factors30. It has been suggested that the initial recovery rate may be dependent on the degree of openness of a wetland and its connectivity to external sources, e.g., sediment, organic matter, seeds, and rhizomes16. The hydrologic open systems such as riverine and tidal wetlands, are linked to larger hydrologic regimes through natural flow variation; therefore, external inputs of mineral sediment and organic particles may accelerate soil development10. In contrast, in relatively isolated systems such as organic flat and depressional wetlands, the dominant sources of water are precipitation or groundwater discharge, with minimal external source inputs; thus, their soil development is considered comparably slow. It would also be expected that warm temperatures could prompt soil development after wetland restoration and creation because of accelerated ecosystem processes31, e.g., increased net primary production (NPP) and plant above- and below-ground C pools in wetland ecosystems. Soil texture has been considered a crucial factor affecting the soil C and N content33. Therefore, it is assumed that soil texture also plays an important role in the process of soil C and N recovery. These factors most likely determine the overall efficacy of wetland restoration and creation projects. However, there is little plot-based data on how soil C and N recover across extensive environment conditions .Research investigating the drivers and constraints of wetland recovery is critical for improving restoration techniques and the management of natural wetlands38. However, the average annual surface loss rate between 2004 and 2009 declined by 97% from the previous period (1950s–1970s), which has been attributed to wetland restoration and creation36. Many individual studies have investigated the trajectories and drivers of soil C and N recovery after wetland restoration or creation, whereas less attention has been paid to an overall evaluation of soil C and N dynamics at a regional scale. Therefore, we compiled data from 48 individual studies to identify the general patterns of soil C and N change after wetland restoration and creation in the United States. These results were obtained from studies of the recovery age, hydrogeomorphic type, soil-sampling depth, soil texture and climate at the study sites. The objective of this study was to address the following questions. First, to what extent do soil C and N approach the levels observed in natural wetlands? Second, how do soil C and N change along the age of restoration and creation? Finally, how do recovery approaches (restoration and creation), hydrogeomorphic type, soil sampling depth and other abiotic factors (climate and soil texture) affect soil C and N recovery?Since the 1950s, an extensive area of wetlands was lost due to silviculture, salt water intrusion, agriculture, and urban and rural development in the conterminous United StatesThe changes observed in 14 variables related to soil properties indicated different responses to wetland restoration and creation Fig. . The totB) = 4.96, p < 0.01 for soil C; QB = 9.07, p < 0.001 for soil N; Fig. Among the four wetland age categories, soil C and N generally increased with increasing recovery age; however, soil C and N in restored and created wetlands were still significantly lower, by 51.7% and 50.3%, than in natural wetlands after 11–20 years Fig. . The rec++) of soil C and N in restored wetland was significantly higher than that in created wetland , but no significant difference existed in restored wetlands  of soil C and N exhibited a significant decreasing trend with latitude Fig. . Among tP < 0.05), implying that the relative change in soil N was larger than that of soil C. Consequently, the lnRR of the soil C:N ratio illustrated a significantly negative relationship with recovery age , except the roots in forested wetlands, and mineral and organic matter inputs occur at the soil surface. Soil N storage could be largely derived from atmospheric deposition, biological N fixation and external inputs through hydrology. Furthermore, several created wetlands were amended with organic-rich soil at the surface, potentially accelerating C and N accumulation in the topsoil42. Therefore, the rate of C and N accumulation decreased as the soil depth increased and exhibited no significant change in the deep soil after restoration43. This implied that more time is needed for the full recovery of C and N in the deep soil layers.The magnitude of soil C and N recovery at ≤10 cm depths were larger than at >10 cm soil depths Fig. . This reWetland restoration and creation are increasingly used as strategies to mitigate the loss of wetland function. In this study, it seemed that wetland restoration was more efficient in soil C and N recovery than creation Fig. . The soi16. However, in this study, restored organic flats and created depressional wetlands seemed to recover more rapidly than riverine and tidal wetlands  and enable soils to provide more available N for plant productivity54. Our study also indicated that, relative to the soil C:N ratio in natural wetlands, the C:N ratio decreased gradually over time in restored and created wetlands  AND (creat* or re-creat* or abandon* or restor* or rehabilitat* or succession or “soil development”) AND (carbon or “organic matter” or nitrogen)”. Additional searches were conducted with Google Scholar. We examined the abstract of each result to assess its potential to meet the selection criteria for inclusion in the review. The selection criteria were as follows: the articles must compare measurements of soil C (N) concentration, or C (N) density together with bulk density in restored or created and reference  wetlands; the articles must contain quantitative information about soil sampling depth and recovery age at the point when the study began; when more than one data-set was published at the same site but in different studies, only one study was chosen or the data sets were integrated with one another. Under these criteria, we selected 54 study sites from 48 articles Fig. . The arthttp://www.wordclim.org/) using the latitude and longitude of the study sites. When only data on SOM concentration were reported, the SOM concentration was converted to an SOC concentration by multiplying it by a conversion factor of 0.5856. When data on SOC density or total N density together with bulk density were reported, the SOC concentration or total N concentration was calculated using the following formula:−1) and total N concentration (g kg−1), respectively. The SOCD and TND are the SOC density (Mg ha−1) and total N density (Mg ha−1), respectively. The BD is the soil bulk density (g cm−3) and H is the soil sampling depth (cm).The data were extracted from the main text, tables, and figures of the articles. The following information was included in the compilation: recovery approaches (restoration and creation), hydrogeomorphic type, recovery age, soil-sampling depth, location (longitude and latitude), climate , C pools , N pools , phosphorus (P) pools , and other soil parameters . Summary information about the data is available in Table et al.57, including depressional, riverine, lacustrine, tidal, mineral flat, organic flat, and slope wetlands. Because of the small number of studies on lacustrine, mineral flat and slope wetlands, these types were not analyzed.The constructed database consisted of 409 lines of observations, which were used to explore the general pattern of soil C and N recovery after wetland restoration and creation. To explore the temporal trends in recovery, the recovery ages were categorized into four classes: ≤5, 6–10, 11–20, and >20 years. The soil sampling depths were divided into two groups: ≤10 cm and >10 cm. We also investigated the effects of recovery approach and hydrogeomorphic type on soil C and N recovery. To reduce the influence of recovery age and to include as much data as possible in the analysis, we chose data with recovery ages ≤30 years. The wetlands were classified as “restored” or “created” and grouped into seven hydrogeomorphic classes according to Smith 58. Bootstrapping (9999 iterations) was used to calculate bias-corrected 95% confidence intervals (CIs) around the mean effect size. If the 95% CI of the effect size for a variable did not include zero, the mean value of the variable in the restored and created wetland was considered significantly different from that in the natural wetland. The percentage difference of a variable between the restored and created and natural wetlands was calculated by the following equation: (eRR++ − 1) × 100%, where RR++ is the mean response ratio. To determine whether there were significant differences in the mean response ratio among various categories, we employed randomization tests to calculate the significance level for between-class heterogeneity (QB). The meta-analysis was conducted using the statistical software Meta-Win59. Linear-regression analysis was performed to establish the relationships between lnRR of SOC  and latitude, climate , soil texture (clay + silt), and between lnRR of the SOC and total N, lnRR of the soil C:N ratio and recovery age.In this study, we employed a meta-analysis to determine changes in soil C and N after wetland restoration and creation. Fourteen variables related to soil C and N pools, and other soil physicochemical properties were selected. One common metric of effect size in a meta-analysis is the log response ratio (RR), which is calculated as the ratio of its value in the experimental treatment (Xe) to that in the control treatment (Xc). In our study, Xe is the value of the measured variable in the restored or created wetland, and Xc is the value of the variable in the reference wetland. To improve the statistical performance, the RR was log-transformed such that lnRR = ln (Xe/Xc) = ln (Xe) – ln (Xc). Among the 409 observations in our dataset, only 173 reported standard errors/deviations and sample sizes of the response variables; thus, we used resampling methods for this meta-analysis. Different from the conventional parametric methods, resampling methods do not require computation of the standard errors of the individual effect size estimatessupplementary information


AbstractEcnomiohylatuberculosa is an Amazonian hylid of uncertain phylogenetic position. Herein DNA sequences of mitochondrial and nuclear genes are used to determine its phylogenetic relationships. New sequences and external morphology of Trachycephalustyphonius are also analyzed to assess the status of Ecuadorian and Peruvian populations. The phylogeny shows unequivocally that Ecnomiohylatuberculosa is nested within the genus Tepuihyla, tribe Lophiohylini. This position was unexpected because the remaining species of Ecnomiohyla belong to the tribe Hylini. To solve the paraphyly of the genus Ecnomiohyla, Ecnomiohylatuberculosa is transferred to the genus Tepuihyla. Comparisons of DNA sequences, external morphology, and advertisement calls between populations of Ecnomiohylatuberculosa from Ecuador and Peru indicate that the Peruvian population represents an undescribed species. The new species is described and a species PageBreakaccount is provided for Ecnomiohylatuberculosa. Trachycephalustyphonius is paraphyletic relative to Trachycephaluscunauaru, Trachycephalushadroceps, and Trachycephalusresinifictrix. The phylogenetic position of populations from western Ecuador indicates that they represent a species separate from Trachycephalustyphoniussensu stricto. We resurrect the name Hylaquadrangulum  for those populations. Amazonian populations of “Trachycephalustyphonius” from Ecuador and Peru are genetically and morphologically distinct from Trachycephalustyphoniussensu stricto and are conspecific with the holotype of Hylamacrotis. Therefore, we also resurrect Hylamacrotis, a decision that results in Trachycephalusmacrotiscomb. n. EcnomiohylaFringe-limbed frogs, genus Ecnomiohylatuberculosa  to amplify DNA fragments for mitochondrial genes 12S rRNA (12S), Cytochrome Oxidase sub-unit I (COI), two overlapping fragments for the last ~320 bp of 16S rRNA (16S), NADH dehydrogenase subunit 1 (ND1) and adjacent tRNAs , and the nuclear gene POMC, using the primers listed in PCR amplification was performed under standard protocols and sequenced by the Macrogen Sequencing Team . The combined DNA matrix had up to 3792 bp.DNA was extracted from muscle or liver tissue preserved in 95% ethanol or tissue storage buffer, using standard phenol–chloroform extraction protocols . We usedHylidae, we blasted our Ecnomiohylatuberculosa 12S and ND1 sequences to the GenBank database (blastn procedure). These searches shown unequivocally that the most similar samples belonged PageBreakto the genera Osteocephalus and Tepuihyla, tribe Lophiohylini. For example, a blastn for the ND1 sequence of EcnomiohylatuberculosaQCAZ 53542 retrieved 50 most similar sequences with identity values ranging from 82.7% to 89.1%. All 50 sequences bePageBreaklonged to the Lophiohylini tribe . To confirm the close relationships between Ecnomiohylatuberculosa and Tepuihyla, we carried out an additional exploratory analysis based on Hylini (including Ecnomiohyla) and Lophiohylini from Agalychnis, Litoria and Phyllomedusa as outgroups. The final matrix had 169 terminals. A maximum likelihood phylogenetic analysis using GARLI 2.0 . GenBank sequences were originally published by Phyllomedusinae were included as outgroups . We also added new sequences from 25 individuals of Ecuadorian and Peruvian Trachycephalus to assess their taxonomic status.For final phylogenetic analysis we included GenBank sequences from all available species of Preliminary sequence alignment was done with MAFFT 7.2 software with the L-INS-i algorithm . All seqnpb), configured with the same settings of the full search, but with three replicates per run.Phylogenetic trees were obtained using maximum likelihood and Bayesian inference. Maximum likelihood searches were carried out with software GARLI 2.0 . We madePageBreak6 generations; each run had six chains with a temperature of 0.08. We used TRACER 1.6 (effective sample sizes (ESS) for all model parameters. The search was considered finished when all ESS were > 200. Each run was sampled every 1000 generations. The first 25% of the samples was discarded as “burn-in”, using the remaining samples to estimate the Bayesian tree, posterior probabilities (pp) and other model parameters.Bayesian inference was performed with MARBAYES 3.2.1 . The anaACER 1.6  to assesFundación Herpetológica Gustavo Orcés, Quito (FHGO); Pontificia Universidad Católica del Ecuador, Quito Museo de Zoología at (QCAZ). Peru: Centro de Ornitología y Biodiversidad, Lima (CORBIDI); Colección Referencial de Biodiversidad del Instituto de Investigaciones de la Amazonía Peruana, Iquitos (IIAP). United States of America: American Museum of Natural History, New York, (AMNH); National Museum of Natural History, Washington DC (USNM); Natural History Museum at the University of Kansas, Lawrence (KU). United Kingdom: Natural History Museum, London(BMNH). Venezuela: Museo de Historia Natural La Salle, Caracas (MHNLS). Our comparisons included the revision of the holotype of Hylatuberculosa  and photographs of the holotype of Hylamacrotis . Examined specimens are listed as Suppl. material SVL; (2) head length; (3) head width; (4) tympanum diameter; (5) femur length; (6) tibia length; (7) foot length; and (8) eye diameter. Additionally, we measured the hand length (HAL) from proximal edge of palmar tubercle to tip of third finger for the new species described herein following Diagnostic characters and comparisons are based on specimens from the following institutions. Ecuador: Ecnomiohylatuberculosa (5), Tepuihylashushupe sp. n. (1), Tepuihylaedelcae (17), Tepuihylaobscura (17), and Tepuihylarodriguezi : or shape . We alsoQCAZ 53699 from Juyuintza, Pastaza province, Ecuador  and CORBIDI 12513 from Ere River, Putumayo Basin, Loreto Department, Peru . Male QCAZ 53699 was recorded at night on 22 June 2012 (23.9°C / relative humidity ~86.0%). Male CORBIDI 12513 was recorded at night of 18 October 2012 (21.3°C / relative humidity ~95.8%). Recordings were saved in PCM format, with a sample rate of 48000 Hz and 24-bits. Call variables were measured with RAVEN PRO 1.4 .Calls were analyzed from two adult males,  PRO 1.4 , under a PRO 1.4  were meaPageBreakEcnomiohylatuberculosa within the tribe Lophiohylini, genus TepuihylaEcnomiohylatuberculosa is sister to all species of Tepuihyla, except Tepuihylawarreni from Ayanganna, Guyana. Tepuihylawarreni is polyphyletic because a sample from the Maringma Tepui, Guyana, clusters with Tepuihylaaecii, Tepuihylaedelcae, Tepuihylaobscura, and Tepuihylarodriguezi instead of the other samples of Tepuihylawarreni  is widely paraphyletic. The new sequences of Trachycephalustyphonius from the Ecuadorian Chocó are sister to a clade composed of Trachycephaluscunauaru, Trachycephalushadroceps, Trachycephalusresinifictrix, Trachycephalustyphonius from Guyana, French Guiana, Venezuela, and the upper Amazon basin in Ecuador and Peru. Populations of Trachycephalustyphonius from Amazonian Peru and Ecuador are sister to a clade composed of Trachycephalusresinifictrix and one sample of Trachycephalustyphonius from Venezuela  is similar to the PCA in not showing interspecific differences. Group assignment proportions are all very low (between 0.02–0.31), indicating very little power of morphometric discrimination among groups, even when attempting to maximize differences with species/lineage priors.Morphometric analyses do not show differences in shape among species Fig. . The onlPageBreakEcnomiohylatuberculosa form three well-supported clades (pp/npb = 100). The sample from Ere River, Peru, is sister to the Yasuní + Juyuintza in Ecuador. The Juyuintza samples are sister to each other. All genetic distances reported below are uncorrected distances for the gene 12S. Genetic distances between Ere River, Peru, and the Ecuadorian populations (Juyuintza and Yasuní) range from 5.3 to 5.9%; distances between Yasuní and Juyuintza are both 1.2%. Samples from Juyuintza are identical to each other. The genetic distances between Ere River and the other populations are above distances between several closely related species in Tepuihyla and Osteocephalus .The four samples of PageBreakPageBreaktwo harmonics with similar energy content while the call from Juyuintza has one well-defined harmonic with high energy and a second harmonic with low energy, barely evident in the power spectra  in having a cream iris with red periphery , therefore we assign them to Ecnomiohylatuberculosasensu stricto. The specimen from Rio Ere belongs to an undescribed species that we describe in the following sections. Our species description is based on a single specimen and therefore we cannot characterize the variation in the new species. However, we examined 11 specimens of Ecnomiohylatuberculosa, which allow us to characterize Ecnomiohylatuberculosa morphological variation and show that the single specimen of the new species falls outside the range of variation of Ecnomiohylatuberculosa. Hence, we demonstrate that the new species is, in fact, distinct from Ecnomiohylatuberculosa. Our morphological evidence is corroborated by genetic and bioacoustic characters.The combined evidence from genetics, morphology, and bioacoustics indicates the existence of two species within “Hylidae HylatuberculosaBMNH 1947.2.13.34Ecnomiohylatuberculosa : Faivovich et al. 2005SVL 67.6 mm  SVL in males 79.3–86.2 mm (n = 5), SVL in females 67.6–85.7 mm (n = 2); (2) skin on dorsum coarsely tuberculate, covered by small tubercles with scattered large tubercles; tubercles without keratinized tips; (3) skin on flanks covered by large tubercles; (4) webbing between fingers extensive but without reaching the proximal border of the disks, hand webbing formula I2 —2 II1 —2 III1½—1IV to I2—2+II1—2III2 —1IV  coloration on flanks similar to dorsal coloration except for greenish yellow axilla and groins and whitish gray ventrolateral area; (9) skin on upper surface of head not co-ossified with underlying cranial elements, cranial crest slightly exostosed; (10) in life, bones green; (11) small triangular dermal flaps form serrate fringe along the ventrolateral margin of the forearm and along the outer edge of Finger IV; heels bearing two or three enlarged fleshy conical tubercles surrounded by few smaller round tubercles; small triangular dermal flaps form serrate fringe along ventrolateral margin of tarsus and outer margin of Toe V; (12) adults, in life, have cream iris with a coppery hue and scattered thin black reticulations; (13) vocal sac is white, single and subgular, (14) a juvenile is similar to adults in coloration but with dorsal background coloration brownish cream and a black medial stripe in the iris; (15) larvae unknown.In this section coloration pertains to preserved specimens unless otherwise noted. A large-sized 1IV Fig. ; webbing0+V Fig. ; (5) dorTepuihylatuberculosa differs from all congeneric species (in parenthesis), except Tepuihylashushupe sp. n., in having a larger size: SVL in males 79.3–86.2 mm (n = 5), SVL in females 67.6–85.7 mm (n = 2) (maximum SVL 59.2 mm in other Tepuihyla), having extensive webbing in the hands  and a serrate fringe along the ventrolateral margin of the forearm (absent). Other differences are listed on Table Tepuihylatuberculosa is most similar to Tepuihylashushupe sp. n. It differs from Tepuihylashushupe sp. n. (character states in parenthesis) in having cream iris in life (iris cream with reddish periphery), dorsum covered with small tubercles intermixed with few large tubercles , smooth skin on dorsum and elongate calcar on the heel. The smaller Dendropsophusmarmoratus exhibits a bright yellow belly with black spots or mottling (uniform yellowish-tan in Tepuihylatuberculosa).QCAZ 55423) and cream suffused with a coppery hue  with brownish cream or creamy coppery enlarged tubercles, bearing scattered dark brown reticulations or marks. In all specimens the belly and ventral areas of thighs are lighter than those of the holotype probably PageBreakPageBreakbecause they were collected more recently. Head is rounded in dorsal view, wider than long; the snout is truncate in dorsal view and truncate (e.g. QCAZ 32716) in profile. The largest male has 86.2 mm SVL  and the largest female 85.7 mm ; lips greenish with scattered small dark brown marks; supratympanic fold delineated by a thin brown edge, tympanic membrane lighter than the background; flanks with coloration similar to dorsum; axilla and groin pale orange, ventrolateral region whitish cream, limbs with faint brown transversal bands; dorsal surfaces of hands and feet, including webbing, greenish cream with scattered dark brown marks; discs pale green. Throat, chest and belly are white but some specimens have an orange hue on the belly (e.g. QCAZ 55423); serrate fringes along forearms and tarsus white; webbing, thighs and concealed surfaces of shanks orange. Iris is cream with a light coppery hue bearing some scattered black venations. Juvenile specimen QCAZ 55413 is similar to adult specimens but has a brownish cream dorsum with scattered faint brown blotches  are detailed in Table PageBreakHz. Interestingly, the call of this species is feared by natives in the lower Pastaza basin , because it is commonly confused with the “calling” of the bushmaster Lachesismuta (Squamata: Viperidae). This belief almost certainly incorrect as Lachesismuta cannot vocalize. See also the Etymology section in Ecnomiohylashushupe sp. n. description.Quantitative characteristics of the advertisement call of sa QCAZ 5699 are dTepuihylashushupe sp. n., but its identity requires confirmation. We recorded five additional localities in the Amazon lowlands of Ecuador and two in Peru  and one from Juyuintza , 145 m above sea level, collected by P. J. Venegas and G. Gagliardi-Urrutia on 19 October 2012.Figs , 6, 10C PageBreakPageBreakPageBreakPageBreakPageBreakTepuihyla differing from other species in the genus by the following combination of characters: (1) SVL in males 85.4 mm (n = 1), females unknown; (2) skin on dorsum coarsely tuberculate, covered by small tubercles intermixed with large tubercles; tubercles without keratinized tips; (3) skin on flanks similar to skin on dorsum; (4) webbing between fingers extensive but without reaching the proximal border of disks; hand webbing formula I2—2II1— 2III1½—1+IV  dorTepuihylashushupe differs from all species of the genus (in parenthesis), except Tepuihylatuberculosa, in being larger, having extensive hand webbing  and a serrate fringe along the ventrolateral margin of the forearm (absent). Other differences are listed on Table Tepuihylashushupe is most similar to Tepuihylatuberculosa. It differs from Tepuihylatuberculosa (character states in parenthesis) in having, in life, a cream iris with red periphery (iris cream without red periphery), dorsum covered by small tubercles intermixed with abundant large tubercles ; in preservative, dorsum cream with a brownish mantle (dorsum light cream or cream suffused with a coppery hue) and the posterior border of large dorsal tubercles dark brown (tubercles brownish cream or creamy coppery). In coloration, size, and texture of dorsal skin Tepuihylashushupe resembles the Amazonian Trachycephalusresinifictrix and Trachycephaluscunauaru. It can be distinguished from both species by the presence of serrate fringes on the limbs and tubercles in the lower jaw  and the iris having red periphery and lacking a vertical black bar below the pupil .CORBIDI 12513), 85.4 mm SVL, head length 26.6, head width 28.9, eye diameter 7.7, tympanum diameter 5.5, femur length 35.9, tibia length 46.9, foot length 35.9. Head rounded in dorsal view, wider PageBreakthan long (HL/HW = 92%); snout truncate in dorsal view and slightly protruding in profile; nostrils are directed laterally, near tip of the snout; top of head tuberculate; canthus rostralis concave in dorsal view, straight in profile; skin on upper surface of head not co-ossified with underlying cranial elements, cranial crests slightly exostosed; loreal region concave; skin on dorsal surface of head, body and limbs coarsely tuberculate, tubercles are either small or large and lack keratinous spines; lower eyelid pigmented; distinct supratympanic fold formed by a row of round tubercles running from midpoint of posterior margin of eye; tympanum prominent, opaque, smooth, 71% of eye diameter, separated from eye by 3.8 mm; small triangular dermal flaps form serrate fringe along the ventrolateral margin of the forearm and the outer edge of Finger IV, fringe more conspicuous on forearm that on finger; hands moderate in length (HAL/SVL = 31.2%); Finger lengths I < II < IV < III, terminal disk on Finger I 63.2% of diameter of disks on Fingers II–IV; diameter of Fingers II-IV ~1.2 times tympanum diameter; distal subarticular tubercles on Fingers I–III large and rounded, on Finger IV larger and bifid; supernumerary tubercles indistinct; prepollical tubercle large, obtuse and elliptical; bony prepollical projection absent; keratinous nuptial pad with dark epidermal projections covering the surface of the thumb up to the distal edge of its subarticular tubercle; fingers considerably webbed although web does not reach base of disks; webbing formula I2—2II1— 2III1½—1¼IV; legs relatively long and slender (tibia length 54.8% of SVL), thigh length 35.9 mm; heels bearing two enlarged conical tubercles surrounded by few lower round tubercles; fleshy, dermal flaps form serrate fringe along ventrolateral margin of tarsus and outer margin of Toe V extending to base of disk, scallops are deeply incised, largest on tarsus, smaller along toe; outer metatarsal tubercle small, inner metatarsal tubercle moderately large, ovoid, and flat; toe lengths I < II < III = V < IV; disks on toes ~70% of diameter of disks on fingers, equal between Toes III–V; subarticular tubercles round; supernumerary tubercles present, ill defined; webbing in toes extensive, reaching the base of disks on at least three toes; webbing formula I1—1II1—1¼III¾—1IV1— ¾V; gular skin smooth with small pointed tubercles along the jaw , venter finely granulate, weak granulation on undersides of arms and finely on ventral surfaces of thighs, smooth skin on anterior surfaces of thighs and ventral surfaces of legs; cloacal opening directed posteriorly at upper-level of thighs, some indistinct small tubercles on the upper edge of vent, skin under the vent covered by small flattened but conspicuous tubercles with two enlarged prominent fleshy tubercles posterolateral to the vent (one on each side); tongue cordiform; vomerine ridges transverse, oblique in the middle, narrowly separated medially, placed between the posterior margins of the large subrectangular choanae; vomerine teeth 6 to 11; vocal slits present.Adult male  are detailed in Table PageBreakand note rate increases markedly along the first half of the call, decreasing again at the end. The call has two harmonics, the first harmonic has slightly more energy than the second . The dominant frequency of the entire call is 515.6 Hz. Midway through the call, nearly 25% of the notes have the greatest energy in the second harmonic (range 1016.6 to 1210 Hz).Quantitative characteristics of the advertisement call ond Fig. . FundameTepuihylashushupe is only known from the type locality in the headwaters of rivers Ere and Campuya, at an elevation of 145 m, PageBreakin the Putumayo river basin near the boundary between Peru and Colombia. According with Oenocarpusbataua; the soil varies between sandy and clayey; most streams have a muddy bottom, few have gravel and sand, and one has big cobbles.Corethrella midges). We recorded its call and immediately made playbacks. The male answered by calling quickly and perching on the hole entrance. We detected at least six individuals during 18 hours of visual encounter surveys  in areas nearby where the holotype was found (primary forest). All individuals were detected by their advertisement calls and none could be collected. No females, amplectant pairs, clutches, or tadpoles have been observed.The holotype was calling at the base of a big tree inside a narrow hole, 150 cm above the ground. The hole had 30 cm of height and had water accumulated. The frog had most of its body submerged Fig.  and the shushupe is used by native people to refer to the bushmaster Lachesismuta (Squamata: Viperidae), the largest viper in the Americas. Our field assistants in Ere river, Alpahuayo Mishana (Peru) and Juyuintza (Ecuador) believed that the advertisement calls of Tepuihylashushupe and Tepuihylatuberculosa were produced by Lachesismuta. The belief that Lachesismuta can sing seems to be widespread among hunters, colonists, and indigenous people from the Amazon basin  from Rio Querari  reported as Ecnomiohylatuberculosa by Tepuihylashushupe, but a taxonomic validation is needed to confirm its identity.The juvenile specimen  and French Guiana (163MC), Clade C in Figure Trachycephalustyphoniussensu stricto. Two linages from Ecuador and Peru are distinct from Clade C and each has an available name. Below we discuss their status.Several authors have discussed the considerable morphological variation in yphonius  and haveyphonius . The parius Fig.  also sugsynonyms . HoweverTrachycephaluscunauaru, Trachycephalushadroceps, Trachycephalusresinifictrix and Trachycephalustyphonius. Their phylogenetic position demonstrates that they are not conspecific with Trachycephalustyphoniussensu stricto or other species of Trachycephalus. One binomial available for Trachycephalus from western Ecuador is HylaquadrangulumHylaquadrangulum under Trachycephaluscoriaceus stating that the holotype is “indistinguishable from Phrynohyascoriacea”. He also questioned its type locality because “several species contained in the Fraser collections supposedly from western Ecuador have been found subsequently only in the Amazon Basin”. Our examination of a large series of specimens from western Ecuador and Trachycephaluscoriaceus from the Amazon basin show unequivocally that the holotype of Hylaquadrangulum . Moreover, the holotype lacks the dark blue blotch above the arm characteristic of Trachycephaluscoriaceus. We conclude that the type locality is correct and Hylaquadrangulum is a valid species. The resurrection of HylaquadrangulumTrachycephalusquadrangulum , Hylapaenulata Brocchi 1879 , and HylanigropunctataTrachycephalusquadrangulum is restricted to the Chocó region. The phylogeny is consistent with a general biogeographic pattern showing that humid lowlands east and west from the Andes do not share amphibian species as result of the vicariant effect of the Andes  shows the clear iris with dark reticulations, also characteristic of populations from French Guyana  from the Chocó region of Ecuador. However, in the phylogeny it is nested within Trachycephaluscunauaru from Amazonian Ecuador. Both the identification and locality are incongruent with its phylogenetic position suggesting that the tissue actually belongs to Trachycephaluscunauaru from the Amazon region. The same error applies to GenBank sequence DQ347027, a sample that was obtained from the pet market. GenBank sequence JQ868532 (field number PS013) is identified as Trachycephalusvenulosus . The specimen is likely lost and its correct identification appears to be Trachycephalusresinifictrix. Despite these potential identification errors, our taxonomic conclusions are only dependent on the correct identifications of the samples PageBreakof Trachycephalustyphonius from Guyana and French Guyana, which are reliable. We obtained photographs of populations from French Guyana , have green discs on the fingers, and a dark brown iris  occur in Central and North America; only Ecnomiohylaphantasmagoria and three species of Smilisca are present in South America and none of them occur in the Amazon basin (most recent common ancestor (MRCA) of Hylini inhabited either Central América or North America while the MRCA of Ecnomiohyla inhabited Central America. Therefore, the assignment of “Hylatuberculosa” to the genus Ecnomiohyla was at odds with the distribution of the rest of the tribe. The tribe Lophiohylini, on the other hand, is primarily distributed in South America. The MRCA of Tepuihyla probably occurred in the Guyana region, the MRCA of Osteocephalus in the Amazon basin and that of Tepuihyla + Osteocephalus + Dryaderces in Guyana-Amazon or Amazon basin exclusively (Tepuihylatuberculosa reported hare imply a more parsimonious biogeographic scenario because they do not require a PageBreakrecent dispersal event between Central America and the Amazon Basin. Further analyses with a more complete taxon sampling are needed to more confidently infer the geographic origin of Tepuihyla.The phylogenetic position of on basin . Accordilusively . The phy


Hypereutectic Al–Si (20 wt.%) alloy parts were fabricated by employing a powder injection moulding (PIM) technique with a developed multi-component binder system composed of high-density polyethylene (35 wt.%), carnauba wax (62 wt.%) and stearic acid (3 wt.%). The feedstocks contained 83 wt.% metal powders. The debinding process was carried out by a combination of solvent extraction and thermal decomposition. The effects of solvent debinding variables such as kind of solvents, debinding temperatures and time, and the bulk surface area to volume ratios on the debinding process were investigated. Thermal debinding and the subsequent sintering process were carried out in a heating sequence under a nitrogen atmosphere. The influences of sintering temperature and sintering time on the mechanical properties and structure were considered. Under the optimal sintering condition, sintering at 550 °C for 3 h, the final sintering parts were free of distortion and exhibited good mechanical properties. Relative sintered density, Brinell hardness, and tensile strength were ~95.5%, 58 HBW and ~154, respectively.  Hypereutectic Al–Si alloys and their composites attract much attention for heat dissipation and electronic packaging applications due to their low density, high wear resistance, low thermal expansion coefficient and excellent thermal conductivity ,2. As elPowder injection moulding (PIM) is a net shape manufacturing technology, which can mass-produce complex, precision, net-shape metal, ceramic or composite materials ,7 and isThere are several methods to remove the binders from the moulded parts, such as catalyst , wickingThe parts are sintered following the debinding process. Appropriate sintering conditions improve the structures of the sintered parts and ensure good mechanical properties ,15. SintTo satisfy the miniaturization, integration and mass production of electronic packaging materials, we developed an injection moulding process for hypereutectic Al–Si alloy. The objectives of the present study were to investigate the influences of solvent debinding variables on debinding processes and the effects of sintering parameters on the microstructure and properties of sintered parts. It was anticipated that the appropriate PIM process could expand the application of hypereutectic Al–Si alloy.D10, D50 and D90 of 2.93, 5.73 and 9.76 µm, respectively. Minor amounts of pure Mg and Sn powders were used as sintering aids. The binder system consisted of 35 wt. % high density polyethylene (HDPE), 62 wt.% carnauba wax (CW), and 3 wt.% stearic acid (SA). The feedstock was prepared with a powder loading of 83 wt.%  based on our previous research.The morphology of Al–Si (20 wt.%) alloy powder and its granular size distribution, as determined using laser granulometry , is shown in −1 respectively.The feedstock was prepared using the hot solvent mixing method. The pre-mixed binder ingredients were added into a flask with xylene at 110 °C and stirred. After all the ingredients were dissolved completely, solid powder was added gradually to achieve the desired powder loading and continually stirred for 1 h. Finally, the xylene was distilled off and homogeneous feedstock was acquired. The injection moulding process was conducted using a reciprocating-screw type injection moulding machine . The green parts with three different shapes, shown in The elimination of binder was performed in two steps: solvent debinding to extract most of the soluble components (CW and SA) and thermal debinding to remove residual binders (HDPE and the little CW and/or SA). In the solvent debinding process, the green parts were immersed into organic solvents  for 1 to 23 h at different temperatures . The ratio of solvent to specimens was 10 mL/g. The parts were taken out from the solvents and vacuum-dried to evaporate the solvents after a predetermined interval to evaluate mass loss. In addition, to investigate the concentration effects of soluble components in the solvent, an experiment was performed in which the solvent was refreshed at predetermined intervals. Thermal debinding and sintering were performed in a tube furnace, which was filled with high-purity nitrogen and the gas flow was maintained at ~0.5 L/min. The heating sequence was established based on the thermal properties of the binder and its ingredients, as well as those of the Al–Si (20 wt.%) alloy powders. The effects of sintering temperature and sintering time on the properties of sintered parts were investigated.The microstructure was examined using a scanning electron microscope  and an optical microscope . Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were performed using a thermal analyzer  and a simultaneous thermal analysis system , respectively, both at a heating rate of 10 min/°C in nitrogen. The density of sintered parts was measured using the Archimedes method. Tensile testing was conducted using an Instron 5969 machine  with a crosshead speed of 0.5 mm/min, utilizing specimens with a polished surface. The Brinell Hardness (HBW) was measured on a polished surface of the sintered parts, which were encapsulated in epoxy resin, using a 2.5 mm steel ball indenter and the load of 62.5 kgf. The phases of alloy powders and sintered parts were detected by X-ray diffraction  using a monochromatic target of Cu-Kα.The solvent debinding process of specimens in different solvents  at 60 °C is shown in The solvent debinded part, C, was chosen on which to perform the subsequent thermal debinding and sintering process. After solvent debinding, ~93 wt. % of the soluble ingredients were extracted. During the thermal debinding process, the remaining binders need to be removed. The thermal properties of binder and Al–Si (20 wt.%) alloy powders are shown in DTA curves of Al–Si (20 wt.%) alloy powders heated from ambient temperature to 750 °C at a heating rate of 10 °C/min in nitrogen are shown in The properties of sintered parts as a function of sintering temperature are shown in The effects of sintering time on relative sintered density, hardness and tensile strength are shown in 2Si.Images of the green parts A and B, and their solvent debinded parts and sintered parts (sintered in nitrogen at 550 °C for 3 h) are shown in Hypereutectic Al–Si (20 wt.%) alloy parts were manufactured by powder injection moulding (PIM). Micron-sized hypereutectic Al–Si (20 wt.%) powder and a multi-component binder system consisting of HDPE, CW and SA were used to prepare the feedstocks. In the solvent debinding process, the influences of the kinds of solvents, debinding temperatures and time, and the bulk surface area to volume ratios on debinding rates were determined. Debinding in xylene had greater debinding efficiency than in hexane and heptane. The increment of debinding temperatures and the bulk surface area to volume ratios obviously decreased debinding time. Sintering temperature and time had a large influence on the microstructure and mechanical properties of the sintered parts. The final parts sintered at 550 °C for 3 h achieved a homogeneous microstructure and high-densification. The relative sintered density, Brinell hardness and tensile strength were ~95.5%, ~58 HBW and ~154 MPa, respectively.


As coprocessed excipients (CPE) gain a lot of focus recently, this article compares three commercially available CPE of Avicel brand, namely, CE 15, DG, and HFE 102. Comparison is based on measured physical properties of coprocessed mixtures, respectively, flow properties, pycnometric density, mean particle size, specific surface area, moisture content, hygroscopicity, solubility, pH leaching, electrostatic charge, SEM images, and DSC. Tablets were made employing three pressure sets. Viscoelastic properties and ejection force were assessed during compression, as well as pycnometric density, mass uniformity, height, tensile strength, friability, disintegration, and wetting times. Avicel CE 15 is of mid-range flow properties, contains mid-size and nonspherical particles, and has high hygroscopicity, growing negative charge, best lubricity, lowest tensile strength, and mid-long disintegration times. Avicel DG possesses the worst flow properties, small asymmetrical particles, lowest hygroscopicity, stable charge, intermediate lubricity, and tensile strength and exhibits fast disintegration of tablets. Finally, Avicel HFE 102 has the best flow properties, large symmetrical particles, and middle hygroscopicity and its charge fluctuates throughout blending. It also exhibits inferior lubricity, the highest tensile strength, and slow disintegration of tablets. Generally, it is impossible to select the best CPE, as their different properties fit versatile needs of countless manufacturers and final products. Tablets are the most frequent dosage form due to their advantages, for example, availability, easy administration, good stability, and low price. The easiest technology of production is still direct compression (DC), but this method requires overcoming many complications. These mostly include a problematic content and mass uniformity and low mechanical resistance. “Coprocessed excipients” (CPE), containing commonly processed blends of fillers, binders, disintegrants, lubricants, and other excipients, are nowadays more and more used and may help to overcome some disadvantages of DC. Using physically and structurally uniform mixture (tableting blend) of usually porous particles with suitable properties and defined particle size distribution enhances flowability, dilution potential, content uniformity, and tabletability, while reducing lubricant sensitivity and enabling better processability of different active pharmaceutical ingredients (API) into dosage forms , 2. ResuCPE are produced by different technologies of which the spray drying (SpD) is the most widely used. SpD forms highly porous particles with good flowability and tabletability ensuring short time of disintegration in the physiological fluids , 7, 8. UThe aim of this work was a comparison of three commercially available binary mixtures of CPE produced by SpD technology. These are Avicel CE 15, Avicel DG, and Avicel HFE 102  with different content of microcrystalline cellulose (MCC) and 15% of guar gum, 25% of dicalcium phosphate (DCPA), and 10% of mannitol, respectively. Contemporary data information coming from a few scientific articles and producer do not provide whole-proof compatibility because of different used methodology . TherefoThe aim of this work was to evaluate the properties of individual CPE. Since the specific properties of these individual excipients can be selectively overlapped by the addition of another substance according to their nature, no model drugs have been added. Obtained data may help producers to choose suitable excipients for tablet formulation and to find out dependency between composition and tablet behavior in oral cavity and/or gastrointestinal tract.Avicel CE 15, Avicel DG, and Avicel HFE 102 were received as a kind gift from FMC Health Nutrition, USA.The flow through the orifice of the CPE was measured on a flowability tester  according to Ph. Eur. with an orifice diameter of 15 mm. Measurements were performed in triplicate and the results are expressed as mean values ± standard deviation.α, according to Ph. Eur. Measurements were performed in triplicate and the results are expressed as mean values ± standard deviation.Angle of repose was determined by measuring the height of the cone of powder (100 g) and calculating the angle of repose, Angle of slide was tested with powder sample (10 g) which was placed on one end of a metal plate with a polished surface. This end was gradually raised until the plate with the horizontal surface formed an angle at which the sample was about to slide . MeasureBulk and tapped volumes were evaluated in a tapped density tester  and subsequently used to calculate bulk and tapped densities, Hausner ratio (HR), and Carr compressibility index (CI) according to Ph. Eur.True density of the CPE was determined by the gas displacement technique using the helium pycnometer , according to Ph. Eur. All density measurements were performed in triplicate and the results are expressed as mean values ± standard deviation. Porosity of CPE was calculated according to the following equation:Particle size distribution was evaluated by a sieve analysis using a set of stainless steel sieves with apertures ranging from 0.025 to 0.800 mm placed on a vibratory sieve shaker . The percentage weight of tablets retained on each of the sieves was determined.D10, D50, and D90 are the diameters of sample at the 10th, 50th, and 90th percentiles of the cumulative percent undersize plot. Measurements were performed in duplicate and the results are expressed as mean values ± standard deviation.Particle size was determined by laser diffraction of dry samples . 2 as the area occupied by one nitrogen molecule. Powdered titan oxide (SSA = 13.76 m2/g) was used as standard. Measurements were performed in triplicate and the results are expressed as mean values ± standard deviation.The specific surface area was determined by nitrogen adsorption . Samples were degassed at 200°C in vacuum for 24 h before the measurement. The specific surface area was obtained from the Brunauer-Emmett-Teller (BET) model  using 0.−1. Measurements were performed in duplicate and the results are expressed as mean values ± standard deviation.The percentage of moisture content in the coprocessed excipients was assayed in a halogen moisture analyzer  under the following conditions: standard drying program, drying temperature 105°C, and switch-off criterion 1 mg·50 sThe hygroscopicity was measured in constant climate chamber  under the following conditions: temperature of 40°C, humidity of 75% RV, and duration of 30 days. 3 grams of the samples were examined in 0.25, 0.5, 1, 3, 5, 8, 24, 72, 120, 168, and 720 hours in a halogen moisture analyzer . Measurements were performed in duplicate and the results are expressed as mean values ± standard deviation.The percentage of soluble fraction of CPE was determined. The first phase was to evaluate the insoluble fraction at physiological amount of saliva (15 mL) in mouth to simulate oral cavity. One gram of the sample was dried in hot air dryer  at 60°C for 4 hours. After drying it was weighed and dissolved in 15 mL of artificial saliva by stirring (600 rounds per minute) in mechanical stirrer  for 3 min. Then it was filtered through a filter paper dried in hot air dryer at 60°C for 4 hours. The filter paper with the captured sample was dried again in hot air dryer at 60°C for 4 hours. The percentage of undissolved fraction was calculated from the weight difference of filter paper. Measurements were performed in duplicate and the results are expressed as mean values ± standard deviation.The second phase was to determine the total soluble fraction. One gram of the sample was dried in hot air oven  and dissolved in 900 mL of artificial saliva by stirring (600 rounds per minute) in mechanical stirrer for 24 hours. Artificial saliva was prepared according to the formula as observed in the study Hobbs et al. . The folpH leaching was determined as pH measurement of 2% solution. The water for measurement was degassed by 1 min of boiling. The pH of solution of CPE was measured using a surface pH microelectrode connected to a pH meter .To evaluate the charge density, 25 g of excipient was blended by a blender  at 40 rpm in a glass container of 2 L volume. After 0, 5, 10, and 20 minutes of blending, excipient was transferred to Faraday pail  connected to calibrated charge measurement unit . Exact transferred mass of excipient was weighted afterwards. Charge density was obtained by dividing the net charge by the mass of excipient. Measurements were performed in duplicate and the results are expressed as mean values ± standard deviation.The surface morphology of the CPE and compressed tablets was examined by SEM. The samples were attached to aluminium stubs with double side adhesive carbon tape and then gold coated with a sputter coater  and examined using a scanning electron microscope . The signals of the samples were produced by backscattered electrons (BSE), at 15 kV voltage and different magnifications.−1 using indium and zinc standard. The heat flow rate was set at 10°C·min−1 and inert nitrogen atmosphere (3.5 Bar) was employed. Approximately 5 mg of every sample was weighed in vented aluminium pan with crimp-on lid. All samples were analyzed over the temperature range 50–250°C except the ones containing guar gum due to early decomposition onset.DSC experiments were performed using testing machine . The heating rate and heat flow were calibrated at 10°C·min−1. 55 tablets were compacted at each compression pressure from each material without any glidants or lubricants. Tablets were stored in a polyethylene bags for 24 hours before testing.Material testing machine  with the compaction punches and die  was used to compress tablets with the cylindrical shape having a mass of 200 mg and the diameter of 7 mm. The used compression pressures were 78, 130, and 182 MPa and the compression rate was 0.5 mm·sE1 (J), the energy consumed for plastic deformation E2 (J), and the elastic energy released during decompression E3 (J) [The force-displacement record was used to evaluate the energetic parameters. During the compression, the computer program testXpert V. 9.01 recorded the energy consumed for friction n E3 (J) . The abon E3 (J) : (2)PL=1−1. The computer program testXpert V. 9.01 recorded the maximal ejection force for 10 tablets from each material. The results of 10 measurements are expressed as mean values ± standard deviation.The ejection force was measured using material testing machine . After the compression, the lower punch was removed and the test was started. The ejection rate was 10 mm·min20 randomly selected tablets from each sample were weighed individually on an analytical balance . The results of 20 measurements are expressed as mean values ± standard deviation.The density of tablets was measured using the gas displacement technique with a helium pycnometer . A completely dry and accurately weighed test cell was filled with the tablets and weighed again. The test cell containing the sample was sealed in the pycnometer and analysis commenced. Measurements were performed ten times and the results are expressed as mean values ± standard deviation.F (N) is the crushing force, d (mm) is the diameter of tablet, and h (mm) is the height of tablet. The results of 10 measurements are expressed as mean values ± standard deviation.The crushing force, diameter, and height of tablets were measured in 10 tablets using the hardness tester . The tensile strength of tablets was calculated using the following equation :(3)TS=2∗V (cm−3) is the tablet volume, r (cm) is tablet radius, and h (cm) is the height of tablet.Vt (cm3) is the volume of tablet and Vb (cm3) is the bulk volume of the powder.The calculation of consolidation behavior of powders was based on the tablet volume (see ) which wApproximately 6.5 g of dedusted tablets was weighed precisely using an analytical balance  and placed into the plastic drum of a tablet friability tester  and rotated for 4 minutes at 25 rpm in compliance with Ph. Eur. Thereafter, the dust was removed and tablets were reweighed. The loss of mass in percent for each tablets' sample was calculated.The disintegration test was performed on six tablets from each sample at 37.0 ± 2.0°C in distilled water using a disintegration test apparatus . The tablets were considered completely disintegrated when no residue remained in the basket. Measurements were performed six times and the results are expressed as mean values ± standard deviation.T1) and the time necessary for complete wetting of the tablet  by the color solution were noted. The weight of tablet in the dry state was noted as m0. The wetted tablet was carefully removed and reweighed using the analytical balance (m1). The water absorption ratio (WA) was calculated using the following equation:Wetting time and water absorption ratio of tablets were determined in a Petri dish using a sponge (5 × 5 cm), impregnated with fifteen grams of water containing a water-soluble blue color for easier identification of complete wetting. The tested tablet was carefully placed on the surface of the impregnated sponge in the Petri dish at the laboratory temperature. The time required to reach the upper surface of the tablet . This energy may be associated with rearrangement, size, and shape of particles and should be as small as possible in favour of energy E2 (energy consumed for plastic deformation) [E1. Particles of Avicel DG have the most irregular shape; however, they are also the smallest ones of used excipients. As a result, the hooking occurs in lesser extent and the energy E1 is smaller than in the case of Avicel HFE 102.Values of energetic parameters rmation) . The lowEnergy E2 represents the amount of energy used for the plastic deformation of particles and friction between the particles and die wall [E2 parameter were measured for Avicel DG and Avicel HFE 102. However, there is no significant difference between these two CPE. They both contain MCC and mannitol or DCPA. DCPA is considered as a brittle material with high fragmentation of particles during compression [die wall . The higdie wall  as well die wall  or mechadie wall , 45. Nevdie wall , granulepression . The compression ; howeverParameter E3 expresses the elastic energy of the compressed material. It is the energy released after compression [E3 parameter can be observed among used excipients. The highest values were measured for Avicel CE 15. This can be caused by good viscoelastic properties of MCC and guar gum. Avicel DG and Avicel HFE 102 have almost the same values of this parameter. Lower E3 than Avicel CE 15 can be given by the presence of mannitol or DCPA, which have poor ability to have elastic deformation [pression , 49. Onlormation , 51.Plasticity (Pl) is calculated according to Stamm and Mathis [d Mathis  and descE2 and hence higher plasticity Pl. Lower plasticity in Avicel DG at higher compression pressures can be also explained by lower E2 values.The values of Pl at compression pressure of 78 MPa decrease in order Avicel DG > Avicel HFE 102 > Avicel CE 15 . HoweverEjection force is the force developed by upper punch required to eject the tablet from the die. It can be affected by several factors, for example, friction between particles and die wall or ejection speed [on speed . For allon speed . In theion speed . Mannitoon speed , 56. Theon speed . The cap uniformity of mass was done only for the control of similar compaction condition, because each tablet was prepared separately using the Zwick/Roell T1-FRO 50 machine without the automatic filling of the die from the hopper. Therefore, deterioration of the mass uniformity of tablets prepared from Avicel CE 15 and Avicel DG without the use of lubricants  can be expected considering their inferior flow properties  of Avicel CE 15 indicate the greatest elastic recovery and hence the highest tablets. On the other hand, Avicel DG and Avicel HFE 102 indicate lower values of E3 and, therefore, lower tablets were obtained.The evaluation ofThe highest percentage loss of volume during compression was observed in Avicel DG tablets . This Av tensile strength of tablet should lie within the range of 0.56 and 1.12 MPa. However, each material has its unique characteristics and therefore even tablets having tensile strength lower than 0.56 MPa or higher than 1.12 MPa can have appropriate properties for manufacturing, handling, and application to patients as was proved in several studies [Tablet hardness serves both as a criterion to guide product development and as a quality-control specification. According to the scientific literature , 61, the studies –64.Tensile strength of all tablets increases with increasing compression pressure as can be seen in The lowest tensile strength was observed in tablets containing Avicel CE 15. This finding is consistent with results obtained in the study of Inghelbrecht and Remon . The addTablets containing Avicel DG and Avicel HFE 102 imply higher values of tensile strength  in compa friability are shown in The measured values of disintegration times of all prepared tablets are shown in TheThe disintegration rate was in order of Avicel HFE 102 > Avicel CE 15 > Avicel DG. The only exceptions were tablets containing Avicel DG prepared using the pressure of 182 MPa, which do not disintegrate within 60 minutes and, therefore, do not meet the compendial requirements for disintegration of uncoated tablets. On the other hand, tablets compressed at lower pressures imply very fast disintegration within 2 minutes and may be described as fast disintegrating tablets. This could be explained by the presence of DCPA, which allows rapid and complete penetration of the liquid into the tablets due to its hydrophilic nature and the high porosity of the tablets. Despite the fast and complete water penetration, tablets containing only DCPA do not disintegrate because the excipient is relatively insoluble in water . Neverth wetting time is closely related to the inner structure of the tablets and to the hydrophilicity of the excipients used. It has also been observed that wetting time often increases with an increase in compression pressure, smaller pore sizes, and decrease in porosity [ water absorption ratio, are presented in It is already known thatporosity . The resAll tablets containing Avicel CE 15 show longest wetting times and highest values of WA in comparison with other formulations. The longer wetting times were observed with increasing compression pressure, while the values of water absorption ratio were similar in all samples. This finding can be explained by the fast water wicking rate of MCC  and swelThe evaluation of wetting time revealed that tablets containing Avicel DG exhibit very fast wetting times. The wetting times are also prolonged with increasing compression pressure due to decreasing porosity of tablets. This observation can be explained by the presence of dicalcium phosphate, which allows rapid and complete penetration of the liquid into the tablet due to its hydrophilic nature and the high porosity of the tablets  as has bThe same dependence of the compression pressure on the wetting time was observed in Avicel HFE 102 tablets. The results also correspond to the prolonged disintegration time and increasing hardness of these tablets. Kalia et al.  comparedThree widely used types of Avicel, based on the spray dried coprocessed mixtures, and the compressed tablets without the addition of lubricant were thoroughly examined for the physical characteristics. Avicel CE 15 has mid-range flow properties and has mid-sized and middle asymmetrical particles, high hygroscopicity, growing negative charge, best lubricity, lowest tensile strength, and mid-long disintegration times. It also has a superior organoleptic performance in chewable tablets. In comparison with other binders, guar gum provides good sensory experience and perceived taste and is not suitable for pH sensitive substances. Avicel DG possesses the worst flow properties, smallest and most asymmetrical particles, low hygroscopicity, stable charge, intermediate lubricity, and tensile strength and exhibits fast disintegration of tablets. It is suitable for dry granulation process of water sensitive substances. Avicel HFE 102 has the best flow properties, large and most symmetrical particles, and middle hygroscopicity and its charge fluctuates throughout blending. It also exhibits the worst lubricity, the highest tensile strength, and slow disintegration of tablets. It offers improvements in flow properties in combination with poorly flowing substances. All types are dedicated for per oral, nonorodispersible administration.


The findings from this work help us to better understand and develop new technical schemes for improving the energy efficiency of WWTPs by repurposing the stream of carbon substrates across systems.Energy neutrality and reduction of carbon emissions are significant challenges to the enhanced sustainability of wastewater treatment plants (WWTPs). Harvesting energy from wastewater carbonaceous substrates can offset energy demands and enable net power generation; yet, there is limited research about how carbonaceous substrates influence energy and carbon implications of WWTPs with integrated energy recovery at systems-level. Consequently, this research uses biokinetics modelling and life cycle assessment philology to explore this notion, by tracing and assessing the quantitative flows of energy embodied or captured, and by exploring the carbon footprint throughout an energy-intensive activated sludge process with integrated energy recovery facilities. The results indicate that energy use and carbon footprint per cubic meter of wastewater treated, varies markedly with the carbon substrate. Compared with systems driven with proteins, carbohydrates or other short-chain fatty acids, systems fed with acetic acid realized energy neutrality with maximal net gain of power from methane combustion (0.198 kWh) and incineration of residual biosolids (0.153 kWh); and also achieved a negative carbon footprint (72.6 g CO As estimated recently, WWTPs in the United States consume ~15 GW annually4, and 50–70% of the electricity consumption is as a result of the aeration processes7, adding an unintended burden that increases energy use and carbon footprints. Yet, carbonaceous substrates could be converted to biomass, and ultimately utilized for power production9. There is therefore a need to better understand the potentials of energy neutrality and carbon reduction in aerobic systems such as activated sludge processes (ASPs); and subsequently, to develop strategies to promote a sustainable reform in the role of WWTPs, from carbon-degradation oriented to carbon-recovery intensive infrastructures10.Energy use and carbon footprints are acknowledged global-scale concerns associated with resource scarcity and climate change. Wastewater treatment plants (WWTPs) can cause an adverse effect because the aerobic removal of carbonaceous organic matters from the waste stream is energy intensive and is also highly related with airborne pollutants such as greenhouse gases11. It was also observed in previous studies that the conversion of short-chain fatty acids (SCFAs) in the aeration processes was dominated by substrate storage and then polyhydroxyalkanoate (PHA) synthesis, rather than aerobic degradation to CO2, thereby promoting energy reduction in the water processing line12. Actually, the carbon sources present in wastewater cover a broad range of macro- and micro-molecules that include proteins, carbohydrates, and SCFAs, among others13. Moreover, sufficient concentrations of carbon substrates in ASPs are needed to enable microbial growth, and the metabolic pathways may vary among different microorganism; thus, this situation is complex. Consequently, little of the literature delivers extended information on the effect and metabolic characteristics of carbonaceous substrates, in relation to the energy and carbon footprints associated with ASPs.Recently, the type of carbon substrate is recognized as one significant factor that affects the energy depletion and harvesting of traditional ASPs17. They provide a useful tool kit for gaining an in-depth understanding of the microbe-mediated processes and strongly support the design, implementation and optimization of the systems. Even so, little effort has been devoted to model and predict the role and behavior of carbon substrates within the context of energy and carbon implications in WWTPs.Moreover, mathematical models have been widely applied to describe, predict, and evaluate the performance of an expanded range of wastewater treatment alternativesAccordingly, this work aimed at providing fundamental information for further efforts on optimizing the current wide-applied technologies such ASPs, by altering and repurposing the influent stream of carbonaceous substrates at the system-levels, and eventually decreasing the expense of upgrading WWTPs with improved sustainability. Thus, it is not surprising that a biokinetics modeling approach was applied initially to describe the aerobically metabolic characteristics of several representative pure forms of carbonaceous source  rather than a complex mixture of different substrates. Afterwards, the calibrated biokinetics data were used to simulate and forecast the transformation of the carbon substrates in a typical ASP. The detailed flows of energy consumed and recovered, and the carbon footprint of the entire treatment system with integrated energy recovery facilities, were then calculated, visualized and explained applying the philosophy of life cycle assessment (LCA). Overall, this work was intended to inform researchers, design practitioners, utility managers, and planners on how the carbonaceous substrate alters the energy use and carbon footprints of wastewater solutions; and on the associated implications for energy recovery practices.18. As the parameters, i.e., substrate adsorption rate (kads), half saturation constant for oxygen (KO), half saturation constant for readily biodegradable substrates (KS), endogenous respiration rate of active heterotrophic organisms (bH), endogenous respiration rate of storage substances (bSTO) and fraction of inert particulate matter (fi) showed low variability in aerobic systems19, their values reported in the literature were directly adopted in this work. Figure XSTO) deficit during the experiments. Though the model used herein to describe the production and consumption profiles of storage substance by starch and protein is relatively robust and rigorous amongst previously reported models, further research efforts are still needed to optimize the model and enhance the capability to trace complicated intermediates in the model. Thus, it is not surprising that a discrepancy existed between measured data and model predictions in the SolS- and BSA-driven systems of this work. Nevertheless, the model captures the parameter trends well, and the good fitting between measured and simulated data in Fig. A sensitivity analysis was extensively applied to reduce the complexity of parameter estimation, determine the significance of model parameters, and pinpoint the dominant parametersTable Obviously, SolS-ASP had a significantly higher substrate metabolization rate than BSA- and HPr-ASP , whereas HAc-ASP represented the lowest metabolization rate (2655.1 mg COD/L d). In this work, substrate metabolization includes three AHO-mediated bioprocesses  synthesis, and degradation of storage IPS for microbe growth). For the SolS-ASP, even though it illustrated the relatively highest IPS synthesis rate (1527.0 mg COD/L d) among the four ASPs, it also presented a greater IPS degradation rate for AHO growth than did BSA-, HPr-, and HAc-ASPs . It was therefore not surprising that a much more powerful carbon substrate metabolism was observed in the SolS-ASP.2/d) and heterotrophic CO2 production (15.2 t CO2/d) present in HAc-ASP. This finding is consistent with results from our previous kinetic tests that indicated that a HAc-driven wastewater treatment system benefits from lower oxygen need and decreased CO2 production due to PHA synthesis12.As further shown in Table Although this work only represents an initial step in determining the amount of energy flows within ASPs and expanded energy recovery systems, the findings confirmed that carbonaceous substrate and its existence forms would indeed significantly affect the energy implications of the investigated ASPs. For a typical ASP consisting of an aerobic bioreactor with recycling and a clarifier designed with nitrification and denitrification uninvolved, its capacity to remove nutrients had been shown to be significantly weak. Thus, it should be noted that the contribution of nutrient removal to embodied energy was neglected in the present work.12, and the consumed energy associated with carbonaceous degradation in the aeration process thus increases with increased carbon degradation and utilization for microbial growth21. Changes in the type of carbonaceous substrate have a larger impact on the unintended energy consumption associated with carbonaceous degradation than on the power demand for aeration. The energy potential consumed in the treatment stages of HAc-ASP and SolS-ASP was 0.367 kWh/m3 and 0.484 kWh/m3, respectively, whereas the energy lost in the HPr-ASP was 0.487 kWh/m3 and the BSA-ASP depleted 0.590 kWh/m3. Obviously, BSA-ASP had greater energy expenditure caused by carbonaceous degradation than did both HPr-ASP and SolS-ASP ; whereas HAc-ASP represented the lowest energy loss from aerobic degradation of a carbon source (47.6%). This result coincides with a formerly shown trend  among the ASPs explored. However, further research is still needed to determine how the offsets of embodied energy for varying energy recovery strategies change in relation to both scale and technologies. This is important because the scale of implementation has been observed to affect the energy consumed or recovered in wastewater treatment with integrated resource recovery23. The residual biosolids released from the anaerobic digestion (AD) systems still have sufficient energy potential for further capture, ranging from 0.099 to 0.153 kWh/m3  exhibited a greater level in HAc-ASP (57.4%) than in HPr-ASP, SolS-ASP, and BSA-ASP . This resulted in the greatest potential for further energy capture and recycling. Despite this, bioenergy harvesting from anaerobic digestion of WAS entirely satisfied the energy offsets for operation and maintenance of the systems in all four ASPs, reaching energy neutrality. Apart from the energy reused for operation of the aerobic and anaerobic systems, all the ASPs had net production of power from each system, whereas HAc-ASP and BSA-ASP, had respectively, the highest and lowest level . These accounted for over 95% of the total carbon footprint . However, this present work excluded the contributions of CH4 and N2O releases during treatment of wastewater; thus, the carbon credit of the BSA-ASP is still much lower than the top end of the range of emissions from historical studies24. This finding implies that reduced carbonaceous oxidation and consequently less energy intensive aeration  could be beneficial for further improving energy efficiency. In activated sludge systems, CH4 contributions can be negligible due to the use of aerobic treatment processes for COD removal; yet, aeration requires additional electricity, highlighting an unavoidable trade-off between aerobic and anaerobic processes25. In this work, the CH4-contained biogas derived from WAS anaerobic digestion can also be overlooked because it was assumed to be collected and combusted to recover energy; nevertheless, it can also contribute significantly to the carbon footprint when emitted directly (0.40–0.95 kg CO2-eq/m3).The overall trend for the carbon footprint is similar to the total embodied energy because the direct emissions associated with carbonaceous substrate degradation in the treatment processes, and indirect emissions linked with electricity, are the dominant contributors . This result aligned with the former outcome that BSA-ASP showed greater degradation of carbon substrates and only a little was saved and converted into WAS for further recovery. In contrast, BSA-ASP did not show an apparently lower capacity to mitigate carbon emission through harvesting energy from incineration of residual biosolids than did the other three ASPs. The main reason is that BSA-ASP had a relatively greater sum of endogenous residue content in its WAS owning to higher microbe metabolism in the systems (data not shown), which coincides with a previous report12. Overall, HAc-ASP was found to be the only alternative that could totally offset the carbon footprint caused by embodied energy through recovering energy from its WAS, achieving maximal level of net energy gain (0.351 kWh/m3), and yielding a negative carbon footprint (72.6 g CO2/m3).Incorporating energy recovery strategies was found to be beneficial for increasing the carbon footprint avoided through delivery of recovered energy products, such as heat and electrical energy. In Fig. The findings of this work highlight that the energy consumed or produced, and the carbon footprint caused, differs dramatically for varying wastewater-derived carbon substrates. The existing form and metabolic characteristics of the carbon source are further recognized as exhibiting a close relationship with embodied and captured energy in conventional wastewater treatment alternatives, such as the explored ASPs without nitrification and denitrification.27. Even if emerging technologies are able to potentially and simultaneously remove carbonaceous substrates and other waterborne substances, shift the energy balance, and empower net power production for other social-economic sectors29. The economic costs and public acceptance, among other critical barriers, are great challenges that must be faced before this subversive reform is ultimately realized31. Thus, this work focused on discussion of another possibility that optimizes the current widely-applied technologies such as ASPs, by altering and repurposing the stream of carbon substrates at the systems-level, providing benefits for extending the service time of widely-accepted ASP technologies, reusing existing infrastructures, and potentially decreasing the expense of upgrading WWTPs.As estimated, many wastewater infrastructures at larger scale (such as city level) will need to be upgraded over the coming 10–15 years, and a new scheme incorporating reuse of wastewater-derived energy is recognized as a promising solution33, whereas primary settling affecting the particulate and dissolved carbon available for the subsequent carbon conversion11, these existing practices along with emerging systems provide numerious potentials to enable the implications of the present work. However, future planning could expand the life cycle framework to clarify how this method, if developed at different scales would influence cost, energy usage, carbon emissions, and other factors of concern for defined relationships. This extensive work will be much beneficial for future larger scale investigation. Further research is also needed to determine how the offsets of energy use and reduced carbon footprints for various wastewater-derived energy harvesting strategies, change with additional environmental impact categories. Moreover, this present work excluded the uncertainty accounting in the quantification of energy use and carbon footprint. It is understandable that this assumption would potentially contribute to the limitation of the work. For many parameters, not accurately knowing their underlying distribution functions, will hamper the smooth use of Monte Carlo simulation. Accordingly, future research efforts should also focus on elucidating sources of uncertainty and reducing uncertainty of the most sensitive parameters, in accounting both for energy use and carbon footprints, enabling robust life-cycle thinking, and determining to assist decision-making for wastewater management solutions with expected energy recovery practices.This work highlights the capacity of acetic acid to facilitate maximal delivery of recovered energy products and to mitigate carbon accounting caused by the wastewater sector. Partial fermentation can occur in sewer systems, increasing the concentration of SCFAs34. The collected sludge was initially filtered using a 0.45 mm mesh to remove small particles, washed twice with distilled water, and finally stored at −4 °C for further respirometry tests. Four sequencing batch reactors (SBR) were started up with the collected activated sludge and were fed with the studied substrates i.e., HAc, HPr, SolS and BSA, respectively, for the enrichment of acclimated biomass for further utilizations. The experimental procedure was similar with a previous literature35.For the present work, the sewage sludge was collected from a secondary clarifier of a WWTP in Northern China. The sludge characteristics are similar to those presented in our previous work36. Specifically, this device consisted of an open aeration vessel (4 L) and a magnetically stirred and sealed respiration chamber (2 L). During respirometry experiments, the activated sludge was continuously recirculated within the device at a flow rate of 0.75 L/min with a peristaltic pump (Longer® BT300-2J). Further, a heating system (COLE-PARMER® 12107-35) was used to maintain the temperature at 20 ± 0.5 °C in the aeration vessel, while a pH controller  was applied to sustain the pH at 7.5 ± 0.5 by the addition of 0.1 M HCl or 0.1 M NaOH. Dissolved oxygen (DO) determination can easily be disturbed by aeration or stirring in respirometry tests37. The online diagnosis and recalibration of DO probes are thus of significance in such tests. Accordingly, two additional measuring vessels (0.05 L each) were developed in the device and both equipped with a DO probe (WTW® CellOx 325). One was connected to the inlet and one to the outlet of the respiration chamber. A flow-switching unit consisting of solenoid valves and a time relay was designed, which allowed exchange of the contents of the two chambers. Finally, the signals of the DO probes were captured and logged on a computer equipped with the MultiLab software package.A schematic overview of the activated sludge respirometer is shown in Fig. 4Cl, 32.0; MgSO4, 10.0; 6EDTA-2Na, 2.0; K2HPO4, 16.0; KH2PO4, 3.8; CaCl2·7H2O, 7.0) was added to the system, whereas 6 mL of allylthiourea  was added to avoid growth limitation and nitrification based on historical literature19. Bovine serum albumin , soluble starch , sodium acetate , and sodium propionate  were used as substrate models for protein, carbohydrate, and common SCFAs, respectively, for the sake of mechanistic exploration. Four sets of the respirometry device were used and a substrate sample  was added into the aeration vessel of each device at 200 mg chemical oxygen demand (COD)/L in 10 mL. The experiments were operated for approximately 6 h to trace and gain the data needed for further analysis. It should be further noted that the stability of the developed experimental system was also verified (p < 0.05) prior to the kinetic tests to ensure the system was robust and rigorous enough to support the whole experiments. The DO probes captured signals every 5 s and sent data to the PC system for quantifying the oxygen uptake rate (OUR) profiles according to Eq. 136, whereas the two measuring vessels would be switched every hour for online diagnosis and recalibration of the probes, based on a previous approach37.SO,in and SO,out were the oxygen concentrations entering and leaving the respiration chamber, respectively.The acclimated SBR sludge  was added to four respirometer, respectively, at 1.0 g/L in 6 L and then aerated until the endogenous respiration phase was reached. At the same time, 20 mL of nutrient solution (g/L: NHSamples (10 mL) of the mixture were taken at set intervals and then immediately treated with 6 M HCl to inhibit biodegradation. Subsequently, the treated mixture was centrifuged at 1000 r/min for nearly 15 min. Afterwards, the suppressant was filtered with 0.45 μm mesh for measurement of soluble matter , while the centrifuged sediment was sampled to measure the glycogen and PHA contents.38, whereas the glycogen and PHA contents were determined based on previous references40. In addition, the conversion factor for BSA, SolS, HAc, HPr, PHA, and glycogen to COD was set as 1.40 g COD/g BSA, 1.12 g COD/g SolS, 1.07 g COD/g HAc, 1.51 g COD/g HPr, 1.67 g COD/g PHA, and 1.15 g COD/g glycogen, respectively43. Moreover, the results from this work are presented as the mean ± standard deviation of triplicate tests performed under the same conditions.Measurements of pH, protein, carbohydrate, SCFAs, COD, and mixed liquid volatile suspended solid (MLVSS) content were performed as historically described44. To this end, a well-known simultaneous storage and growth (SSAG) model was used and calibrated subsequently for SCFAs45. However, since SSAG model was not develped to model and trace the conventional characteristics of both protens and strach, a specific model reported previously was selected and modified for the metabolism of proteins and starch39. Note that the nomenclature of all model components and parameters is the same as reported. The model used to describe the kinetic and stoichiometric behaviors of active heterotrophic organisms (AHOs) consists of several key processes: adsorption of slowly biodegradable substrate ; hydrolysis of adsorbed substrates (XSads); storage of intracellular polymeric substances (XSTO) on readily biodegradable substrates ; aerobic growth of AHOs (XAHO) on SS; aerobic growth of AHOs on storage products (XSTO); endogenous respiration of XSTO; and decay of AHOs. Note that the nutrients profile and its relevant mechanisms were not considered in the model. The stoichiometrics and kinetics of the modified model are summarized in SI  with the obtained best-fit parameter values with 95% confidence intervals.The model includes seven specific biochemical processes and 27 stoichiometric and kinetic parameters, as given in Table 49. ASPs were simulated and fed with the four different carbonaceous substrates . Each ASP had the same influent COD level (200 mg COD/L). The hydraulic retention time of the aerobic reactor was fixed at 2 h, and the DO set point was 2.0 mg/L. Furthermore, the removal rate of initial carbon substrate greater than 97% in the ASPs was set as a benchmark for evaluation. All the simulations were run continuously in AQUASIM for over 2000 h to ensure the model components reached stable conditions (p < 0.05); next, the flow and composition details throughout the ASPs were acquired for further calculation.To mimic a representative ASP, an aerobic bioreactor with recycling and a secondary clarifier was modeled using our previous protocol50, a functional unit of 1 m3 of treated water was selected in this work, and treated water from all ASPs meet the same discharge requirement . The operation and maintenance phases for treatment and integrated energy recovery stages were mainly included in the system boundary. The power production avoided by energy recovery was considered via system expansion, in which coal for electricity production was considered an avoided product.As recommended by LCA literature and ISO 14040 guidelines51. The SSAG model was applied to determine the oxygen transfer rate (kg CO2/m3 of treated water), and the electricity consumed for aeration could be quantified by employing an aeration efficiency of 2 kg O2/kWh12. Energy demands to operate the sludge anaerobic digester included the requirements for heating. The amount of heat (kWh) required per 1000 kg of wet sludge was calculated from the difference between the assumed initial (20 °C) and desired (35 °C) temperatures, multiplied by the specific heat capacity of sludge with 6% solids (4.18 kJ/kg °C)48, and the heat loss from the AD system with available heat transfer coefficients52. The energy requirement for stirring in the AD system was excluded because it required much less than that for the heating of sludge or heat loss from the AD system53. Furthermore, it should be noted that the biogas mostly containing CH4 and CO2 generated from AS system will be considered as a whole for onsite recovery of energy (including heat and electricty). Consequently, the energy recovery (kWh/m3 of treated water) from biogas combustion was determined from the biogas yield (m3) multiplied by the heat value of biogas (23 MJ/m3)54, and the total energy conversion rate 48 in a combined heat and power (CHP) system. The biogas figure was gained from the modeled sludge yield (kg/m3 of treated water) multiplied by the modeled volatile solids (VS) and the biogas yield rate (0.65 m3/kg of VS)54.A life cycle inventory of embodied and recovered energy was compiled for a comprehensive substance flow analysis of systems and key operating factors, which differ among the four ASP alternatives. The influent and effluent COD concentration of each ASP was employed to estimate the total energy contained in the influent and effluent stream by assuming a conversion factor of 3.86 kWh/kg COD2 emission factor of bioenergy conversion was still involved herein. Specifically, the CO2 production factors from coal-based energy production and bioenergy recovery were 877 and 353 g CO2/kWh, respectively55. The CO2 emission factor of sludge incineration was assumed to be 0.415 kg CO2/kg of sludge56. The on-site carbonaceous degradation and consequent CO2 release in ASPs was obtained from the SSAG model, whereas the contribution of excess carbonaceous degradation from effluent discharge to aquatic systems was determined by the effluent COD concentration and an assumed conversion factor of 1.5 kg CO2/kg COD57.It should be noted that the carbon emission in this work was estimated from energy consumption, on-site and excess carbonaceous degradation, and carbon release avoided through harvesting of bioenergy from biogas combustion and incineration of residual biosolids. As the biogas derived from WAS anaerobic digestion was viewed as a whole for energy recovery, it can be overlooked in carbon footprint accounting; however, a COSupplementary Information