Olea europaea species. A complete survey of the variability across the nuclear and plastid genomes of different genotypes was studied through single nucleotide polymorphisms, indels (insertions and deletions), and length variation.Olive is one of the most cultivated species in the Mediterranean Basin and beyond. Despite being extensively studied for its commercial relevance, the origin of cultivated olive and the history of its domestication remain open questions. Here, we present a genealogical and kinship relationships analysis by mean of chloroplast and nuclear markers of different genera, subgenus, species, subspecies, ecotypes, cultivated, ancient and wild types, which constitutes one of the most inclusive research to date on the diversity within Oleaceae family including Olea europaea, other species and genera. The chloroplast genome evolution, within Olea europaea subspecies, probably started from subsp. cuspidata, which likely represents the ancestor of all the other subspecies and therefore of wild types and cultivars. Our study allows us to hypothesize that, inside the subspecies europaea containing cultivars and the wild types, the ancestral selection from var. sylvestris occurred both in the eastern side of the Mediterranean and in the central-western part of Basin. Moreover, it was elucidated the origin of several cultivars, which depends on the introduction of eastern cultivars, belonging to the lineage E1, followed by crossing and replacement of the autochthonous olive germplasm of central-western Mediterranean Basin. In fact, our study highlighted that two main ‘founders’ gave the origin to more than 60% of analyzed olive cultivars. Other secondary founders, which strongly contributed to give origin to the actual olive cultivar diversity, were already detected.Fifty-six different chlorotypes were identified among the Olea europaea species but also provides original insights into other elusive evolutionary processes, such as chloroplast inheritance and parentage inside olive cultivars, opening new scenarios for further research such as the association studies and breeding programs.The application of comparative genomics not only paves the way for a better understanding of the phylogenetic relationships within the The online version contains supplementary material available at 10.1186/s12870-023-04440-3. Olea europaea subsp. europaea is a distinctive element of the Mediterranean Basin (MB) flora and it can be found either as cultivated (subsp. europaea var. europaea) or as wild olive (subsp. europaea var. sylvestris) tree. Five other subspecies have been recognized [laperrinei ; subsp. guanchica (Canary Islands); subsp. cerasiformis (Madeira archipelago); subsp. maroccana (South West Morocco) [cuspidata .cognized , with a Morocco) ; and subOlive is among the oldest woody crops of the MB. Several theories have been proposed over the last decades to track its origin. Molecular and archaeological data have given new insights concerning its domestication and cultivation diffusion –8. The pO. europaea subspecies and olive cultivars [Some studies focusing on the analysis of chloroplast polymorphisms , 19, havultivars , have alultivars , 30 and ultivars . Besnardultivars  detectedIn recent years, single nucleotide polymorphisms (SNPs) at nuclear level have been developed in olive –36. ConsO. europaea accessions, including subspecies samples, olive cultivars from the main producing countries, ecotypes, which are probably related to the remnants of ancient cultivars as well as to feral forms disseminated by seeds; and, for the first time, ancient olive trees, were deeply examined by whole chloroplast genome polymorphisms. Furthermore, nuclear EST-SNPs were applied to establish the genetic relationships, rebuild the direct parentage of the most representative olive cultivars and reconstruct their origin and diffusion along the MB. The combination of chloroplast and nuclear polymorphisms can pave the way to unravel the genetic origin and diffusion of this important crop.The combination of chloroplast polymorphisms and EST-SNPs can allow tracing back the maternal origin of the current cultivars, point out the routes of migration and, by highlighting the admixture and relationship among them, the genetic kinship of the cultivars. In the present work, hundreds of O. europaea subspecies, among them 45 chlorotypes were reported for the first time in the present study and were indicated with ‘N’ in the Supplementary Table The results from length polymorphism and SNP analyses revealed 51 different chlorotypes within the The analysis of non-linked chloroplast polymorphisms, including insertion/deletion, showed that the number of alleles (Na) varied between two and four, with an average of 2.28. Values of Shannon’s information index (I) ranged between 0.09 and 0.89, with an average value of 0.41. The average of calculated diversity index (h) was 0.25, with the highest value in position 40, the diversity and unbiased diversity were 0.54 and 0.55, respectively , and one from India. The second most represented was CUS.N6, with 15 samples from Iran and one from China. Two genotypes from Nepal, carrying the same CUS.N10 profile, while all the other chlorotypes were represented by a single tree  in three samples.O. europaea subsp. cerasiformis. The four analyzed samples, collected in the Portuguese Madeira and Porto Santo Islands, were found to belong to four different chlorotypes: CER.N1, CER.N 2, GUA.N2 and MAR.N1. In fact, one of cerasiformis sample (S42) was found to share the same chlorotype (GUA.N2) of two samples belonging to subspecies guanchica and coming from the Spanish Tenerife and La Palma islands. Similarly, the sample S57 in which was detected the the chlorotype MAR.N1.O. europaea subsp. guanchica. High level of variation was detected among the 13 samples from the Canary Islands that showed six different chlorotypes derived from few mutation points.O. europaea subsp. laperrinei. Three samples of this subspecies had E1.1 chlorotype, mostly diffused in the olive cultivar, while the fourth one showed a slightly different profile and it was called Lap.N1.O. europaea subsp. europaea. Chloroplast data showed that the three lineages typical to the subspecies europaea were geographically separated, placing E1 throughout MB, while the other two (E2 and E3) both in central and western side of Basin. In particular, lineage E2 was found in genotypes from Italy, Spain, France and western part of Greece. While the E3 lineage was mainly present in Italy, Greece, Algeria, France and Spain  followed by E3, where 36 samples out of 43 were identified in the Iberian Peninsula  were identified as the most abundant. Finally, 15 samples, collected as wild types in Morocco, were belong to lineage MAR.N2 or N5.Some chlorotypes are diffused in wild olive genotypes from restricted geographical zones such as E2.1 (mostly found in Sardinia and Corsica Islands), E2.3 on south-west of Spain and E2.N17 only in Morocco. Contrariwise, E2.N14 was detected in wild olive genotypes from five different places in Morocco, together with the Spanish regions of Extremadura, Cadiz and Palma de Mallorca Island; the E2.N5 in Corsica Island, central Italy and Montenegro; and finally, the E3.1 and E3.N2 were detected in wild olive genotypes from five different countries and locations such as France, Italy, Spain, Portugal and Morocco.O. europaea subsp. europaea, the subgroup formed by ecotypes and ancient trees, both probably representing remnants of ancient olive cultivation, the identified chlorotypes showed different scenarios. In fact, while a single molecular profile (E1.1) was found in the Iranian country, within ‘ancient olive’ group collected from other countries eight different chlorotypes were discovered. E1.1 was the most abundant chlorotype being shared by 95 out of 131 monumental trees. The second most occurred chlorotype was E3.1 individuated in nineteen genotypes from the western Mediterranean Basin (WMB) countries such as Spain (13 out of 19), which were genetically different from known cultivars belonging to the same chlorotypes, and Portugal. Among the eight different chlorotypes observed in the ancient plants, two were private to 98 canopy samples E1.N7 and E2.N11 exclusively present in Italy; and two, E1.N4 and E2.N12, were private to 38 rootstocks, found in Crete and Sardinia Islands, respectively. The highest variability was disclosed inside E2 with four different profiles, three of them belonging only to Italian genotypes, while the E2.N5 chlorotype was shared among Italy, Montenegro and Algeria ancient olive trees.Furthermore, within O. europaea subsp. europaea var. europaea), eleven different chlorotypes were ascertained. Six within lineage E1 , four in lineage E2  and one (E3.1) in E3  and the diploid subspecies guanchica, a network analysis ; in Jordan (cv. Kanabisi); Lebanon ; Turkey ; Greece (cv. Throubolia ‘Throumbolia’); Albania (cv. Marksi); Italy (cvs. Grossolana and Sant’Agatese); and in Spain . The second main founder, cv. Gordal Sevillana showed close genetic relationship with some EMB cultivars as Uslu, Kiraz and Izmir Sofralik from Turkey and Toffahi from Egypt. These mentioned cultivars, sharing the same chlorotype (E1.2) of their founder, could represent seedlings originated from the cv. Gordal Sevillana in that area. In addition, this founder is called with different names as Giarraffa, Bella di Spagna, Pizzo di Corvo and Santa Caterina in Italy, Grosse du Hamma in Algeria, Boube in France, Ters Yaprak ‘Tavsan Yuregi’ in Turkey, and Gordal Valle de las Palmeras in Mexico. Another significant aspect was the close relationships between the cv. Gordal Sevillana with cultivars showing the E3 lineage. In fact, considering that this lineage (E3) was found in the cultivated, ancient, and wild germplasm of the WMB, a trace of the possible crossing between the ‘eastern founder’ with the ‘western’ and autochthonous germplasm could be still present in the cultivated germplasm. Moreover, in addition to the main founders, well-known Italian cultivars, such as Maurino, Pendolino, Coratina and Leccino were directly connected to the Italian cultivar Frantoio, which can be classified as secondary founder for this group. Another cultivar from the Spanish Andalusia region, Manzanilla de Sevilla, was a secondary founder of some important Spanish and Moroccan cultivars  , with the exception of cvs. Arbequina and Arbosana, which were placed in the central Mediterranean group with an admixture value that was related to the cultivars from the eastern side of Mediterranean Basin and beyond, as observed also after the phylogenetic analysis. Finally, another group is formed by cultivars originating from the Middle and Near East which clearly clustering together.Olea subspecies. Moreover, our study proposes a detailed reconstruction of the current cultivar assortment along the MB, starting from those that may represent the main and secondary founders of the olive cultivars actually under cultivation. Olive represents one of the few cultivated fruit trees that has not undergone significant breeding programs. In fact, only a very few of the current cultivars has been obtained by systematic breeding, while most of them, still under cultivation, were likely selected hundreds or thousands of years ago, a few generations away from the original ancestors [By assessing the genetic diversity at plastidial and nuclear levels on a large set of olive genotypes, here we have traced the origin and diffusion of ncestors  as demoncuspidata was placed immediately after the Olea related species and before all the other Olea europaea subspecies. Following these results, the subspecies cuspidata, represented by ten different chlorotypes, three of them related to the subsp. ferruginea , could be considered as the first step of evolution from what the other subspecies have been originated, including the cultivated ones. This finding is in accordance with recent reports in which subsp. cuspidata is the early diverging taxon of the Olea complex [laperrinei, here analyzed, showed a strong similarity at chloroplast level with the subspecies europaea belonging to lineage E1 and therefore to the cultivars, as already described in previous studies [O. europaea subsp. laperrinei, restricted to Central Saharan mountains of Algeria, Niger, and Sudan, generally with small population size [guanchica to the present cultivars’ diversity and distribution considering the gene flow between subsp. guanchica, western wild olives and cultivars [maroccana and cerasiformis, subspecies guanchica and laperrinei are mostly diploid, which gave more possibilities of intercrosses with the local wilds and cultivars [laperrinei played an important role in the origin of olive genotypes, both wild types and cultivars, belonging to the E1 lineage, especially in the EMB. On the other side of MB, the subsp. guanchica may had a key role especially in the origin of the subspecies europaea var. sylvestris and few remaining cultivars, belonging to E2 and E3 chloroplast lineages of the central-western sides of Mediterranean Basin (CWMB), which were not undergone the crossing with the eastern olive germplasm, also considering their nuclear genetic affinity reported here and in already published paper [guanchica and the results obtained by network analysis confirmed the important relationship among lineage E2, E3 and subsp. guanchica. A new scenario on the origin, diffusion and differentiation of ‘western’ wild types could be hypothesized, their diffusion could start from the long-term refugia during the Quaternary [In our network analysis, based on chloroplast polymorphisms, the subsp.  complex –43. Five studies , 44, 45.ion size , similarion size , 40, 42.ultivars , 47. Thiultivars , 42. Based paper , 43. Theaternary  and subsaternary , 49, 50.laperrinei and the genotypes of the E1 lineage, the present study highlights that some chloroplast profiles were shared among different subspecies and var. sylvestris. Two sample of subsp. cerasiformis had the same chlorotype of subsp. guanchica (GUA.N2) and subsp. maroccana (MAR.N1). Moreover, some var. sylvestris samples from Morocco, had chloroplast profile of subspecies maroccana. These results could derive from possible misidentification of samples during the surveys considering that some places of diffusion are the same, especially for subsp. maroccana and var. sylvestris. At the same time, it was confirmed the presence of different chlorotypes belonging to different subspecies in the same area [Olea, the genomic evolution [In addition to the already discussed similarity between the subsp. ame area , openingvolution , 47, 51 europaea. The presence of chlorotype E1.1 among the studied wild samples from CWMB could correspond to the seedlings of var. europaea, and not to the var. sylvestris [sylvestris in this area, which, therefore, could be considered the original one.This study helps also to clarify the relationships between wild and cultivated olive populations according to their lineages and chlorotypes , 53. Thelvestris , which aMoreover, our study represents the first effort for chlorotyping several ancient olive trees; in fact, the new detected chlorotypes shed light on their unexplored genetic richness. Rootstocks and canopies of monumental olive trees, not genetically related to known cultivars , shared Among the cultivated olives, analyzed in the present study, even if eleven different chlorotypes were found, 80% of them were belong to E1.1, confirming that the E1 lineage is the most diffused worldwide , 29. Thesylvestris belonging to E1 lineage in the EMB, and the second from the var. sylvestris belonging to E2 and E3 lineages, in the CWMB. What happened later could be described as improvement and selection by introducing allochthonous cultivars mostly coming from EMB, which have been replaced the genuine ‘western’ olive trees by both crossing and grafting.The answer should be ‘yes, but…’. In fact, our results showed a clear separation at chloroplast and nuclear level between genuine wild types and cultivars collected from East, Center, and West MB, suggesting at least two possible areas of domestication from the wild types diffused in each area , 43, 55.In olive, the first step of domestication would seem to have been forest clearing and maintenance of promising wild trees, which, together with the pressure by animals feeding, may have favored vigorous olive genotypes . Moreoveeuropaea is located in the Near East [The olive cultivars were analyzed to detect their ancestry and explain how crossing, selection, plant material exchanges and displacements may have shaped the present distribution of olive variability. Recently, it was hypothesized that the original center of domestication of subsp. ear East , 6, 60, ear East . In the ear East , 60. Theear East , 5. In mear East , 62. In ear East , 58. Farear East , 56, 57.ear East , 63, 64.ear East , and amoear East , 68. Theear East . In Corsear East  were graear East , 71. Allear East . Few culear East , 70, 72.Olea pollen remains showed an expansion starting from Middle Neolithic  [vice versa. Our study highlights the importance to study the ancient olive trees especially in the regions, such as Sicily, Sardinia, Andalucía, Malta, western side of Morocco and Syrian-Lebanon-Turkish borders, where wild types, monumental trees and cultivated forms are still present close each other. The mentioned approach could help to clarify the role of autochthonous versus allochthonous olive genotypes in different introgression events, uncovering possible ancient selection from wild types, crosses between eastern and western germplasm which help to understand how much has been lost and how much has been gained during the domestication and selection scenarios.The main factors, that made Sicily a key territory for cultivar differentiation after the introduction of cultivars from EMB, were probably related to: (i) the Sicilian ancient history, well known for the commercial routes of MB; (ii) its geographic location, as previously described, which is also connected to favorable climatic conditions for olive growing; and (iii) the different civilizations who migrated and were established there –75. Oliv,200 BP) . Moreove,200 BP) . In anti,200 BP) . This ev,200 BP) , as well,200 BP) , it is p,200 BP) . In the Basing on a deep genetic survey involving hundreds olive genotypes and a wide set of chloroplast and nuclear polymorphisms answers to some long-time discussed questions regarding the origin of olive cultivars and their kinship were formulated. As for other crops, olive is undergoing to a human-driven selection process. During the first selection from wild to cultivated plants, the variability was enriched probably thanks to the high number of wild types still present along MB and beyond. After the domestication events, the genetic diversity was decreased due to farmers’ selection, but lately, ‘new’ crosses between native and allochthonous, olive accessions provided an increase of variability and diversity. Whereupon, genetic diversity decreases rapidly by grafting with only few genotypes, a method largely used by the ancient Greeks and Romans.ex situ collections. Since many of these genotypes could represent an essential reservoir of genetic diversity for many traits particularly those linked to environmental adaptation, abiotic  and biotic  stress resistance, and/or fruit and olive oil quality. The implementation of specific policies for their protection will be a crucial challenge for the next future.Along the last century, the preservation of olive germplasms resources has been mined by the anthropization of many habitats, leading to the explantation of native trees. It is mandatory to retrieve the remnants of ancient genotypes starting from the study of monumental olive trees and their rootstocks as well as the preservation of the ‘real’ wild types constituting protected natural areas and Xylella fastidiosa). Population and comparative genomics may also reveal which traits have been selected during the early steps of domestication and which were selected during the modern breeding decades, to move from an era of big data into the era of better data.Further application of population and comparative genomics to subspecies, wild types, ecotypes, ancients and cultivated olives will provide new opportunities to investigate the molecular basis of (i) agronomic traits potentially selected and introgressed during domestication , and (ii) ecological traits contributing to the adaptation of wild populations and ancient olive trees to environmental and biotic factors ; 78 genotypes of O.europaea subspecies, including four samples of subsp. laperrinei from Algeria, 13 of subsp. guanchica from the Spanish Canary Islands, 52 of subsp. cuspidata , four of subsp. cerasiformis from the Portuguese Madeira and Porto Santo Islands and five of subsp. maroccana from Morocco. Moreover, two Olea species were included in the study: Olea capensis, belonging to Subgenus Olea, Section Ligustroides, and Olea brachiata, from Subgenus Tetrapilus [Phillyrea angustifolia, Forsythia spp. and Sirynga vulgaris , with 93 canopies and 38 rootstocks; 180 wild olives . DNA quantity and quality were controlled by using NanoDrop spectrophotometer .All accessions were analysed by 42 chloroplast markers, including SNPs, INDELs and length polymorphisms extending over 37 chloroplast genomic regions. Nineteen length polymorphisms were previously selected  and amplPositive controls were included in all experiments, using DNA of cv. Frantoio, because the complete sequence of its plastid genome is already available , 83. Pol− 1. Two technical replications and two independent runs were performed for each reaction. The melting curves were normalized using the software provided by HR1 instrument and visualized using Derivative and Difference Plot tools.For the first time in olive chlorotyping, the detection of polymorphisms was performed also through the high-resolution melting (HRM) analysis , 85. A nTo confirm the correspondence between lengths and SNP polymorphisms, all different fragments, detected by the above-cited methods, were sequenced on an ABI PRISM 3130 Genetic Analyzer . BigDye PCR was performed on the amplicons (without fluorescence) by adding 0.2 µl BigDye Terminator v1.1 Cycle Sequencing Kit (ThermoFisher Scientific), 0.6 µl of 2 pmol primer , 1.8 µl of 10X buffer and 0.3 µl of PCR product in a 10 µl final volume. The following conditions were used: 96 °C for 1 min; 25 cycles: 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min. Purified BigDye PCR products were sequenced.All plastid polymorphisms, highlighted by length and HRM analyses, and confirmed by sequencing analyses, were merged to build a single polymorphic fragment for each sample. Considering all positions of the final set, sequences were aligned, and the evolutionary analyses were conducted through MEGA7 software . The phyguanchica, wild types and cultivars [A median-joining network was created using the default parameters in PopART version 1.7 . Data prultivars , 39, werCoancestry between cultivars and their inbreeding coefficients were calculated using COANCESTRY software using the triadic likelihood estimator (TrioML) . TrioML Furthermore, to explore the possible direct parentages of the above mentioned 171 cultivars, EST-SNP data were analyzed by using the maximum likelihood-based method described in Kalinowski et al.  and implThe 171 cultivars genotyped by 1,040 EST-SNPs , 39 wereBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8Supplementary Material 9


In the published article, there was an error regarding the affiliation for Haïm C. Joseph. Instead of having affiliation [2], they should have had [3].In the published article, there was an error in the author list, and authors Luke Gamble, Frederic Lohr were erroneously excluded. The corrected author list and appears below.1, 2*, Pierre Dilius3, Andrew D. Gibson4, 5, Kelly Crowdis6, Natael Fénelon7, Yasmeen Ross1, Sarah Bonaparte1, Luke Gamble4, Frederic Lohr4, Haïm C. Joseph3 and Ryan M. Wallace1Caroline A. SchrodtIn the published article, there was an error. Luke Gamble and Frederic Lohr should have been included in the Conflict of interest and Author contributions sections.A correction has been made to the Conflict of Interest section. This sentence previously stated:“AG was project lead for the development of the electronic application as a part of employment for Mission Rabies.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.”The corrected sentence appears below:“AG was project lead for the development of the electronic application, with LG and FL supporting the development, as a part of their employment for Mission Rabies.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.”A correction has been made to the Author Contributions section. This sentence previously stated:“RW and AG conceptualized the project. RW acquired funding and supervised the project. RW, CS, YR, and SB synthesized and analyzed study data. AG provided resources and software programming. PD, KC, NF, and HJ managed coordination/execution of activities. CS wrote original draft with review and editing by RW. All authors contributed to the article, reviewed, and approved the submitted version.”The corrected sentence appears below:“RW, AG, LG, and FL conceptualized the project. RW acquired funding and supervised the project. RW, CS, YR, and SB synthesized and analyzed study data. AG, LG and FL provided resources and software programming. PD, KC, NF, and HJ managed coordination/execution of activities. CS wrote original draft with review and editing by RW. All authors contributed to the article, reviewed, and approved the submitted version.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Vafaee et al., RSC Adv., 2021, 11, 22661–22676, https://doi.org/10.1039/D1RA02780H.Retraction of ‘Synthesis and characterization of AFe RSC Advances article due to concerns with the reliability of the data.The Royal Society of Chemistry, with the agreement of the authors, hereby wholly retracts this 1 a new concern has been identified with the published article. The XRD patterns in Fig. 2b and both the original Fig. 4a and b contain multiple overlapping sections. In addition, new information brought to the attention of the Editor undermines the integrity of the authors' original explanation, meaning that the Editor has lost confidence that the findings presented in this paper are reliable.Following the publication of a previous correction to replace the XRD patterns in Fig 4a and b,Omid Amiri, Mansour Jahangiri, and Masoud Salavati-Niasari wish to state that they had no role in preparing the XRD data.1 related to this article.This retraction supersedes the information provided in the correctionSigned: Fahimeh Vafaee, Samira Mandizadeh, Omid Amiri, Mansour Jahangiri, Masoud Salavati-NiasariDate: 24/7/2023RSC AdvancesRetraction endorsed by Laura Fisher, Executive Editor,


Incidental durotomy is the most common intraoperative complication of spine surgeries. Our main goal is to report a case of a postoperative postdural puncture headache following an incidental durotomy successfully managed with a sphenopalatine ganglion block. A 75-year-old woman, American Society of Anesthesiologists physical status II, proposed for a lumbar interbody fusion. During surgery, an incidental durotomy with cerebrospinal fluid leak occurred, being repaired with muscle and DuraSeal® Dural Sealant System. In the recovery room, 1 hour after the end of the surgery, the patient developed a severe headache associated with nausea and photophobia. A bilateral transnasal sphenopalatine ganglion block with 0.75% ropivacaine was performed. Immediate pain relief was verified. The patient reported only mild headaches on the first postoperative day, feeling progressively better until discharge. The sphenopalatine ganglion block may be an effective alternative treatment for postdural puncture headache following an incidental durotomy during neurosurgeries. Sphenopalatine ganglion block may be a safe, low-risk alternative in the treatment of postdural puncture headache after an incidental durotomy that can be used in the immediate postoperative period to enable early recovery and return to day-to-day activities, which will hopefully lead to better surgical outcomes and patient satisfaction. Pain in the immediate postoperative period leads to late recovery and late return to day-to-day activities, which will lead to worse surgical outcomes and poor patient satisfaction.Sphenopalatine ganglion block may be a safe, low-risk alternative in the treatment of postdural puncture headache after an incidental durotomy.We suggest that in cases of incidental durotomy, a sphenopalatine ganglion block can be performed at the end of the surgery even before the excruciating pain occurs.1Incidental durotomy (ID) is the most common intraoperative complication of spine surgeries. The incidence of ID ranges from 0.3% for lumbar discectomy to 11% in spinal deformity correction surgeries. Higher incidence of ID is associated with age, obesity, diabetes mellitus, surgical invasiveness, number of levels, revision surgeries, and less experienced surgeons. Incidental durotomy is associated with short- and long-term sequelae such as persistent cerebrospinal fluid (CSF) leaks, headaches, pseudomeningocele formation, dural cutaneous fistulas, meningitis, epidural abscess, and new-onset neurological deficits. It ultimately affects the surgical outcome and patients’ postoperative quality of life.2 After an ID, CSF’s volume decreases, and compensatory vasodilation is seen. This vasodilation is parasympathetically mediated by neurons in the sphenopalatine ganglion (SPG), and it is responsible for the headache felt after an ID. Traction on pain-sensitive intracranial structures also contributes to the pain. By attenuating the parasympathetic activity with an SPG block, the vasodilation is addressed, and the patient achieves symptomatic relief.3 Another possible effect is the modulation of sensory processes in the trigeminal nucleus caudalis via afferent sensory fibres, which is responsible for a change in pain modulation and reduced central sensitisation to pain.4According to the Monro–Kellie hypothesis, the sum of the volumes of the brain, CSF, and intracranial blood compartments is a constant value. If the volume of one of these reduces, the others must increase to maintain an equilibrium.5The standard postdural puncture headache (PDPH) treatment consists in adopting a supine position, hydration, analgesics, caffeine, sumatriptan, laxatives, and abdominal binders which are rarely effective and the epidural blood patch which is demanding, invasive, and often painful.Sphenopalatine ganglion block has emerged as a noninvasive alternative to this treatment. Our main goal is to report a case of a postoperative PDPH following an ID successfully managed with an SPG block.-2) and a previous surgical history of removal of a left L4–L5 disc herniation and posterior fixation.After patient’s informed written consent, we report the case of a 75-year-old woman, American Society of Anesthesiologists (ASA) physical status II, with a past medical history of dyslipidemia and obesity  access, the patient was monitored according to ASA standards. A bilateral L3–L4 ultrasound-guided erector spinae plane block was performed with the patient in prone position. A total of 40 mL of 0.5% ropivacaine (100 mg) was injected deep into the erector spinae muscle. Following this, balanced general anaesthesia was performed, with no intercurrences.The surgical technique consisted of the placement of pedicle screws in L3, L4, and L5 and removal of the L4 and L5 spinous apophysis as well as the pre-existing interlaminar stabilisation dispositive. An L3 and L4 laminectomy and flavectomy and an L3–L4 facetectomy were performed followed by decompression of the recesses and foramina, bilaterally from L3 and L4. During this step, an incidental anterior durotomy occurred. Due to suturing difficulties, the tear was repaired with muscle and DuraSeal® Dural Sealant System.-1 IV were administered. By the end of the surgery, the patient awoke with no complaints of low back pain or headache.The laborious surgery lasted for 5 hours with an estimated blood loss of 1000 mL. In the intraoperative period, paracetamol 1000 mg IV, tramadol 100 mg IV, ketorolac 30 mg IV, and ketamine 0.25 mg kgIn the recovery room, approximately 1 hour after the end of the surgery, a new onset of a severe occipital headache  associated with nausea and photophobia was objectivated. There was no neck stiffness, dysphasia, low back pain, motor deficits, or any other relevant findings on clinical examination.Since an ID occurred during the surgery, a PDPH was assumed and an SPG block was performed, guided by anatomical references, after discussion with the neurosurgeon. Life-threatening aetiologies like haemorrhage, thrombosis, vasculopathy, and meningitis were less probable since there were no focal neurologic deficits.A transnasal approach with 0.75% ropivacaine, 2.5 mL in each nostril, was used. Two cotton tip applicators were placed in the nasopharynx’s posterior wall at the level of the middle turbinate for 12 minutes. The technique was well tolerated, causing no discomfort, oropharyngeal paresthesia, bleeding, or any other side effects. Pain relief was immediate, resulting in a Numeric Rating Scale of 3. The patient reported no nausea, vomiting, visual symptoms, or any other complications. New onset of neurological signs, changes in the headache nature, or conscious level fluctuations should prompt further investigation and imaging.During the next 5 days, the patient received paracetamol 1000 mg IV every 8 hours, ketorolac 30 mg IV every 8 hours, tramadol 100 mg IV every 12 hours, and diazepam 5 mg IV once a day. She reported only mild headaches on the first postoperative day , feeling progressively better until discharge.6 newer and less invasive treatments like the SPG block are gaining popularity.Severe PDPH is debilitating, preventing early mobilisation and rehabilitation and may mask signs of serious complications. Although conservative measures and epidural blood patch are presently considered the therapeutic gold standard,3The SPG is a parasympathetic ganglion, triangular-shaped, located in the pterygopalatine fossa, posterior to the middle nasal turbinate and anterior to the pterygoid canal. Postganglionic parasympathetic and sympathetic neurons and the somatic sensory afferents can be blocked by an SPG block.3 Sphenopalatine ganglion block can also be performed under direct endoscopic view for a more effective block. This technique consists of SPG bilateral direct injection with a local anaesthetic, via an endonasal endoscope, allowing the needle to directly penetrate the pterygopalatine fossa minimising extraneous absorption through the nasopharynx.7To perform an SPG block, a cotton swab soaked in ropivacaine or lidocaine is inserted parallel to the floor of the nose until resistance is encountered. At this point, the swab will be at the posterior pharyngeal wall superior to the middle turbinate. The applicator should be retained for 5-10 minutes.3 The SPG block effective duration is approximately 8 hours, and it should be considered as a temporary measure to relieve symptoms until the dural puncture site generates sufficient granulation tissue to obstruct the outflow of CSF.8Complications are usually rare and minor , and there are almost no contraindications .3 Recently, it proved to be an effective technique in reducing early post operative nausea and vomiting in endoscopic sinus surgery patients.7The transnasal SPG block has been traditionally used to treat acute and chronic medical conditions, like migraine, cluster headache, trigeminal neuralgia, and atypical facial pain. It has also shown good results in recent case series to treat acute PDPH headache in obstetric patients and in other cases of neuraxial anaesthesia and diagnostic lumbar punctures.6However, there are no reports of the SPG block applied to ID as a neurosurgical complication. To our knowledge, there is only one report of an SPG block in a neurosurgical case: it reports successful use of an SPG block in a PDPH secondary to a lumboperitoneal shunt performed to treat intracranial hypertension due to a venous sinus thrombosis.In our case, a transnasal approach with a total of 5 mL of 0.75% ropivacaine applied for 12 minutes provided immediate and sustained pain relief without any complications. In addition, our patient presented various risk factors for ID like age, obesity, previous spine surgical history, and surgical invasiveness. We suggest that in cases like this if an ID occurs, the possibility of an SPG block at the end of the surgery should be discussed, even before the excruciating pain occurs.In conclusion, our clinical case suggests that sphenopalatine ganglion block may be a safe, low-risk alternative in the treatment of PDPH after an ID that can be used in the immediate postoperative period to enable early recovery and return to day-to-day activities, which will hopefully lead to better surgical outcomes and patient satisfaction.Future randomised, case-control studies are needed to ascertain SPG block efficacy in PDPH associated with neurosurgical complications.


Rumex acetosa L.) leaves. Based on 16S rDNA gene sequence comparison, and biochemical and physiological characteristics, this strain was formally identified as Bacillus mojavensis. Bacillus mojavensis UTF-33 was sensitive to most of the antibiotics tested except neomycin. Moreover, the filtrate fermentation solution of Bacillus mojavensis UTF-33 had a significant inhibitory effect on the growth of rice blast and was used in field evaluation tests, which reduced the infestation of rice blast effectively. Rice treated with filtrate fermentation broth exhibited multiple defense mechanisms in response, including the enhanced expression of disease process-related genes and transcription factor genes, and significantly upregulated the gene expression of titin, salicylic acid pathway-related genes, and H2O2 accumulation, in plants; this may directly or indirectly act as an antagonist to pathogenic infestation. Further analysis revealed that the n-butanol crude extract of Bacillus mojavensis UTF-33 could retard or even inhibit conidial germination and prevent the formation of adherent cells both in vitro and in vivo. In addition, the amplification of functional genes for biocontrol using specific primers showed that Bacillus mojavensis UTF-33 expresses genes that can direct the synthesis of bioA, bmyB, fenB, ituD, srfAA and other substances; this information can help us to determine the extraction direction and purification method for inhibitory substances at a later stage. In conclusion, this is the first study to identify Bacillus mojavensis as a potential agent for the control of rice diseases; this strain, and its bioactive substances, have the potential to be developed as biopesticides.Biological control has gradually become the dominant means of controlling fungal disease over recent years. In this study, an endophytic strain of UTF-33 was isolated from acid mold ( Oryza sativa L.) is one of the world’s major food crops and also serves as a staple food for more than half of the world’s population (Magnaporthe oryzae). The disease affects rice all year round, producing lesions on leaves, leaf necks, culms, culm nodes, spike nodes and spike necks. These lesions cause leaf blast, neck blast, and spike blast and the development of spots. The color and shape of the spots vary depending on environmental conditions and the developmental stage of the plant  plates on a shaking platform. In order to exclude the non-endogenous bacteria adhering to the surface of plant leaves from interfering with the test results, sterile distilled water from the last rinsing of leaves was collected and plated on PDA, and then plates were incubated for 48 h. The bacterial growth on the surface of the plates was observed to determine whether all the non-endogenous bacteria on the surface of plant leaves had been killed.The leaves of fresh and healthy acid mold, clover, hoodia, dong quai, cactus, goosefoot, double-podded cassia, leek, and coronary, were collected and placed in anhydrous ethanol, soaked and disinfected for 2–3 min; the mixture was then poured out and the procedure was repeated 2–3 times. Then, the leaves were placed in 1% sodium hypochlorite solution, soaked for 30 s, rinsed with sterile distilled water, dried and grinded by homogenization. After gradient dilution of 10, 10The antifungal activity of endophytic bacteria was investigated using Guy11, a standard strain provided by the Rice Research Institute of Sichuan Agricultural University. First, 5 mL of sterilized fermentation broth was filtered by a 0.22 μm filter and added to 100 mL of cool uncoagulated PDA medium. This was shaken well and used to create a drug-containing plate. Then, we inoculated a rice fungus cake on the drug-containing plate and incubate it in a constant light incubator for 7 days at 28°C with alternating light and dark period. The inhibition rate of each endophytic bacterial filtrate ferment was counted, and the most active endophytic strain was selected as the antagonist UTF-33 as the reference strain for further study. The formula used to calculate inhibition rate was as follows:M. oryzae in the treatment group – diameter of M. oryzae cake in the treatment group)/(diameter of M. oryzae in the control group – diameter of M. oryzae cake in the control group)] × 100.Inhibition rate (%) = [1− and 1492R (5-TACGGCTACCTTGTTACGACGACTT)]. PCR (polymerase chain reaction) was then performed according to previous literature . PCR ampThe paper agar diffusion method was used to detect the sensitivity of UTF-33 to various antibiotics. Fresh endophytes were diluted 100 times and applied evenly on the surface of PDA plates. After uniform coating, drug-sensitive tablets containing different antibiotics were placed in the center of the medium, and the plates were placed in an incubator at a constant temperature of 28°C for 24 h. Finally, susceptibility was determined by measuring the size of the corresponding inhibition circle according to clinical and laboratory standards .2 blocks of 15 plots  with a 40–50 cm interval between each plot. Dewy LTH (Lijiangxintuan) seeds were distributed in the field blocks, covered with film until the rice seedlings grew to 15–20 cm, and were cultivated for approximately 15 days after the film was removed. The leaves of LTH in the field were sprayed with 150 mL of sterile water, LB (Luria-Bertani) medium, carbendazim, UTF-33 bacterial solution, UTF-33 filter fermentation solution, and 1 × 105 CFU/mL fungus conidia suspension, mixed in equal amounts. All of these solutions contained 0.1% Tween-20. After treatment, the rice was left to grow naturally for 5–7 days, and the disease susceptibility of each treatment group was observed and quantified by the random sampling of each leaf intercepted 5 cm for the number of disease spots.A trial was conducted to evaluate the control of UTF-33 filtrate fermentation solution on rice blast in the field at Wenjiang District, Chengdu, Sichuan Province, China . The paddy fields were divided into 1 m5 CFU/mL of fungus conidia suspension, followed by leaf collection at 0, 24, 48, 72 and 96 h, respectively  until the three-leaf stage. Then, the plants were sprayed with equal amounts of sterile water, UTF-33 filtered fermentation solution, and 1 × 10ectively . Total RLTH leaves were treated as described above and leaves were collected at 0, 24, 48, 72 and 96 h post-treatment, respectively. Then, we weighed 50 mg of DAB  powder and dissolved this with a small amount of concentrated hydrochloric acid. The volume was then made up to 100 mL with distilled water to 100 mL and the pH was adjusted to 3.8. Then, the leaves were treated for different times in DAB staining solution, placed under vacuum for 30 min, and then placed in the dark at room temperature for 12 h . Next, t5 CFU/mL with a hemocytometer under a microscope (Conidia were inoculated onto CM (complete medium) plates with Guy11, incubated at 25°C for 7–11 days with alternating light and dark, and then scraped and filtered through non-woven fabric to remove the impurities left by the mycelium and medium. The filtered spore solution was then centrifuged at 10000 r/min for 2 min; then, the supernatant was removed and the procedure was repeated twice. Next, we added an appropriate amount of sterile distilled water to resuspend the spores, mixed thoroughly, and adjusted the concentration to approximately 1 × 10croscope . Then, wsfp, ituD, fenB, bioA, and bmyB genes , as shown in s 99.43% ; thus, tAnalysis  showed tAs shown in OsActin gene was used as an internal reference to detect the normalized expression of genes related to the rice defense response, which was divided into three main categories: (1) genes related to disease course, including OsPR family genes such as OsPR1a, OsPR10a and OsPR5  genay genes ; (3) genfamilies .OsPR1a and OsPR5 genes, peaked at 72 h. Compared with the control group, the expression of OsPR1a was upregulated by approximately 90-fold while that of OsPR5 was upregulated by approximately 30-fold. The expression of OsCEBiP and OsLYP6 peaked at 72 h while that of OsLYP4 peaked at 24 h. Compared with the control group, the expression of OsCEBiP was up-regulated by approximately 11-fold, OsLYP6 by approximately 3-fold, and OsLYP4 by approximately 15-fold. The expression levels of both the salicylate signaling receptor gene OsNH1 and the OsMAPK6 signaling pathway gene peaked at 24 h. Compared with the control group, the expression of OsNH1 was up-regulated by approximately 3-fold while that of OsMAPK6 was up-regulated by approximately 4-fold. The expression of genes encoding transcription factors  peaked at 24 h (the expression of OsWRKY89 at 24 and 48 h was very similar), while the expression levels of OsEREBP were up-regulated by approximately 7-fold, OsWRKY53 by approximately 2-fold, and OsWRKY89 by approximately 6-fold when compared with the control group. In summary, the antagonistic bacterium UTF-33 promoted the expression of rice defense genes, and the concentrated response was expressed in the early and mid-late stages after rice treatment.Analysis  indicate2O2 accumulation (as detected by DAB staining); the most intense DAB staining was observed in leaf material at 72 h, thus showing the maximal accumulation of H2O2.Analysis  showed tAs shown in bioA, bmyB, fenB, fenD, ituD and srfAA obtained after sequencing analysis were 187 bp, 332 bp, 1,306 bp, 252 bp, 1,108 bp and 187 bp, respectively, corresponding to the number of encoded amino acid residues of 54, 156, 433, 83, 366 and 65, respectively. The nucleic acid sequences of each gene were compared by NCBI; analysis showed that these functional genes had very high and uniform similarity with Bacillus velezensis, Bacillus sp. and Bacillus amyloliquefaciens strains featuring corresponding genes, with 97.24, 99.07, 99.92, 99.58, 99.91 and 98.21% similarity, respectively. The nucleic acid sequence comparison with common strains is shown in bioA, bmyB, fenB, fenD, ituD and srfAA genes corresponded to adenosylmethionine - 8-amino-7-oxononanoate transaminase in Bacillus velezensis, bmyB in Bacillus subtilis, fengycin synthetase in Bacillus subtilis, FenD in Bacillus subtilis, bacillomycin D biosynthesis malonyl-CoA transacylase BamD in Bacillus velezensis, and surfactin non-ribosomal peptide synthetase SrfAA in Bacillus velezensis, with similarities of 98.15, 99.02, 99.77, 91.57, 98.91 and 97.06%, respectively. In summary, the functional genes involved in the synthesis of lipopeptides were successfully detected in the antagonist bacterium UTF-33.Bacillus, as one of the most important microbial pesticides, exerts inhibitory effects through mechanisms such as competitive action, antagonism, and the induction of plant resistance  production. ETI is triggered by the recognition of effector proteins produced by pathogenic microorganisms by the plant’s disease-resistant R proteins, including the production of resistant SAR (systemic acquired resistance) and the activation of various protein kinase cascades (The plant’s innate immune system consists of two immune responses: PTI (PAMP-triggered immunity) and ETI (effector-triggered immunity) . PTI is cascades . The defcascades .Bacillus mojavensis UTF-33, PR1a and PR5 (genes related to the course of disease) reached maximal expression levels at 72 h. The genes associated with the titin signaling receptors (OsCEBiP and OsLYP4 and OsLYP6) also reached maximal levels during the same period. OsCEBiP is known to play a key role in the perception (OsLYP4 and OsLYP6 encoding proteins that are capable of sensing PGN (peptidoglycan) and fungal titin as bifunctional PRs (pathogenesis-related proteins)  also reached maximal levels at this time; this is consistent with the timing and characteristics of the rice autoimmune response when analyzed together with the H2O2 accumulation results. It was evident that the immune response of the treated rice was concentrated in the early and mid-late stages, with the initial defense against infestation in the early stage; this was due to pathogenic infection stimulating PRS and up-regulating the expression of the fungal titin recognition pathway. The mid-late stage mainly targeted the formation of the infestation pegs, mediating the internal synthesis of SA, and upregulating the expression in concert with multiple transcription factor-related pathways to complete the immune response. In recent studies  . The gen studies  identifiBacillus velezensis (Bacillus mojavensis UTF-33 was clearly more effective. Considering both the control effect of biopesticides and the economic effect at the same cost, Bacillus mojavensis UTF-33 is more advantageous.In addition, fermentation products were extracted with n-butanol to obtain crude extracts. We found that these crude extracts had a significant effect in delaying and inhibiting the germination of rice fever conidia and the formation of attachment cells; furthermore, some of the treated conidia showed distortion after 12 h. Most of the mycelium that could germinate also swelled and broke and could not form a normal attachment cell structure. This indicates that these extracts can directly inhibit the developmental growth process of rice fever conidia, as described previously by Chen et al. who treated conidia using the fermentation product of lezensis ; howeverBacillus mojavensis UTF-33 expressed genes required for the synthesis of various lipopeptides, including bioA, bmyB and fenB, ituD, srfAA. fenB, ituD, and sfp. Synthesis products are useful as Bacillus extracts are often used in antifungal studies and have recognized potential for use in biocontrol (B. velezensis 9D-6 produces surfactins C14 and C15 to antagonize fungal pathogens such as Fusarium oxysporum, Gibberella zeae, Pyrenochaeta terrestris (Bacillus subtilis WL-2 antagonizes potato blight by producing Iturin A (Bacillus subtilis Z-14 and Bacillus amyloliquefaciens FZB42 to destroy the structure of Gaeumannomyces graminis var. tritici, and Fusarium graminearum to achieve a suppressive effect (Bacillus mojavensis UTF-33 has the potential to synthesize a variety of lipopeptides and is presumed to have direct antagonistic effects on a variety of plant pathogens. It is necessary to further purify and investigate the metabolic activity of a variety of antibacterial active substances to investigate their mechanisms of action and broaden the scope of application.Functional gene amplification experiments were also conducted; we found that ocontrol . For exarrestris  and BaciIturin A . In a pre effect . In contBacillus mojavensis UTF-33 as a biopesticide was further evaluated. Leaf disease and spot statistics of the fermented filtrate spray treatment were significantly lower than those of the water and LB treatment groups, and were similar to those of the carbendazim treatment group. Analysis showed that Bacillus mojavensis UTF-33 also exhibited excellent bacterial inhibition activity in practical use and could be a beneficial tool for controlling rice blast in the field.In the field trials, the control effect of Bacillus mojavensis against Fusarium verticillioides. They also used the metabolites of this species to reduce the lesions of maize stalk seedlings, and developed it as a biopesticide for disease control in maize (Bacillus mojavensis to extract lipopeptides such as surface activator and eugenol (Bacillus mojavensis on rice diseases. Our findings illustrate the direct inhibitory effect of Bacillus mojavensis UTF-33 on rice blast and the indirect induction of the rice defense gene expression mechanism. These data confirm the potential of Bacillus mojavensis as a biopesticide and advance the progress of research on the disease suppression mechanisms in plant endophytes.In recent years, Snook et al., Bacon et al., and Hinton et al. used maize as experimental material to study the antagonistic mechanism of in maize ,b. Hanen eugenol , and als eugenol , or opti eugenol . The preThe original contributions presented in the study are included in the article/ZX, RC, YZ, and YY contributed to conception and design of the study. YZ, YY, LZ, and ZZ organized the database. YZ, JZ, XG, and JY performed the statistical analysis. YZ wrote the first draft of the manuscript. YY, YH, ZH, and LL wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Periodic health checkups (PHCs) represent a unique system in Japan that is useful for the early detection of lifestyle-related diseases and cardiovascular diseases (CVDs). This study aims to investigate the association of PHCs with the hospitalization risk of patients with type 2 diabetes mellitus (T2DM).A retrospective cohort study was conducted from April 2013 to December 2015 and included participant information such as CVD history, lifestyle, and whether PHC was conducted in addition to regular medical examinations. Difference in clinical data between patients with and without PHC was examined. Furthermore, Cox regression analysis was performed to investigate the independent association of PHCs with hospitalization.Herein, 1,256 patients were selected and followed up for 2.35 ± 0.73 years. In the PHC group, body mass index, waist circumference, proportion of patients with a history of CVD, and number of hospitalizations were lower than those in the non-PHC group. Furthermore, the PHC group exhibited a significant association with lower hospitalization risk  in the Cox model.This study revealed that PHCs minimized the risk of hospitalization in patients with T2DM. Furthermore, we discussed the effectiveness of PHCs in enhancing health outcomes and reducing health care costs in such patients. The rate of cancer detection, including that of suspected cancer cases and confirmed cancer diagnoses in examinees, has been estimated as follows: lung cancer, 0.41% and 6.65%; gastric cancer, 0.7% and 11.36%; and breast cancer, 1.34% and 16.23%; respectively  that are usually conducted annually represent a unique system in Japan created for the early detection of lifestyle-associated diseases and cancer . The goaCost escalation of health care is a serious problem worldwide. The global economic burden of T2DM will increase to >$2 trillion by 2030 . The trePatients with T2DM exhibit physically weakness , suscept22.1This retrospective cohort study was conducted at National Center for Global Health and Medicine (NCGHM), Kohnodai Hospital, a public, secondary care hospital in Japan. Between April 2013 and December 2015, patients who came to the outpatient department and were diagnosed with T2DM were enquired regarding their medical history. Furthermore, data regarding diabetes and lifestyle-related behaviors after obtaining informed consent from the patients were collected from the Biobank for Metabolic Disorders in the NCGHM Kohnodai Hospital  and retrThe research design was certified by the Medical Ethics Committee of the NCGHM (Reference No. NCGM-G-002052), this research was conducted as per the Declaration of Helsinki.2.2PHCs are performed voluntarily and separately from regular medical examinations for patients with T2DM as well as healthy individuals. In Japan, company employees and students have the provision of availing PHCs annually. In addition, Japanese individuals aged between 40 and 74 years are encouraged to undergo annual health checkups that include physical body measurements and examination, BP measurement, blood examination  and urine examination (protein and glucose) to prevent the incidence and progression of lifestyle-associated diseases, such as diabetes mellitus, hypertension, and dyslipidemia . Althoug2.3via the standard formula: BW (kg) divided by the square of individual height measured (m). Waist circumference (WC) was measured in centimeter using an inch tape at the umbilical level at the end of expiration from a standing position. A standard automatic sphygmomanometer  was used to measure the BP in a seated position and sedentary state.A rigid stadiometer  was employed to document the height of the participants and a standard calibrated scale  was used to determine the body weight (BW). Body mass index (BMI) was calculated 2.4A trained professional from the Clinical Research Center of the NCGHM at Kohnodai Hospital recorded the CVD history of the patients, comprising information such as stroke, nonfatal coronary artery or peripheral artery disease, and foul habits, including smoking and alcoholism, during the baseline evaluation. To quantify the smoking habits of the participants, their Brinkman index was calculated as the number of cigarettes smoked per day multiplied by the number of years of smoking . Alcohol2.5We measured the blood glucose and HbA1c of the participants at the time of enrollment. The glomerular filtration rate (eGFR) of the participants was documented using the revised equation exclusively adjusted for the Japanese patients .2.6post-hoc sample size estimation using a command comparing survival curves between two independent groups in EZR . Continuous variables were presented as the mean ± standard deviation (SD). Student’s t test or the Mann–Whitney test, depending on whether the variables followed normal or non-normal distribution, was preferred to find differences between patients who did and did not undergo PHCs, respectively. Categorical variables were expressed as numbers and compared using χ2 test. The Cox proportional hazard equation was used to examine the independent associations of hospitalization, CVD occurrence, and death with PHCs. We included age, gender, BMI, CVD history, AC, Brinkman index for smoking habit, exercise time, systolic BP (SBP), diastolic BP (DBP), blood glucose, HbA1c, and eGFR in the Cox model. The significance level for the study was fixed at <0.05.32, respectively. Patient characteristics are presented in The study recruited 1,256 patients with T2DM, of which 695 were men, 561 were women, and 557 (44.3%) had undergone a PHC. The mean age and BMI were 63.7 ± 13.9 years and 25.5 ± 5.5 kg/mBMI, WC, number of patients with CVD history, and number of hospitalizations during the study period were lower in the PHC group than in the non-PHC group. Furthermore, AC, exercise time, and walking time were higher in the PHC group than in the non-PHC group. On an average, patients without PHC were admitted once to our hospital during the study period Table 2.During a mean follow-up of 857 ± 267 days, 20 patients (1.6%) died, 14 (1.1%) sustained cardiovascular events, and 550 (43.8%) were admitted to our hospital. The number of patients who were hospitalized at least once was 213 (38.2%) in the PHC group and 337 (48.2%) in the non-PHC group. The total number of hospitalizations was 1092, of which 382 belonged to the PHC group and 710 to the non-PHC group. Of these, 378 (34.6%) belonged to the diabetes, endocrinology, and metabolism ward, 165 (15.1%) to the surgery ward, 135 (12.4%) to the internal medicine ward, 85 (7.8%) to the ophthalmology ward, 79 (7.2%) to the gastroenterology ward, 65 (6.0%) to the hepatology ward, 36 (7.5%) to the cardiology ward, 27 (2.5%) to the respiratory medicine ward, and 122 (11.1%) to other wards, such as neurology, rheumatology, and psychiatry.Furthermore, Cox proportional hazard analyses with adjustment for age, gender, BMI, CVD history, drinking habit, smoking habit, exercise time, SBP, DBP, blood glucose, HbA1c, and eGFR showed that PHCs exhibited a significant impact on patient hospitalization risk ; however, there was no significant association of PHCs with CVDs and all-cause mortality Table 3.4We demonstrated that PHCs can remarkably lower the hospitalization risk of patients with T2DM. Although PHCs were not correlated with CVDs and all-cause mortality, the primary result of the study suggests that PHCs improved health outcomes in patients with T2DM. To the best of our knowledge, this is the first study to demonstrate that all-cause hospitalization could be prevented in patients with T2DM who have undergone a PHC in addition to regular medical examinations.A previous systematic review and meta-analysis concluded that general PHCs were not beneficial for reducing all-cause mortality and cardiovascular events; however, the studies included were from Western countries  . Ethnic Generally, patients who regularly visit hospitals for chronic diseases undergo PHCs voluntarily in Japan; thus, health consciousness and literacy could also influence their behavior. A systematic review reported that health literacy was a significant determinant of obesity . MoreoveA higher AC in the PHC group than in the non-PHC group is inconsistent with the finding that the patients who underwent PHCs were less obese and more active than the patients who did not undergo PHC. However, previous studies have indicated that AC impacted the levels of physical activity in a positive manner . AdditioTreatment costs vary depending on the cause of hospitalization, type and severity of disease, patient condition, and type of health care organization. For example, hospitalization due to uncontrolled diabetes costs ~¥40,000 ($296 at the current exchange rate) per day in our organization . ConsideHowever, PHC is not necessary for every healthy individual. Current evidence suggests that regular nationwide health checkups are neither beneficial nor cost effective to improve health outcomes . CompareReducing inequities in health and raising health awareness across all social strata are critical for improving health in patients with T2DM. In Japan, company employees and students can avail PHCs every year at the expense of the company or school, respectively. However, the unemployment rate has been positively associated with reduced PHCs . Furthervia a specific tool . Fourth, the socioeconomic status and educational level of the participants were not investigated. Individuals with high risk factors, such as smoking, physical inactivity, and low education and socioeconomic statuses, are less likely to attend PHCs than others . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.HH conducted the study, performed the data analyses, drafted the manuscript, and revised the manuscript. HY critically reviewed the manuscript and the scientific interpretations of study results. All authors contributed to the article and approved the submitted version.


Configurations and operations of microbial fuel cells are discussed.Bioelectrochemical system performance depends on the type of design and electrode materials.Microbial fuel cells are a feasible alternative for fuel production and wastewater treatment.Advances in electrode materials are summarized.Techno-economic and life-cycle assessments of microbial fuel cells are highlighted.2) and fuels . Furthermore, this review highlights practical applications, challenges, and the life-cycle assessment (LCA) of MFCs. Despite the promising biotechnological development of MFCs, great efforts should be made to implement them in a real-time and commercially viable manner.The over-exploitation of fossil fuels and their negative environmental impacts have attracted the attention of researchers worldwide, and efforts have been made to propose alternatives for the production of sustainable and clean energy. One proposed alternative is the implementation of bioelectrochemical systems (BESs), such as microbial fuel cells (MFCs), which are sustainable and environmentally friendly. MFCs are devices that use bacterial activity to break down organic matter while generating sustainable electricity. Furthermore, MFCs can produce bioelectricity from various substrates, including domestic wastewater (DWW), municipal wastewater (MWW), and potato and fruit wastes, reducing environmental contamination and decreasing energy consumption and treatment costs. This review focuses on recent advancements regarding the design, configuration, and operation mode of MFCs, as well as their capacity to produce bioelectricity (e.g., 2203 mW/m In response to the environmental pollution caused by conventional energy sources, researchers worldwide have searched for reliable and sustainable energy production sources. In this line, bioelectrochemical systems (BESs) capable of transforming chemical energy into electrical energy aided by microorganisms as catalysts have been developed . BESs ar−), protons (H+), and carbon dioxide (CO2) [− are transported toward the cathode chamber [2O). Equation (3) indicates the complete chemical reaction in an MFC. Additionally, the electron transfer mechanism at the anode surface of an MFC is presented in The components of an MFC basically include an anode compartment and a cathode compartment a, both sde (CO2) ; then, e chamber ,18. Subs2 was reached in a dual-chamber MFC inoculated with an excellent mixed culture [2 was achieved in an MFC operated with atomically dispersed Fe–N4 moieties as an excellent cathode catalyst [4 moieties were considered a perfect option to enhance the performance of the MFCs.Since the implementation of MFCs, wastewater has been used as an excellent organic substrate for these systems, improving their efficiency in both bioenergy generation and waste management ,23,24,25 culture . This hicatalyst . The atoAdditionally, the performance of an MFC system depends mainly on the electrode materials, anolyte/catholyte, pH of the medium, bacterial communities , type ofSince the implementation of MFCs, different types of designs and configurations have been proposed to improve their efficiencies, including single-chamber MFCs SC-MFCs; A, dual-cFurthermore, much research concerning the design and configuration of MFCs has focused on improving the performance of these systems by modifying the anode surface area ,39,40,41Many studies on different types of MFC designs have been carried out over the last decade. According to the ScienceDirect database (up to 1 June 2023), a total of 9470 research articles have been published in different Elsevier journals, of which 4519 focused on SC-MFCs, 2546 on DC-MFCs, 2054 on Stacked-MFCs, and 351 on TC-MFCs. Pseudomonas aeruginosa-MTCC-7814 had been applied in an MFC with the same characteristics. The system operated for 15 days, and the highest power density achieved was 105 mW/m2 with a current of 313 mA/m2. This performance was obtained when the SC-MFCs were connected in series, producing a maximum voltage of 1025 mV. The above results were interesting, and the authors hypothesized that adding the pure strain Pseudomonas aeruginosa improved power production in the SC-MFC. However, when the structure of the MFC was changed , a maximum power density of ∼104 mW/m2 was observed [Shewanella putrefaciens [2, respectively, due to modification of the electrode. These results were higher than those reported in the above-discussed studies. This achievement in the SC-MFCs was due to the anode electrode modification [In one experiment by Choudhury et al. , two SC-observed . This reobserved . Therefoefaciens  and pureefaciens , the higfication ,50. The fication .The above results demonstrated that most approaches for generating electricity using BESs have been limited to laboratory research, and the scaling up of these systems for practical application still needs to be improved. Hence, commercial and real-time application use must be the focus of further progress. In addition, there is a need to scale MFC systems to a practical level, in order to understand the usefulness of the bacteria’s electroactivity concerning the sustainable recovery of electricity, organic nutrients, and value-added products from wastewater with zero contamination. However, scaling up MFC systems requires searching for cheaper and more suitable materials than conventional ones .2) was used to build an air-cathode electrode. Meanwhile, for the anode electrode, bamboo charcoal (four pieces connected in series) was used. Peruvian clay ceramic was used as a membrane, due to its excellent surface porosity. Subsequently, the air-cathode MFC-Hyps with different working volumes were operated in a batch mode at room temperature  for 49 days. With the idea of evaluating the system’s performance, MFC-Hyps were assessed with and without the presence of plants (Allium tuberosum). The obtained data indicated that, when the MFC-Hyp system was embedded with a plant, the power generation efficiency was improved and more stable, indicating a higher power density of 130.2 mW/m2 with the highest COD removal of 96.3%, compared to those without plants. Furthermore, one of the advantages of the MFC-Hyp system is its positive effect on plant growth parameters, and agricultural farmers can adopt this kind of technology for the sustainable production of aliments. Another advantage of this system is the production of organic fertilizers, such as nitrogen (N), potassium (K), and phosphorous (P), thus reducing the risk of GHG emissions into the atmosphere compared to conventional agriculture. However, more studies in MFC-Hyp systems are required to shift this technology from lab-scale  was placed in an air-cathode MFC to improve the system performance. The air-cathode MFC was operated during two stages  and showed great potential to produce power, as well as presenting high contaminant removal efficiency, with a biochemical oxygen demand (BOD) and COD of 96% and 94%, respectively, at 75 days. At the same time, the air-cathode MFC indicated high removal efficiencies of 100% for NO3−, 95% for P, 75% for sulfate, and 47% for organic N. The system’s performance was considered to be due to the consortium and efficiency of the air-cathode operating conditions. In another recent study, an SC-MFC based on an air-cathode was operated in an open field for 25 days, using urine as a substrate, and showed excellent performance regarding power generation with cow urine [More recently, a novel SC-MFC based on a hydroponic system (MFC-Hyp), consisting of a plastic container with a practical water volume of 2500 mL, has been presented . Three uo et al. ) to praco et al. , dairy eow urine . This pe+2) were achieved along with the highest power density. Furthermore, in this comparative Table, each reactor’s working volume and operating time were considered.On the other hand, DC-MFCs, like other types of BESs, have been implemented for wastewater treatment, elimination of contaminants in the soil, nutrient recovery for sustainable agriculture, and bioelectricity generation. Researchers have used different methods to improve these systems in terms of power efficiency, working hard to manufacture prototypes which are capable of continuously generating bioelectricity with high power. 2, and SnO2) were applied in a DC-MFC as cathodic catalysts in order to recover phenolic compounds and generate bioelectricity using industrial wastewater. Apart from using these types of catalysts, the system was configured with carbon felt and carbon plate as anodic and cathodic electrode materials, respectively. Once the system configuration was completed, it was operated for 168 h. The findings of this exciting study demonstrated the feasibility and potential of DC-MFCs to generate bioelectricity while removing phenolics in wastewater. It has been observed that parameters such as pH and temperature influence the performance of DC-MFCs. For example, when the catholyte was pH 8 , the system produced a maximum power density of 29.24 mW/m2; in contrast, at pH 11, the bioelectricity production decreased by 43.50%  [An MFC with suitable electrode materials can power a digital clock, an LED , and bio2 mW/m2) . This st4 emissions by MFCs installed in waterlogging and paddy fields [−2, and mWm−3, making these systems challenging to compare [The utility of MFCs can be expected to bring about a paradigm shift built on sustainability principles, encompassing the closed-loop biorefinery approach. Such efforts would help to manage waste and generate renewable fuel and value-added products, thus fostering sustainable development . On the y fields , due to  compare .2, 97.6% lower than the bioelectricity generation reported in the previous study [Furthermore, in another study , a DC-MFus study . Due to us study .2 in the cathode chamber [In summary, the above studies demonstrated that improved power generation using BES systems  can be attained by coupling two inter-related systems (hybrids). Comparing the type of configuration of MFC technologies, it is not easy to say that one system is more effective than another if it presents a high efficiency rate. For example, the SC-MFCs based on an air-cathode configuration play a significant role in the implementation and performance of MFCs, considering different advantages such as robustness, low cost, simplicity in terms of structure and use, and low/less internal resistance , as has been previously reviewed ,62. Howe chamber ; when th chamber .A wide range of materials for electrodes have been used to assess the efficiency of MFCs. Finding efficient materials for the design of MFCs presents great challenges for researchers working in this field. While the operation and implementation of MFC technology have been limited by various factors, the electrodes are the primary aspects of the MFC. Different electrode materials have previously been investigated and tested to enhance MFCs. Fortunately, the test results were encouraging, as the materials showed potential to drive MFC technology forward. In this section, an overview of the different electrode materials that have been used most frequently is provided, as they can be considered to be suitable materials. For an electrode material (anodic or cathodic) to be suitable, it must possess high conductivity, anticorrosive, biocompatible, chemically stable, and high surface area and porosity characteristics, as described in . Using sIn addition, an appropriate material allows for the formation of biofilms in the anode region, and the generated bacterial community should remain in contact with the anode surface. However, if the anode material is not suitable , the electrons may decrease considerably; this would also imply a decrease in bioenergy production, as comprehensively reviewed in . Other cYaqoob et al.  have conThe membrane is an essential component in the configuration of an MFC. The use of a membrane in an MFC is inevitable, as it prevents the mixture of reactions coming from the cathode and anode compartments; it also prevents the transport of oxygen . In addiA variety of separators (membranes) have been used in MFC systems to date . Nafion Geobacter spp. (including Geobacter sulphurreducens), which have great potential. Due to their metabolic pathways, Geobacter sulfurreducens can produce high current densities in MFCs. Furthermore, Proteobacteria, Pseudomonas, and Shewanella are other groups of microorganisms that have been widely investigated in mediator-less MFC systems [Microbes are key drivers in the performance of BESs such as MFCs. The microbes are responsible for directly transporting the electrons through oxidation of the substrate. For successful electron transfer to occur, the electroactive microbes must be in contact with the anode surface via the extracellular membranes of the EABs. They (microbes) may also interact with electrodes through nanowires or electron media. In addition, cytochrome proteins from the extracellular membrane are responsible for the electron transfer process . From a  systems , as rece systems . To unde systems .Mixed cultures of bacterial communities forming a biofilm (from wastewater) and pure cultures have been widely used in MFCs to increase their working performance regarding power density . HoweverMFC technologies have received significant attention, due to their extraordinary capacity to produce bioenergy without harming the environment. Great strides have been made in improving bioelectricity production by MFCs. Many recent studies have reported high power generation efficiency using MFCs inoculated with different types of waste . 2 was obtained using an SC-MFC inoculated with anaerobic sludge as an outstanding inoculum [2  in an SC-MFC using carbon cloth as an anode and cathode, separated by a Nafion 117 membrane. This value was almost four times lower than that reported with Pt/C [2) reported in a tubular MFC (T-MFC) [2) as a cathode electrode, and nanocomposite as a proton exchange membrane. The findings indicated that the best option for improving the performance of an MFC is modifying its electrode  and using suitable configuration materials. In the previous study by Bensaida et al. [2 was obtained in a prototype MFC inoculated with Mg(OH)2-coated iron nanoparticles. This value was 29.19 and 83.34% superior to that in agro-waste and synthetic wastewater, respectively; however, it was approximately three times inferior to that reported in anaerobic sludge elsewhere [The highest power density of 2203 mW/minoculum . The SC-inoculum  obtainedith Pt/C . In the ith Pt/C . The resith Pt/C  was 76.6a et al. , the higlsewhere .Anaerobic sludge appears to be the most suitable inoculum for use in MFCs to improve the power density efficiency, compared to others. This kind of inoculum (sludge) has tremendous potential to increase the performance of MFC systems . This pe2 have been recorded in an SC-MFC inoculated with butyrate (1000 mg/L) and acetate (800 mg/L), respectively [2 was reported, approximately 9 times and 5 times less than those derived for butyrate and acetate, respectively [2 in a DC-MFC when removing hazardous materials from wastewater. This yield in MFC was due to the addition of 350 μmol/L of C6N6FeK3 (potassium ferricyanide) in the cathode compartment. Concurrently, 180 μmol/L of CH2 (methylene) was applied to the anode compartment. As mentioned above, a high concentration of the chemical compounds is an excellent option to increase the performance of MFCs while treating wastewater.On the other hand, in their quest to increase the amount of OM and keep bacterial populations active on the anode surface, researchers have investigated other types of substrates with great potential, such as acetate, butyrate, glucose, cellulose, and sucrose. These substrates have all been used in MFCs in an attempt to improve their output power, as described in . The maiectively . It was ectively . Hashmi ectively  have rec2-coated iron nanoparticles, rGOHI-AcOH, and acetate as excellent substrates (with excellent carbon sources to improve or stimulate EAB) for MFC technologies [According to the above studies, modifying the electrode material is the better or cheaper option to improve MFC performance, in terms of power density, compared to conventional electrodes. The configuration of an MFC using a conventional electrode is expensive, making its large-scale application much more costly. Furthermore, another alternative to improve the performance of MFCs is the use of organic substrates, which are excellent electron donors. Finally, anaerobic sludge has been reported to be one of the best inoculums  for improving power generation in MFCs, followed by Mg(OH)nologies ,119. Furnologies ,123, incnologies , bioremenologies ,126. Acc2 (within 20 days of operation) using potato wastewater as an electron donor for the biodegradation of pollutants in a benthic microbial fuel cell (BMFC). This MFC performance was 82.28% less than that previously reported in a DC-MFC operated with potato waste as the substrate [Yaqoob et al.  recordedubstrate . The stuubstrate  demonstrubstrate , the pH ubstrate . In addi2 was observed. The current density achieved by Sato et al. [Furthermore, in another investigation, an SC-MFC was configured using bamboo charcoal (BC) for the anode electrode and a Pt-coated carbon cloth for the cathode . The woro et al.  in potato et al. .In summary, the above findings indicated that potato waste—like other organic substrates—is potentially an excellent alternative, allowing for enhanced power generation and wastewater bioremediation performance when using MFCs. Furthermore, the electrode materials are other aspects to be considered when implementing MFC technology. However, more investigations are required to evaluate the influence of potato wastewater in MFC technological development.4 and H2 has captured the attention of researchers worldwide. Apart from the fact that this technology is environmentally friendly, it is also an excellent option for producing fuels while eliminating pollutants in wastewater. Furthermore, MFC technologies are less expensive, when compared to conventional methods. 2.In recent years, great strides have been made regarding the use of BESs, such as MFCs, for the simultaneous treatment of wastewater and production of fuels. This progress has been achieved by combining BES prototypes to improve their process performance. In this line, the use of MFCs for the production of both CH4 production. While 40 cm2 of carbon felt was utilized as an anode, the cathode was constructed from various materials . Before assembly of the SCMFC-AnMBR, the carbon felt was sterilized using 1 mol/L HCl and NaOH. The SCMFC-AnMBR was inoculated with synthetic wastewater (SWW) and compared to the conventional one (C-AnMBR). The SWW was added to SCMFC-AnMBR with a concentration ratio of 205:5:1 (C:N:P), and the main components of SWW were: (1) 3000 mg/L of glucose (C6H12O6), (2) 120 mg/L of monopotassium phosphate (KH2PO4), (3) 120 mg/L of ammonium chloride (NH4Cl), (4) 112 mg/L of iron chloride (FeCl2), and (5) 30 mg/L of magnesium sulfate (MgSO4). The SCMFC-AnMBR indicated high recovery efficiencies of 38.89 mg/L (COD) and 67.84 mg/L (CH4) and presented a maximum voltage of 107 ± 14 mV. This study demonstrated that the SCMFC-AnMBR is better than the conventional system, as it was stable in terms of the micro-bioelectric field environment [4 production. The prototype SMFC-AD was inoculated with glucose at different concentrations . The authors compared the performance of the combined system and the single one (AD). The results indicated that, at a concentration of 2 g/L of glucose, the SMFC-AD reached a higher CH4 production of 320 mg/L , when compared to the other concentrations used in this study. The CH4 production achieved [4 production is 2 g/L. Furthermore, high glucose concentrations negatively affected the reactor’s performance, in terms of fuel production.Hao et al.  have recironment . Meanwhiironment , a submeachieved  was 8 anSaccharina japonica (sDFMEC), in order to improve fuel production and accelerate the process. The sDFMEC recorded a maximum production of H2 of 438.7 ± 13.3 mL/g-TS, outperforming the control . At the same time, the sDFMEC system reached maximum CH4 production and COD removal of 63.1 ± 3.4 mL/g-TS and 75.6 ± 1.4%, respectively. Nonetheless, DF-MEC showed an efficient recovery of 32.2%, achieving a maximum H2 production of 403.4 mL/g-TS [2 production of 110 mL/g-VS was reported in a similar sDFMEC. This yield was 3.66 times lower than that reported by Nguyen et al. Later, in an sDFMFC constructed using a surface-modified stainless steel mesh cathode, a higher H2 production of 408 mL/g-TS was recorded [S. japonica into the sDFMEC reactor. However, all of these methods can feasibly improve CH4 and H2 production while conducting bioremediation of contaminants from wastewater.Additionally, in a previous study by Nguyen et al. , single- mL/g-TS . In cont mL/g-TS  a maximurecorded . The aut2 and CH4) include the use of other types of substrates, such as biochar, which led to increases in H2 production of up to 118.5% and in CH4 of up to 14% [2 [2 by 52% [4 injection on anaerobic oxidation (AO)-coupled MFCs (AO-MFCs), in terms of power generation [2 (13 × 6.5 cm) of breathable cloth was used as an anode, while the cathode was made of 81 cm2 (9 × 9 cm) of carbon felt, which were separated by an ultrafiltration membrane. The anode chambers were inoculated with 40 mL (0.04 L) of anaerobic sludge. The obtained results indicated that the specific EACs were responsible for converting CH4 to CO2 while producing bioelectricity.Other strategies used to improve fuel production , multi-layer Ti3C2TX MXene, and MAX phase titanium aluminum carbide (MAX). Subsequently, the reactor was inoculated with cattle manure (CM) and sewage sludge (SS). Furthermore, graphite rods were used for the anode and cathode materials. The MEC-AD operated with a 0.035 wt% of ML-MXene showed a maximum CH4 production of 358.7 mL/g VS. This result was superior to that reported in MECs (38.4 ± 1.7 mL/g TS) elsewhere [2 and CH4 production.Liu et al.  have conlsewhere . The recMFCs have been explored for their real-time application, similar to other bioelectrochemical systems. The main idea of research in this field is the scaling up of MFCs toward their commercialization. MFCs play a fundamental role in the bioremediation of contaminants, such as azo dyes, heavy metals , sulfides and sulfates, nutrients (nitrates and phosphates), antibiotics, and petroleum, from wastewater and soil ,160,161.2Cr2O7 or potassium permanganate, KMnO4) or anyolite  in an MFC could be an excellent alternative to improve its power density, as described in [Furthermore, the challenges faced by MFC technologies include low power density, the high cost of electrode materials, continuous electricity generation, low conductivity of electrode materials, and membrane fouling, as described in . In contribed in . Human uribed in ,169,170,ribed in  have recribed in , the Intribed in , and digribed in , as wellribed in . NeverthIn summary, the application of MFCs in different fields, including wastewater treatment, bioremediation of pollutants, and nutrient recovery while producing bioelectricity, is still in its beginning stage. Thus, there is a need for more research on the scaling of MFCs from laboratory to practical application, despite their limitations in terms of scaling up due to membrane fouling, voltage losses, and low power output. In addition, the knowledge gaps in terms of the designing, configuring, and operating conditions of MFC technologies should be addressed as soon as possible. It is worth mentioning that more studies on different designs of prototype MFCs are required, in order to carry out the design of such systems for large-scale and commercial applications. In previous critical reviews ,175, sevBESs such as MFCs have been a key area of research, regarding their commercialization worldwide, due to the high global energy demand and accelerated population growth and industrialization. The literature has reported that 80% of the world’s energy generation depends mainly on the exploitation of fossil fuels . HoweverAs MFCs are devices that recycle waste materials for energy generation  and improve water quality, they have great promise to become feasible bio-economic systems; however, wastewater substrates containing buffer salts , either in the anode or cathode of the system, are not economically or environmentally practicable . Additio−4/FU) and carbon paper (5.89 × 10−4 g/FU) as an anode electrode [−3 kWh/FU (profitability of 1.68 × 10−5 kWh/FU) [−3 kWh/FU when using graphite felt as an anode and carbon brush as a cathode material [LCAs are an emerging approach used to assess both bioenergy production and carbon sequestration through all the stages of a product’s life cycle. Additionally, an LCA is a well-known standardized technique that systematically and quantitatively evaluates the potential risks and impacts of a product or system’s environment throughout its life cycle. According to the International Standards Organization (ISO 14040: 2006), an LCA includes the following stages: (i) goal and scope definition, (ii) life-cycle inventory analysis (LCI), (iii) life-cycle impact assessment (LCIA), and (v) interpretation of results . The foulectrode , the ene kWh/FU) ,181. In material . In turnmaterial , comparematerial . Accordimaterial .−9 and 4.62 × 10−9 kWh/FU, respectively. These values represent the impact category during the LCIA study  [Furthermore, the third stage of the LCA (LCIA) involves evaluating the environmental impacts of MFC technology during its operational stage. This could include simultaneous wastewater treatment and electricity production. The LCIA comprises two steps: (1) midpoint impact categories, such as the source of emissions and resources ; and (2) health) .2O2 (hydrogen peroxide) in the MEC. Nevertheless, the low power generation of 10 W m−3 (volumetric power density) recorded in both the MFC and MDC systems was correlated with the external operating conditions, as described in [Zhang et al.  have devribed in . Low levribed in . This reIn another LCA study, Zhang et al.  assessedOn the other hand, an auto-circulating bioelectrochemical reactor (AutoCirBER) was designed at the laboratory scale to treat 8.4 L of DWW continuously . An expe−1 HCOOH [The environmental impact assessment of MFCs is essential, which has received much research attention compared to other aspects, such as the design, configuration, operation, and performance evaluations . Previou−1 HCOOH .2 and CH4). Thus, using an LCA allows researchers to assess the environmental impact of MFCs and propose possible solutions to mitigate the potential risks associated with implementing the technology [According to the discussion above, both techno-economic assessments and LCAs may be conducted to determine the economic viability and environmental impact of emerging technologies such as MFCs during their implementation. Before its use, every emerging technology must be evaluated in terms of GHG emissions , which have mainly been used for wastewater treatment. MFCs have great potential to produce bioelectricity while treating wastewater through activated sludge, as well as removing pollutants. Nevertheless, these systems have faced significant limitations and challenges since their implementation. The major limitations of MFC technologies are high internal resistance, unstable current, biofouling, low power output, and mass transport loss, and the primary challenges faced by MFC researchers include expensive electrode materials, scaling up to large volumes, and energy losses due to the resistance of the conducting materials when compared to conventional systems. It will be necessary to address these challenges in order to overcome the limited real-time application and commercialization of MFCs. Despite these limitations, MFCs present various advantages, such as the ability to power BOD biosensors, digital clocks and LEDs, and sensors integrated into the Internet of Things (IoT).Furthermore, MFCs have a significant advantage in terms of reducing important greenhouse gas emissions. Hence, it is necessary to perform an LCA of MFCs in order to mitigate their environmental impacts. On the other hand, idealized design and operating conditions, such as the smaller electrodes, reactor volumes, and static conditions commonly used in laboratory-scale setups, often result in better performance than in real-time large-scale application systems. Further research is required to address the limitations and challenges of MFCs, including scaling up using modified electrode materials, which may make this technology practicable and commercial.