AGEM = 8;0) living in the UK who have Romanian as heritage language (L1) and English as majority language (L2) and 32 Romanian monolingual children aged 6;11 to 9;8 . We examined whether differences emerge between heritage and monolingual children in the online comprehension and in the production of multiple interrogatives in Romanian, which requires fronting of all wh-phrases, contrary to English. The main aim was to uncover to which extent similarities or differences in morphosyntactic properties between the L1 and the L2 systems affect the acquisition and processing of the heritage language/L1. Online comprehension was assessed in a self-paced listening task, while production was assessed using an elicitation task. The results reveal that Romanian heritage children show similar online comprehension patterns to monolingual children for multiple interrogatives in Romanian. A different pattern emerges for production as heritage children produce less complex multiple questions in Romanian and avoid movement of two wh-phrases in all elicited structures. Given that their predominant responses for multiple interrogatives only make use of the structural option present in English, namely one fronted wh-phrase and one in-situ, we take this to show that there is transfer from the majority language to the heritage language. Thus, language production in the children’s L1 seems to be affected by properties of the dominant L2, under cross-linguistic influence. Taken together, the results for both comprehension and production suggest that heritage children are able to establish the underlying representation of multiple wh-movement structures, similarly to monolinguals, but have difficulties activating the more complex structure in production.This study compared the online comprehension and the production of multiple interrogatives in 18 Romanian-English bilingual children aged 6;0–9;2 ( Various studies on heritage language (HL) acquisition have investigated the end-state grammars of adult HL speakers ,b and hain situ. By investigating performance under different modalities, we aimed to get a more straightforward glimpse at the nature of the differences between child heritage speakers and child monolingual L1 speakers and how this relates to cross-linguistic influence , and also wh-in-situ questions as in *Jij doe wat giraffe? (lit. You do what giraffe?). According to Strik and Pérez-Leroux, complexity is a trigger for cross-linguistic influence such that structures involving less derivational complexity in one language  may impact structures which are derivationally more complex in the other language .The acquisition of wh-dependencies in the HL has also received little attention. These previous works reporting different performance patterns in heritage compared to monolingual children assessed only children’s productive skills in their heritage language/L1. Various studies with monolinguals and bilinguals have revealed asymmetries between comprehension and production  and althwho-questions and (2) exemplifies which-questions in Romanian.Full acquisition of multiple wh-questions involves various aspects that are subject to cross-linguistic variation. We will briefly outline the properties of multiple wh-questions that Romanian-speaking children need to acquire, by putting emphasis on differences with English. (1) illustrates multiple 1.a.  Cine    pe cine  acoperă?    who.Nom PE who  covers    ‘Who is covering whom?’ b.  *Pe cine  cine  acoperă?    PE who  who covers    *‘Whom is covering who?’j îlj   acoperă?2.a.  Care  fată  pe care  băiatj    himj  covers    which girl  PE which boy    ‘Which girl is covering which boy?’j care  fată  îlj   acoperă?  b.  Pe care  băiatj  which girl himj  covers    PE which  boy    ‘Which boy is which girl covering?’lexical properties of wh-words, wh-objects in Romanian are marked with a differential object marker pe, similar to a in Spanish. Although in the prescriptive use of English, who shows overt case-assignment in the form of whom, increasingly native English-speakers use who instead in informal spoken contexts (care (‘which’)-phrases in Romanian are doubled by a co-indexed clitic pronoun îl (‘him’) for masculine and o (‘her’) for feminine.In terms of contexts . Additiomovement properties, wh-words move overtly, the difference with respect to English being that multiple wh-words in Romanian move together to a clause-initial position, as shown by (1) and (2) above. This is a property that Romanian shares with Bulgarian and other Slavic languages. According to via a ‘tucking in’ mechanism , as indicated by the asterisk in example (1b). Movement of wh-words in both languages obeys Superiority  is “The girl covers the dog and the boy covers the cat.” in which the exhaustive sets of who and which are pairwise linked.The wh-questions. The acquisition of multiple wh-questions has received relatively little attention in the literature. -in-situ language. Grebenyova’s findings demonstrate that English- and Malayalam-acquiring children have adult-like knowledge of the syntax of multiple wh-questions, whereas Russian-speaking children allow fronting of only one of the wh-phrases, following an English-like structure.Children’s experience with such sentences is extremely reduced. wh-questions . wh-element present in the question. In the same vein, wh-word, which was also the subject.To our knowledge, three studies so far investigated the acquisition of multiple wh-questions in Romanian and they all looked at how Romanian-speaking monolingual children ranging in age from 4 to 9 years old comprehend this type of question. who and which-questions, who- compared to which-phrases, as measured by reaction times (RTs) in a self-paced listening task. However, only adults seem to show an online sensitivity to the ordering constraints in who-questions illustrated in (1b) above. Bentea & Marinis also report higher accuracy scores with multiple who- than which-questions and show that the latter pose more difficulties for comprehension, particularly in the object-subject order , where participants  show a preference to interpret the first wh-element as agent, along the lines of what has been reported for the processing and comprehension of simple which-questions. Bentea & Marinis also found that children even at the age of 6 and 7 answered only one of the wh-phrases, similarly to exhaust the question domain and also pair the two wh-elements. Therefore, the question that arises is whether bilinguals, who receive less input than monolinguals and are often not tutored in the L1, converge on the correct syntactic structure for multiple wh-questions and attain knowledge of the grammaticality distinctions among who and which-multiple questions, especially when such wh-dependencies display a different structure in the L2, the societal language.In a recent study on the processing of Romanian multiple To sum it up, multiple wh-interrogatives allow to explore the extent to which bilingual children’s language comprehension and production are affected by cross-linguistic influence, as they vary across languages and display language-specific syntactic and semantic properties that children need to acquire although these structures are not frequent in the parental input. In this study, we compare for the first time both the on-line/off-line comprehension and the production of these structures in Romanian heritage children in order to get a clearer picture of the way in which the societal language (here English) influences the acquisition of morphosyntax in the HL.The present study investigates the early stages in the acquisition of the HL to examine whether the differences that emerge between HL children and monolinguals hold not only for production, as has been shown by the previous studies examining the acquisition of simple wh-questions in HL children, but also for comprehension. We postulated that the use of a more sensitive and implicit on-line comprehension task, like the self-paced listening task used in this study, might offer a more straightforward glimpse into underlying language representations that are accessed for real-time processing. Together with production tasks, on-line comprehension might help to better understand what differentiates between HL children and monolingual children. The study focused on Romanian as heritage language and addressed the following research questions:1. Do Romanian HL children and Romanian monolingual children differ when processing questions with multiple wh-fronting in an on-line processing task?Previous studies with L2 children looking at real-time sentence processing report qualitatively similar processing patterns in bilinguals and monolinguals for tense , article2. Do Romanian HL children and Romanian monolingual children differ with respect to the production of interrogatives with multiple wh-movement and how does this compare to comprehension?in-situ, under cross-linguistic from English, the majority language. Moreover, if asymmetries arise between production and comprehension, then we expect the results to show a similar pattern to that reported for other bilingual populations in which comprehension of multiple interrogatives outpaces their use in production  living in the United Kingdom (Greater London area and South-East England) and 30 Romanian monolingual children aged six to nine  living in Romania, participated in the studyEighteen 6- to 9-year-old Romanian heritage children with English as L2  and the mean length of exposure (LoE) to English was 6;0 years . The language background data for the heritage children illustrated in t-test , whereas English is the dominant language outside the home , as well as when it comes to children’s current expressive language skills, as reported by the parents .Details regarding the bilingual children’s language history, including information about their current use of and exposure to both Romanian and English, were collected using a modified version of the Questionnaire for Parents of Bilingual Children PABIQ; . Three pChildren’s ability to comprehend Romanian multiple wh-questions in real-time was assessed with an on-line SPL task with picture verification see . In this-phrases in which we crossed two factors: the order of the wh-constituents [either the wh-subject preceded the wh-object (SO) or vice-versa (OS)] and the type of wh-phrase (who vs. which). There were 40 test items in total, with 10 items per condition. The sentences included stories about superheroes  and princesses  engaging in imaginary activities with different people or animals. At the beginning of every trial, children listened to a lead-in sentence, introducing the characters. This was then followed by the test sentence  segmented into 8 parts, each part containing one to three words. No images appeared on the screen while children listened to the lead-in and the test sentence. The slashes in the examples signal the end of each segment when children had to press the Space bar to hear the next segment. After the final segment of each test sentence, a picture with three pairs of characters appeared on the screen. All the visual stimuli used in the study can be provided upon request. The position of the pairs and the direction of the actions varied between pictures. Children had to verbally identify all the pairs of characters performing the action described in the sentence. An answer was coded as “correct” when all the relevant pairs were identified. After hearing the sentence in (3), for example, the expected answer was The fireman is splashing Superman and the elephant is splashing Batman. This is because Romanian only allows exhaustive pair-list interpretations for multiple wh-questions like the ones exemplified in (3) to (6) below.The self-paced listening task in the current study was presented as a computer game with a puppet (Paddington the Bear). The children were told that they have to listen very carefully to Paddington’s questions in order to be able to identify the correct characters in the picture that followed each question. The experimental items contained embedded questions with two fronted whLead-in:    This is an image of Spiderman, Superman, Batman, a boy, a fireman and an elephant.Test sentence: Paddington/ wants to know/SO who  cine/pe cine/ stropește/în joacă/seara/           la circ/ 3.         who PE whos plashes jokingly in the evening        at the circus        “who is splashing whom jokingly at the circus in the evening.”OS who  *pe cine/ cine/ stropește/în joacă/  seara/          la circ/ 4.         PE who   who splashes  jokingly    in the evening       at the circus        “who whom splashing jokingly at the circus in the evening.”Lead-in    This is an image of Anna, Elsa, Jasmine, and three monkeys.Test sentence: Paddington/ wants to know/SO which  care  prințesă/pe care  maimuță/  o/  acoperă/  dimineața/      la zoo/ 5.         which princess  PE which monkey      her   covers    in the morning    at the zoo        “which princess is covering which monkey at the zoo in the morning.”OS which  pe care prințesă/care    maimuță/  o/  acoperă/  dimineața/      la zoo/ 6.         PE which princesswhich monkey        her  covers     in the morning    at the zoo        “which princess is which monkey covering at the zoo in the morning.”who-questions and the other half were simple subject which-questions. They included both transitive actions  in which the agent was always animate and the patient inanimate, as well as intransitive actions .Apart from the 40 test items, 10 fillers were also included as distractors. Half of the fillers were simple subject who-questions with a SO order (7), 12 items elicited multiple which-questions (six with a SO order as in (8) and six with an OS order as in (9)), while six more items were designed to elicit multiple wh-questions with an OS order (10), in which the object was a which-phrase and the subject a who-phrase, a grammatical option in Romanian.For the elicited production task, children played a guessing game with Paddington the Bear in which they were prompted to produce 24 multiple wh-questions with a subject-object and an object-subject order. Six items were designed to elicit multiple SO who     Cine  pe   cine  a  mângâiat?7.          who   PE   who  has patted          “Who patted whom?”SO which     Care  fată  pe   care  pisică  a   mângâiat-o?8.          which  girl  PE   which  cat    has  patted-her          “Which girl patted which cat?”OS which     Pe  care   pisică  care  fată   a    mângâiat-o?9.          PE  which   cat    which  girl   has   patted-her          “Which cat did which girl pat?”OS which-who  Pe  care  pisică  cine   a    mângâiat-o?10.          PE   which  cat     who   has   patted-her          “Who patted which cat?”Children interacted with a Paddington the Bear puppet for the whole duration of the task (see details in the Procedure section below).Here are two girls, a boy, two cats and a monkey, which helped them to familiarise themselves with the characters in the image. The experimenter then covered Paddington’s eyes and ears so that the puppet could not see or hear anymore what was happening next in the image. At this point, the child saw another image and heard Look! This girl is patting the black cat and this girl is patting the white cat. The boy is taking a picture of the monkey. Then the image presented at the beginning of the trial appeared again on the screen. Paddington’s eyes and ears were uncovered and afterwards the experimenter would tell the puppet Paddington, we can tell you that the boy did not pat anyone, but each girl patted a different cat. The experimenter then prompted the child to ask Paddington a question about this  and then Paddington made his guess. In order to make sure that children produced questions with two wh-phrases, they were told that they need to ask questions about two things at the same time. The task also included six filler items. These prompted children to produce simple wh-questions (three argument questions with a mismatch in animacy like “What did the father wash? and three adjunct questions such as Where did the queen sit?). Children’s responses were coded as “felicitous” when they produced a target question with multiple wh-movement like in examples (7) to (10) above. Other responses were coded as “infelicitous,” alongside the type of error produced.The structure of each scenario was as follows. First, both the child and Paddington saw an image of Lego figures and heard The study was approved by the Ethics Committee of the University of Reading. Informed parental consent was obtained for each child prior to the testing sessions, as well as the oral consent of the child. Each participant was tested individually in a quiet room either at their school or in their home. The experimenter gave oral instructions for both tasks. These were administered at least 1 week apart and in different orders, such that half of the children first saw the SPL task, followed by the elicited production task, while for the other half we first assessed production and then comprehension. Each testing session lasted around 30–40 min, but children could take breaks whenever they felt tired. They received stickers and certificates of participation after each task and at the end of the study they received as well a voucher which they could use to buy books at local bookstores.what-questions  and two multiple wh-questions with an animate subject and an inanimate object . Each participant then listened to a total of 50 sentences during the test phase and these were randomized in PsychoPy. The task instructions and all the sentences were pre-recorded by a native speaker of Romanian.The SPL task was programmed and administered using PsychoPy . The tasSO who questions) and the order of the blocks was randomized across four lists, each containing 30 items. The task began with four practice trials which elicited multiple wh-questions with a mismatch in animacy . The audio presentation of the items was also pre-recorded by a native speaker of Romanian. The questions that the children produced were recorded on answer sheets and then coded for analysis.The elicited production task was administered as a PowerPoint presentation. The task started by introducing each child to the Paddington puppet. The experimenter explained that Paddington wanted to become a magician and for this he had to improve his guessing skills. The child’s task was to help Paddington by playing a game with him in which the child asked Paddington questions about various images and the puppet had to guess the correct answer. For each correct guess, the child gave Paddington a smiley face sticker and if Paddington had at least 20 correct guesses by the end of the task, the child gave Paddington his Magician Diploma. Given the complexity of the target questions, these were presented in four blocks and interspersed with fillers. Each block contained 6 items of the same type , but show lower accuracy for multiple which-questions.The fireman is splashing Superman, the elephant is splashing Batman, and the boy is pulling Spiderman although the question only refers to the action of splashing. List answers are cases in which children only answer one of the two wh-words by exhaustively listing all the characters involved in the action. That is, they would answer question (5) with Jasmine and Elsa. Role reversals are answers in which the Agent-Patient roles are reversed. For instance, children would answer the question in (6) with Anna is covering the black monkey and thus assign the wrong thematic role to which princess. There were also very few instances in which children simply identified the wrong action, for example the pulling action when the question was about splashing. who-questions are providing list answers to only one of the wh-words, while role reversal errors are the most frequent with multiple which-questions. This suggests that children find it harder to assign the correct thematic roles in a patient-before-agent structure when the question contains two which-phrases.The two groups also show similarities in the errors they produce, which are (i) over-exhaustive answers, (ii) list answers, and (iii) reversed role answers. For over-exhaustive answers children name all the pairs in the image, even when one pair performs a different action. For example, children would answer a question like in (3) or (4) above with lme4 package (emmeans package (Given the binary nature of the data (Correct/Incorrect), we analyzed the accuracy results using a binomial generalized linear mixed model with group , wh-type (who vs. which), wh-order (SO vs. OS), and their interaction as fixed factors. The fixed factors were coded using repeated contrast coding which tests the difference between the mean of the dependent variable for one level of the categorical variable and the mean of the dependent variable for the adjacent level . The ran package  in R (R  package . Planned package .p = 0.988). The summary of the final model is given in who-questions were comprehended better than which-questions) and a significant wh-type*wh-order interaction. No differences in performance emerged between the monolingual and the heritage group , while there was a significant difference between the two wh-orders in the case of questions with two which-words, with SO which questions yielding significantly better comprehension scores than OS which questions .We followed-up on the significant interaction between wh-type and wh-order with a pair-wise comparison. This showed that response accuracy for questions with two online processing of multiple wh-questions are illustrated in SD above or below the mean per condition per participant and per item were considered as outliers and therefore replaced with the mean per condition per participant and per item. The total proportion of extreme values and outliers was 3.2% of all data points.The segment-by-segment residual reaction times (RTs) for the p were calculated by way of Satterthwaites’s approximation to degrees of freedom with the lmerTest package , wh-type (who vs. which), wh-order (SO vs. OS), as well as their interaction, were included in the fixed effects structure for each model. All fixed effects were coded using repeated contrast coding. Values of  package .Segment 1 (the first wh-word) Results attested to a significant effect of group , indicating that the monolingual children had overall longer RTs than the heritage children. There was also a significant effect of wh-type , with longer RTs for who-phrases (M = 482 ms) than which-phrases (317 ms), as well as a significant effect of wh-order , with shorter RTs for subject (M = 371 ms) compared to object wh-words (458 ms). None of the interactions was significant.Segment 2 (the second wh-word) Results revealed a significant effect of group , with the monolingual children displaying longer RTs than the heritage children, and a significant effect of wh-type , with longer RTs for who-phrases (M = 484 ms) than which-phrases (352 ms). No other effect or interaction were significant.Segment 3 (the verb in multiple who-questions and the clitic in multiple which-questions) There was a significant effect of group , with longer RTs for the monolingual group compared to the bilingual group, and a significant effect of wh-type  showing that the verb segment in the multiple who-conditions yielded shorter RTs (M = 386 ms) than the clitic segment in multiple which-conditions (M = 638). There was also a significant interaction Group*WhType . As a follow-up on the significant interaction, pair-wise comparisons revealed that the heritage group showed significantly shorter RTs than the monolingual group for the who-conditions, , while no significant differences surfaced between the two groups in the which-conditions.Segment 4 (the adverb in multiple who-questions and the verb in multiple which-questions) No effect was significant.Segment 5 (in the morning) Results attested to a significant effect of group , as the monolingual children had longer RTs than the heritage children. No other effect was significant.Segment 6 (at the zoo) No effect was significant.To summarize, the results of the comprehension task show similar response accuracy and a similar pattern during online comprehension of multiple wh-questions in Romanian-speaking monolingual and heritage Romanian children.The results of the elicitation task showed that children do not only produce questions with multiple wh-fronting, the expected target structure based on the syntax of Romanian multiple wh-questions, but that they often produce other structures as well. To reflect this variability in the children’s answers, four scoring categories were used, each corresponding to the four main question types that children produced and which we classified as follows:who or which, and with both wh-words fronted, as in (11). a. MWH_MULTIPLEMOVE: when children produced a question with two wh-words, either Cine pe cine   a mângâiat ?11.   who PE whom has patted  “Who patted whom?”b. MWH_SINGLEMOVE: when children produced a question containing two wh-words in which only one wh-phrase is fronted and the other one appears in-situ, like in (12). Cine a mângâiat pe cine ?12.   who has patted PE whom  “Who patted whom?”c. SIMPLE_WH: when participants produced a grammatically correct question but only with one fronted wh-word (13). Care  fată a mângâiat pisica?13. F.SG  which girl  has patted  cat.the.  “Which girl patted the cat?”d. COORDINATED_WH: when children produced a question with two coordinated wh-words (14). Cine și  pe cine a mângâiat ?14.   who and PE who has patted?  “Who and whom is punching?”The distribution of responses differs between the Romanian monolingual and Romanian heritage children, as can been seen in SO which and OS which questions and thus report the data for three types of multiple wh-questions with two who-phrases (who), with two which-phrases (which), and with one which and one who phrase (which-who). in-situ) represent the preferred produced structure for the heritage group across all types of elicited questions, irrespective of whether these contained only who, only which, or both which and who phrases. The monolingual group produce more MWH_SINGLEMOVE structures of the type illustrated in (12) with questions containing which-phrases, whereas they produce more MWH_MULTIPLEMOVE structures like in (10) in the presence of two who-elements.A finer characterization of the results  gives anwho vs. which vs. which-who), we fitted a generalized liner mixed model for each of the four scoring categories outlined above, namely MWH_MULTIPLEMOVE, MWH_SINGLEMOVE, SIMPLE_WH, COORDINATED_WH. The dependent variable in each model was response accuracy, that is, the correct production of questions within each scoring category. We analyzed the production data using the lme4 package (who vs. which vs. which-who) as well as their interaction. We used a repeated contrast specification for the fixed factors. The random effect structure included by-participant random slopes. We only report the significant effects and interactions.In order to uncover whether differences emerge between monolingual and heritage children in their productions and whether these differences are modulated by the type of elicited question . Overall, the heritage children produced significantly fewer questions with multiple wh-fronting compared to the monolingual children. The analysis also revealed a significant difference between which and who questions . Multiple wh-fronting was most frequent when children had to produce questions with two who-phrases compared to two which-phrases. The interaction Groupvs MonolingualHeritage *QuestionTypevs whichwhich-who  was also significant . As a follow-up on the significant interaction, pair-wise comparisons with an adjusted alpha level using the Tukey method showed that the monolingual group produced significantly fewer questions with multiple wh-movement when the target items only contained which-phrases compared to when they contained one who-phrase and one which-phrase .The statistical analysis revealed a main effect of Group . This means that, overall, heritage children produced significantly more multiple wh-questions with one element fronted and one in-situ than the monolingual children. There was also a significant difference between which and who questions , indicating that there were overall more multiple wh-questions with single wh-fronting for which-questions relative to who-questions.The statistical analysis revealed a main effect of Group . This means that children produced less coordinated wh-questions when both wh-words elements were which-phrases. There was also a significant Groupvs MonolingualHeritage *QuestionTypevs whowhich  interaction . Subsequent pairwise comparisons show that the heritage group produces significantly fewer coordinated wh-questions when the target items contained two which-phrases compared to when these contained two who-phrases  and this difference is more pronounced in heritage children than in monolinguals.The statistical analysis revealed a significant difference between in-situ compared to the monolingual children.Thus, heritage children display a different pattern than monolinguals in production, as they produce significantly fewer questions with multiple wh-fronting and significantly more questions with one fronted wh-phrase and one who- and which-questions. The on-line task investigated how Romanian heritage children and Romanian monolingual children process wh-dependencies with two fronted wh-words, while they were listening to sentences on-line for comprehension. This task also aimed to find out whether Romanian-speaking children were sensitive to the asymmetry in object-over-subject movement between multiple who- and which-questions in real-time comprehension. The combination of production and on-line comprehension tasks can help to get a better picture of heritage language development and better understand the relationship between performance in production and real-time processing.The main questions guiding the present study were whether differences emerge between Romanian-English bilingual children, for whom Romanian is the heritage language, and Romanian monolingual children in the on-line comprehension and production of multiple wh-interrogatives and whether these differences are due to cross-linguistic influence from the dominant English. To address these questions, we used an on-line comprehension task and an elicitation task targeting multiple who-subject and who-object well, irrespective of the order in which the two wh-phrases occur. In contrast, they have more difficulties with the comprehension of multiple which-question, particularly when the wh-object precedes the wh-subject. This is in line with cross-linguistic findings reported for the comprehension of simple which-questions showing that children find object which-questions harder to comprehend than subject which-questions and that the type of wh-element also affects comprehension of wh-dependencies, with object who-questions being acquired earlier thn object which-questions , but also questions in which only one wh-phrase is fronted, the other one remaining in-situ. This option exploited in production surfaces mostly in questions with two which-phrases. In addition, monolingual children also produce a significant number of simple wh-questions, that is, questions with only one wh-word. While heritage children also produce simple questions, they produce significantly more questions with one fronted wh-phrase, one in-situ than monolingual children and, with the exception of one child, avoid multiple wh-movement, contrary to monolinguals. Importantly, there were no instances of multiple who-questions with an ungrammatical object-subject order in any of the children’s productions. This indicates that their grammatical system does not allow this option and further reinforces the idea that their lack of sensitivity to the object-over-subject ungrammaticality in multiple who-questions in on-line comprehension is not due to a different grammar than that of adults, but it most likely stems from the processing load associated with such complex structures involving multiple movement dependencies.Let us now turn to the elicitation task which examined whether Romanian heritage and monolingual children are able to produce questions with multiple wh-movement and whether they have fully acquired the specific syntax of multiple interrogatives in Romanian, which requires fronting of all wh-phrases. Contrary to comprehension, we found differences in the production of multiple wh-questions between Romanian heritage and monolingual children. Monolingual children produce questions with multiple wh-fronting  are acquired earlier and also that they are more likely to influence structures which require more complex derivations (like multiple wh-fronting), in line with a complexity-based theory of transfer , indicating that they treat multiple wh-questions as simple questions. These errors in comprehension, together with children’s production patterns, show that even monolingual Romanian-speaking children take longer to produce structures with multiple wh-fronting and that they also make use of the option of having one wh-fronted and one in-situ. This corroborates An interesting observation here is the fact that children’s production patterns mirror the errors that they produce in the comprehension task. For example, reversed role errors are linked to children’s preference to mainly produce questions with a subject-object order for the conditions containing Our study makes a substantial contribution to the understanding of child HL development, as it investigates the production and comprehension of the same phenomenon in HL speakers and provides insight into the language development stages of both monolingual and heritage bilingual children. The current study is among the first to investigate cross-linguistic influence in bilingual children during real-time sentence processing and the first to use the self-paced listening paradigm with heritage children. However, the study also paves the way to future questions that remain unaddressed, such as when the syntax of multiple interrogatives is fully acquired as well what happens with adult heritage speakers or child heritage speakers whose dominant language(s) also have multiple wh-movement or lack this type of questions entirely. Studies that compare multiple bilingual groups will further increase our understanding of the impact of cross-linguistic influence on heritage language development.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University of Reading. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AB carried out the data collection and was responsible for data analysis. AB and TM shared responsibility for the conception of the work and the interpretation of results. AB wrote the manuscript with input from TM. All authors contributed to the article and approved the submitted version.This research was supported by an Early Postdoc. Mobility grant for AB from the Swiss National Science Foundation (grant number 174870).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.


The role of the pulmonary microbiome in sarcoidosis is unknown. The objectives of this study were the following: (1) examine whether the pulmonary fungal and bacterial microbiota differed in patients with sarcoidosis compared with controls; (2) examine whether there was an association between the microbiota and levels of the antimicrobial peptides (AMPs) in protected bronchoalveolar lavage (PBAL).Thirty-five sarcoidosis patients and 35 healthy controls underwent bronchoscopy and were sampled with oral wash (OW), protected BAL (PBAL), and left protected sterile brushes (LPSB). The fungal ITS1 region and the V3V4 region of the bacterial 16S rRNA gene were sequenced. Bioinformatic analyses were performed with QIIME 2. The AMPs secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 and hBD-2), were measured in PBAL by enzyme-linked immunosorbent assay (ELISA).Aspergillus dominated the PBAL samples in sarcoidosis. Differences in bacterial taxonomy were minor. There was no significant difference in fungal alpha diversity between sarcoidosis and controls, but the bacterial alpha diversity in sarcoidosis was significantly lower in OW (p = 0.047) and PBAL (p = 0.03) compared with controls. The beta diversity for sarcoidosis compared with controls differed for both fungi and bacteria. AMP levels were significantly lower in sarcoidosis compared to controls (SLPI and hBD-1: p < 0.01). No significant correlations were found between alpha diversity and AMPs.The pulmonary fungal and bacterial microbiota in sarcoidosis differed from in controls. Lower antimicrobial peptides levels were seen in sarcoidosis, indicating an interaction between the microbiota and the innate immune system. Whether this dysbiosis represents a pathogenic mechanism in sarcoidosis needs to be confirmed in experimental studies.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-022-01362-4. Sarcoidosis is a systemic inflammatory disease that can be challenging to diagnose and treat. Disease manifestations are heterogeneous, with symptoms varying considerably . The disMycobacterium and Propionibacterium has been widely studied [The causative agents inducing the immune response in sarcoidosis are yet somewhat of an enigma, but the immune system seems to respond to different substances by producing inflammatory cells organized in granulomas. It has long been recognized that the immune response could reflect an infectious etiology and scientists have for decades searched for that one pathogen that might cause sarcoidosis , 2. Desp studied .Aspergillus [Novel culture independent techniques as high-throughput 16S rRNA gene sequencing has revealed the presence of a diverse bacterial microbiota in the lungs . In sarcergillus , 6. A smergillus , 7, 8 orergillus –12. The ergillus , 12 and ergillus , 10.Antimicrobial peptides and proteins (AMPs) are key effector molecules in the innate immune system against respiratory pathogens , 14. TheThe aim of this bronchoscopy study was to examine whether the fungal and bacterial pulmonary microbiota differed in patients with sarcoidosis compared to healthy controls, and if there was an association between the microbiota and the AMPs.runiform  function in Stata 14.2 [Participants were recruited from our outpatient clinic at the Department of Thoracic Medicine, Haukeland University Hospital, Norway, participating in The Microbiome in Interstitial Lung Disease study (MicroILD)  and The ata 14.2 . MetadatThe participants underwent bronchoscopy with oral access and topical anesthesia in a supine position. Intravenous alfentanil (0.25–1.0 mg) was offered. To minimize contamination, no suctioning was performed above the vocal cords. During bronchoscopy we collected 3 protected sterile brushes from the left upper lobe (LPSB) and 3 × 50 ml (2 × 50 ml for controls) of bronchoalveolar lavage from the right middle lobe through a sterile protected sheath (PBAL). Sterile phosphate-buffered saline (PBS) was used for all fluid samples, including 10 ml of oral wash (OW). A negative control sample was obtained from the PBS bottle used for the procedural samples of each participant, without being exposed to the participant or the bronchoscope.The 16S rRNA sequencing was performed during the course of the MicroCOPD and MicroILD studies and included sequencing of more than 2500 samples from almost 400 bronchoscopies, with up to 8 sampling sites per participant. For the ITS sequencing which was performed afterwards, we chose to limit the sequencing to PBAL, OW, and negative control samples, due to cost restraints.The detailed protocol DNA extraction, PCR and sample preparation for MiSeq sequencing for the 16S rRNA gene analyses is previously published . The AddThe bioinformatic analyses were performed with QIIME 2 .Cytospin slides were made from PBAL and stained with May-Grünwald-Giemsa. A minimum of 300 cells were counted for differential cell counts by an observer blinded for the patient’s data. The AMPs, SLPI , and hBDFor statistical analyses we used Stata  and QIIMThe demographics of the participants are shown in Table Saccharomycetes was the most abundant fungal taxa in the OW samples for both study groups and PBAL samples for controls. However, in PBAL samples for sarcoidosis patients, the taxa were more evenly distributed, with the most frequent classes being Eurotiomycetes and Malasseziomycetes. At genus level, Candida was most abundant in the OW samples for both categories, while being less abundant in PBAL samples for patients compared to controls. Aspergillus was the most frequent genus measured in PBAL samples for sarcoidosis patients but was undetectable in PBAL and OW samples for controls. ANCOM analysis at class level showed significantly less Saccharomycetes in PBAL samples and less Sordariomycetes in OW samples from sarcoidosis patients compared to controls , there were significantly less Abscondibacteria in PBAL and Fusobacteria in OW for sarcoidosis patients compared to controls at phylum level, and significantly more Stomatobacteria in OW and Synergistia in LPSB in patients compared to controls at genus level  and Bray-Curtis (p = 0.03), and in OW by Jaccard (p = 0.04) , in PBAL by unweighted UniFrac (p = 0.02); and in LPSB by weighted UniFrac (p = 0.02), unweighted UniFrac (p = 0.03), Bray-Curtis (p < 0.01), and Jaccard (p < 0.01)  and hBD-1 (p < 0.01) (Table The levels of AMPs in the PBAL samples were significantly lower in sarcoidosis compared to controls for SLPI p < 0.0 and hBD-1) Table .This study demonstrated that stable sarcoidosis patients have a different fungal and bacterial microbial composition in the lower airways and oral cavity compared with healthy controls, being most pronounced for fungi. Differences between sarcoidosis and controls were found when examining differential abundances of taxonomy and differences in microbial diversity. The lower airways levels of the antimicrobial peptides SLPI and hBD-1 also differed between sarcoidosis and controls, with lower levels in sarcoidosis. However, we did not find a statistically significant association between the alpha diversity and the examined antimicrobial molecules.Aspergillus nidulans when examining BAL fluid in 9 patients with Lofgren syndrome, compared with 3 controls, together with increased serum antibodies, and the authors suggested that Aspergillus nidulans had a potential pathogenic role [Aspergillus  in BAL fluid but questioned this to be contamination [Fusobacterium spp and Atopobium spp in sarcoidosis compared to healthy controls [Erythrobacteraceae family in sarcoidosis patients [Actinobacteria and Proteobacteria to be significantly more abundant in sarcoidosis patients compared to ILD patients [Pneumocystis pneumonia patients and 9 healthy controls [Since the airways are not sterile in healthy individuals, a potential hypothesis is that a dysbiosis of the lower airways microbiota is a causal factor in the aberrant immune response seen in several chronic lung diseases. The role of the microbiome in the pathogenesis of pulmonal sarcoidosis has only been examined in a few studies, without consistent results. A recently published paper by Greaves et al. found enrichment of a peptide from nic role . Clarke mination . In anotcontrols . Fukui epatients . Gupta epatients . Three opatients , and a scontrols . All thrCandida was the most abundant genus in the OW samples for both groups and in the PBAL samples from healthy controls. Candida was significantly lower in PBAL samples for sarcoidosis patients compared to controls, possibly driven by Aspergillus, which was the most frequent taxon in this group. This was in line with the two other known studies on the fungal pulmonary microbiota in sarcoidosis patients as previously described [Candida and Aspergillus are the most prevalent and well-known fungal pathogens [ladosporium and Penicillium [In our study, the differences between patients and controls were more striking for the fungal taxonomy compared with the bacterial taxonomy. escribed , 6. Candathogens . Increasathogens . Antifunathogens , but neeicillium , 26, whiFirmicutes and Bacteroidetes, while Streptococcus, Prevotella, and Veillonella dominated the genus level, all shared by both study groups. These taxa are described as the most common bacteria in both the lower and upper airways in a healthy lung [The most abundant bacterial phyla we detected were thy lung . DiffereAntimicrobial peptides are produced by immune and inflammatory cells in the lungs and other mucosal tissues, and exhibit broad spectrum antimicrobial, immune modulatory, and wound repair features . These mInterestingly, we found significantly lower values for SLPI and hBD-1 in our study for sarcoidosis patients compared to controls. Agerberth et al.  examinedSince our study is cross-sectional, we cannot differentiate between lower levels of anti-microbial peptides leading to a microbial dysbiosis, or vice versa. Low levels of antimicrobial peptides in the airways could be a pathogenic factor, or a sign of an overwhelmed innate immune response. In any event, the persistence of granuloma and inflammation in sarcoidosis point to activation of stimulating antigens driving the disease process, and our study indicates that the fungal microbiota may be part of this process.Accurately measuring the low biomass microbiota in the lower airways is challenging. The bacterial DNA density in the upper airways is at least 100-fold higher than in the lower airways . Low bioHowever, our study has some shortcomings. First, BAL yield will differ within both the patients and control groups, which could influence microbial composition and measured levels of the AMPs. Mean BAL yield was 47.2% (SD 11.6) for controls and 48.3% (SD 17.7) for the patients, thus reasonably similar but with larger spread for the patients. Second, our study lacks patients with acute sarcoidosis in the early stage of disease. Third, fungi are even more delicate to examine with next-generation sequencing compared to bacteria due to its low abundance in many samples, high levels of fungal DNA from contamination sources and finally lack of standardization in primer designs, reference databases, and analytic methods , 37. TheAspergillus genera dominated the lower airways in sarcoidosis. In addition, sarcoidosis patients had lower levels of antimicrobial peptides in the airways. These findings could indicate the presence of a microbial dysbiosis in the airways in sarcoidosis. Future research should address whether this dysbiosis has a pathogenic role, and thus be a potential target for new treatment principles.In this study, we found differences in fungal and bacterial diversity between sarcoidosis patients and controls, and a more clearly distinct fungal taxonomy in the lower airways compared with controls, where Additional file 1. Supplementary Methods.Additional file 2: Supplementary Table 1. Comparison of beta diversity by pairwise PERMANOVA between the sarcoidosis patients and controls for fungal and bacterial taxa sampled by different methods* and assessed by different beta diversity metrices. Supplementary Table 2. Spearman's correlation coefficients (rho) and p-values for differences in alpha diversity correlated with antimicrobial peptides.


The major bottlenecks in the development of highly productive MES systems are the low bacterial loading, low electron transfer rate and low production of relevant chemicals, which limit the future potential for scaling up this process. Graphene has excellent electrical conductivity, remarkably high carrier mobility, special intrinsic mechanical strength, chemical stability, outstanding specific surface area, and biocompatibility. Therefore, in this regard, graphene can overcome these challenges and provide new opportunities. Graphene is suited for use as a cathode for increasing the bacterial loading and boosting the performance of MES. Over the last decade, graphene has been extensively developed and explored in MES. Graphene incorporation in cathodes can augment the surface area, reduce the resistance, and increase the electron transfer rate; thus, high current density, high coulombic efficiency, and high chemical production can be achieved. To better understand and further explore the modification of graphene-based materials as cathodes in MES systems, it is quite necessary to review and summarize recent developments in this field. Therefore, in this report, we briefly survey and discuss the up-to-date research activities regarding graphene in cathode modification and fabrication, with particular emphasis on their fabrication strategies and characterization, highlighting their key roles in MES systems, as well as presenting the challenges and the future prospects.Microbial electrosynthesis (MES) is a potential energy transformation technology for the reduction of the greenhouse gas carbon oxide (CO 2) into commercial chemicals.Microbial electrosynthesis (MES) is a potential energy transformation technology for the reduction of the greenhouse gas carbon oxide (CO CO2-capturing technologies and a replacement for fossil energy sources with sustainable energy systems are necessary.2,3 The rapid development of green energy technologies such as renewable energy has become a research focus in recent years.4,5 Microbial electrochemical synthesis is a novel strategy in which electroactive microorganisms use electrons obtained from solid electrodes to convert waste organic matter or CO2 into organic compounds and store them in the form of chemical energy.6,7 Microbial electrochemical synthesis is an attractive method to generate renewable fuels, and is perhaps the most promising way to meet future human energy demand.8–10 The electrochemical synthesis of microorganisms can reduce the greenhouse effect and eliminate CO2 emissions, which can minimize the CO2 content in the atmosphere.11–13 By converting abundant and inexpensive CO2 into valuable chemicals, we can create a new sustainable energy transformation system. Microbial products synthesized with CO2 have the following advantages: they do not rely on limited arable land and precious water resources, and they are virtually non-toxic and harmless to living organisms. If the electrical energy required for microbial electrochemical synthesis comes from photovoltaic power generation systems, MES may represent an artificial form of photosynthesis with high efficiency.14 Microorganisms can use solar energy to convert CO2 and water into organic compounds with oxygen as a byproduct, and solar energy can thus be stored in the form of chemical energy. Organic matter can provide fuel for transportation or it can be extracted and used at any time when urgently needed within existing infrastructures. A diagram of CO2 capture by the microbial electrosynthesis of biochemicals from renewable energy and chemical energy utilization is shown in 15Due to the rapid development of human civilization and the economy, the increasing demand for energy has mankind facing huge challenges in the 21st century. The dependence on fossil fuels and continuous growth in annual carbon oxide  and graphene have been explored in MES systems.35–38 A schematic of the MES cathode material development history and the various related biochemicals produced is shown in In early cathode research on MES systems, a negatively charged solid-state graphite block was used as the electron donor source.2-bonded hexagonal pattern.39 It is currently known as the thinnest material in the world. Ever since it was discovered in 2004,40 studies on graphene quickly became an international hot topic.41,42 Particularly in the last decade, it has been extensively studied in the scientific and engineering fields.43,44 Owing to its distinctive properties, graphene has excellent electrical conductivity, remarkably high carrier mobility, special intrinsic mechanical strength, chemical stability, outstanding specific surface area, and biocompatibility. Due to these special characteristics, in recent years, graphene has also been extensively studied and explored in the environmental, medical and electrochemical fields, especially in bioelectrochemical systems.45 In order to better understand and outline future prospects for the modification of graphene and graphene-based composite/hybrid material cathodes in MES systems, it is quite necessary to review and summarize recent developments in this field. Therefore, in the present report, we briefly discuss and outline the up-to-date research activities on graphene in the modification and fabrication of microbial electrosynthesis cathodes, with particular emphasis on their fabrication strategies and characterization, highlighting the key roles of graphene in MES systems, and finally the challenges faced and their future prospects.Graphene is a two-dimensional atomic lattice structure in which carbon atoms are arranged in an sp2.46 Through GO reduction to reduced graphene oxide (RGO), RGO can be decorated on the cathode. GO reduction methods can be used, such as thermal annealing, chemical reducing agents, and bacterial reduction such as self-assembly .assembly . Further2.1.in situ fixed on the cathode materials, and the surface of the cathode can be enhanced. The chemical reducing agents used include sodium borohydride, ascorbic acid, and hydrazine. For example, a very simple method involves dripping GO on the carbon felt surface and then directly reducing it to RGO using the reducing agent of sodium borohydride.80 The larger specific surface area of RGO infiltrated into the carbon cloth lattice can enrich microorganism attachment and result in the high production of acetate and butyrate. Using ascorbic acid as a chemical reducing agent, a carbon felt cathode soaked with GO was directly reduced to RGO via a solvothermal reduction process.47 Using scanning electron microscopy (SEM)  , it can via the reduction of GO sheets to RGO, GO can be prepared as a paper and then directly reduced and fabricated as freestanding and flexible RGO paper, which can then be exploited as a novel bioelectrochemical cathode to enhance electron transfer between the cathode and microbe.48 As shown in In addition to its infiltration into and immobilization on the cathode of MES 2.2.49,50 In microbial electrosynthesis, electrostatic repulsion can inhibit interfacial electron transfer and reduce the bioelectrocatalytic activity between the electrode and bacteria.51 Additionally, the inherent hydrophobicity of RGO can reduce its affinity for bacterial attachment.52 Thus, the novel strategy of functionalizing RGO with positive charges can solve this problem, strengthening the interaction between bacteria and the cathode and substantially augmenting the biofilm density at the surface of the electrode. The conducting polymer of poly  (PEDOT) with a positively charged backbone covered on RGO has been constructed and used in a high-performance E. coli-driven MFC.53 The negatively charged bacteria can easily interact with the positively charged backbone of PEDOT coated on RGO and form a denser biofilm, which can augment the electron transfer. In early research on MES, chemical agents with a positive charge such as chitosan were used to modify the surface of the cathode to reinforce this interaction.33 Based on the electrostatic interaction between the bacteria and graphene, RGO was functionalized with positive charges of tetraethylenepentamine (RGO-TEPA), and a carbon cloth cathode was then modified with RGO-TEPA.54 Therefore, negatively charged S. ovata strains can be easily incorporated into the positively charged matrix of RGO-TEPA through electrostatic interaction via a simple self-assembly method  with a rough surface can provide extra room for microorganisms to colonize; therefore, the bioconversion ability was activated. Graphene modification on the cathode can also improve the available surface area and create more electroactive sites for the microbially catalyzed reaction in MES, resulting in an increased electron exchange capacity with the microbes. The high specific surface area of the graphene-based cathode can maximize the direct mass transfer from the cathode to the microbes for CO2 conversion. Taking RGO paper as an example of a cathode, its specific surface area is 5.4 times higher than that of a carbon paper cathode with a similar diameter and thickness.48 Thus, the high surface area of the electrode substantially increased the electrode surface area available for electronic interactions with the microbes. This can be confirmed by confocal laser scanning microscopy (CLSM)  . A dense2 delivery and adsorption, which can improve the bacterial growth and enhance the conversion rates in MES systems. Nanocomposites of porous copper ferrite/RGO were fabricated and applied in MES.72 The overall surface area of copper ferrite/RGO increased significantly, as verified by SEM  modified on a CF cathode showed a lower charge transfer resistance (12.1 Ω) than CF (83.3 Ω).38 The inexpensive metal oxide nickel ferrite (NiFe2O4) coated on conventional CF possessed a lower electron transfer resistance (714 Ω) than CF (898 Ω).94 In the case of the above-mentioned modified cathodes, not only was there a charge transfer resistance reduction but the current output, acetate production rate, and Faraday efficiency also increased.A low charge transfer resistance can speed up electron delivery and bring about higher electron uptake, which can boost the performance of MES. A low charge transfer resistance can result in better electrocatalytic activity due to low charge transfer between the microbes and the electrode. High-conductivity materials in cathodes can promote electron transportation and speed up the extracellular electron transfer capacity, which can achieve higher chemical production. In recent years, in order to reduce the electron transfer resistance, conductive materials or nanocomposites such as conductive polymers, metal sulfides and metal oxides have been commonly used as ideal modification materials to lower the electron transfer resistance. The charge transfer resistance of a cathode coated with the high-conductivity polymer polypyrrole (PPy) was reduced by 33–70% compared to that of the uncoated one.84–86 Due to the synergetic interaction of nanocomposites compared to their individual components, the improved electrical properties of graphene-based composites or hybrids can facilitate faster electron transfer kinetics.87–90 The low charge transfer resistance of graphene or graphene-based nanocomposites/hybrids can also boost the reduction current density and bring about excellent electrochemical properties to enable high-performance MES.91–94 In an example using only the introduction of graphene as a cathodes in MES,60 the whole-cell resistance was much smaller (12.3 Ω) than that of the control CF cathode (456.8 Ω). The introduction of graphene in the biofilm greatly reduced the cathodic charge transfer resistance and boosted the electron transfer rate. In an example with a nanocomposite of graphene and magnetite, the incorporated graphene reduced the electron transfer resistance and increased the extracellular electron transfer.73 The EIS data for an RGO with magnetite nanoparticles (RGO-MNPs) modified electrode and unmodified CF electrode with or without an attached biofilm were plotted as Nyquist curves (Rct) of the biofilm-enriched CF/RGO-MNPs cathode was low compared to that of the unmodified CF with the biofilm, which verified that it could improve the extracellular electron exchange, resulting in a higher electron uptake and high chemical production in MES. Due to the lower charge transfer resistance, the faradaic efficiency increased two times.Due to its exceptional electrical conductivity, chemical stability, and biocompatibility, graphene can serve as a desirable conductive element between microbes and bacterial electrodes for MES. When graphene is involved in microbial electrochemical systems, its superb electrical properties can speed up electron transfer and improve the overall system properties.t curves . The cha3.3.2, formate, Fe(ii) and ammonia as redox mediators or when electrophilic microorganisms release or excrete their own soluble redox mediators to carry electrons from the cathode, as shown in 2 as the sole carbon source; therefore, EET is a very important procedure for MES.95–98 Accelerating the EET rate is critical for the optimization of MES performance.99,100 A high EET rate can result in better carbon dioxide reduction, high biochemical production, high current density, and high coulombic efficiency in MES.101,102 The EET rate is associated with the cultivation medium, surface area and roughness of the cathode, interaction between the microbes and cathode, and transfer resistance.103 Increasing the interfacial area using a porous three-dimensional scaffold electrode, augmenting the catalytic activity, reducing the electron transfer resistance, and incorporating positively charged species can greatly improve the EET rate.Extracellular electron transfer (EET) is a vital pathway to transport electrons between the bacteria and electrodes in MES. According to the literature, EET can be divided into the two mechanisms of direct electron transfer and indirect electron transfer . Direct 54,73,110 The high conductivity of graphene can promote electron transfer and further accelerate the EET. The high biocompatibility and permeability of graphene together with its improved bioelectroactive sites can speed up the EET. The synergetic effects of graphene-based nanocomposites can also promote the enrichment of electroautotrophic microbes for biomass clogs, which can reduce the resistance and facilitate the EET. Particularly, the high surface area-to-volume ratio of the porous three-dimensional hierarchical structure of composites based on graphene can further increase bacterial attachment and enhance the microbial EET rate.104,105 For example, the fabrication of a novel high-surface-area three-dimensional graphene-nickel foam cathode was reported.46 The hierarchical porous graphene-nickel cathode possessed a high specific surface area, which can be beneficial to bacteria entering the inner structure of the 3D electrode to form an electrocatalytically active biofilm. The high conductivity of graphene can form multiplexed conductive pathways, which can expedite electron transfer between the microorganisms and electrode. At the same time, the graphene decorated on nickel foam can be beneficial for hydrogen generation, which can indirectly increase the indirect electron transfer rate and CO2 reduction. A volumetric acetate production rate of 3.11 mmol L−1 day−1 was achieved and 70% of the electrons consumed were recovered.The high surface area and very thin layer of graphene can favor greater bacterial adhesion and intimate contact, which can increase the direct electron exchange. The functionalization of graphene with positive charges can strength its interaction with the negatively charged bacterial cell surface, which can further increase the EET and significantly speed up acetate production. At the same time, the functionalization of graphene with positive charges can make electrophilic microorganisms release or excrete their own soluble redox mediators, which can bring about indirect electron transfer.Due to their unique physicochemical characteristics, graphene and functionalized graphene-based nanocomposites have been developed to promote the accelerated proliferation of electroactive bacteria and their strong interaction with the surface of electrodes. The enlarged surface area, low conductive resistance and high electron transfer rate can boost the capability of MES. 50,106,107 However, it needs to be clarified whether the antibacterial properties of graphene come from specific aerobic conditions. Graphene can induce oxidative stress with reactive oxygen species using molecular oxygen. The antibacterial properties of graphene have also been used in water treatment, metal recovery, medical equipment, and tissue engineering scaffolds. However, in the case of graphene in the MES of biochemicals from CO2, the growth conditions are usually maintained under strict anaerobic conditions to remove O2, with constant gas flushing. In the absence of O2, the graphene-coated bioelectrode can promote bacterial proliferation. This is why graphene can exhibit bacterial growth-promoting activity in some reports and antibacterial properties in other reports.In recent years, a vast number of studies have reported that graphene has antibacterial properties.4.2 into high-value multicarbon biochemicals using renewable sources of electricity such as solar and wind and effectively store electrical energy in the form of chemical bonds.108–111 For purpose of obtaining highly productive MES systems and speeding up their potential scaling up, the low bacterial loading, low electron transfer rate and low productivity of chemicals are great challenges that need to be overcome.112–114 High active surface area, highly hierarchical porous networks, and high electronic conductivity in materials are essential to develop highly productive MES systems. Therefore, in this regard, graphene and graphene-based composites can help overcome the challenges and provide opportunities in the future research on MES. Graphene is suited for application as a cathode to increase the bacterial loading and boost MES performance due to its high active surface area, chemical stability, high electrical conductivity, corrosion resistance and biocompatibility. Due to their additional positive synergistic effects over individual graphene, graphene-based nanocomposites have even better functional properties, which can improve the EET and increase the microorganism density due to their exceptional electrical conductivity and high surface area. Therefore, graphene and graphene-based nanocomposite bacterial electrodes possess excellent electrochemical properties for high-performance microbial electrosynthesis. Graphene-modified cathodes increase the electrosynthetic rate of chemicals from CO2 and significantly improve the biofilm density and current consumption.MES is a promising way to drive the reduction of the greenhouse gas CO115,116 Carbon nanotubes exhibit high biocompatibility, and can thus promote bacterial metabolism and growth. Therefore, carbon nanotubes have become extremely attractive for applications in MES. In the last decade, some researchers have reported carbon nanotube-based biocathodes for the EMS of chemicals from CO2.36,37,83 The main approaches for carbon nanotube-based cathode fabrication include electrophoretic deposition (EDP) and CVD. The EDP method is suitable and attractive for large-scale MES-based cathode fabrication. The CVD method is expensive, complicated, and energy and time consuming. We believe that in the near future, graphene has sufficient advantages to tackle the challenges associated with carbon nanotubes in MES. Graphene has a higher specific surface area  than carbon nanotubes. Compared with carbon nanotubes, the larger specific surface area of graphene can result in more sites available for microbial interaction and adhesion. The rough surfaces and numerous irregularities in graphene are beneficial for microbial colonization. Graphene has a higher conductivity (108 S m−1) than carbon nanotubes. The high conductivity of graphene can promote the electron transportation and speed up the extracellular electron transfer capacity. Due to its easy synthetic process, graphene has a lower fabrication cost than carbon nanotubes. Graphene-based cathode fabrication is inexpensive, simple, and can be up-scaled for industrial-scale production.Carbon nanotubes have a high aspect ratio, high surface area, good chemical stability, and excellent electrical conductivity.Over the last decade, a variety of methods have been developed for designing and fabricating graphene and graphene-based composite cathodes for MES. Although some considerable achievements have been made in this area, such as high current density, high coulombic efficiency, and high chemical production, some problems such as the high cost of graphene, the complicated process, energy/time-consuming process and the small lab-scale synthesis have been encountered, which limit their wide application. The low-cost, high efficiency, pilot-scale, high quality, and high yield synthesis of these products is still a challenge in expanding their fundamental performance and potential practical applications. The key considerations of graphene research are presented as follows:(i) The EET between microorganisms and cathodes should be further understood through theory and experimentation. Till now, a deeper understanding of the mechanism behind electron transfer and exchange is limited. When graphene is introduced in MES systems, connecting the microbes and cathode, it can accelerate electron transfer and reduce electron transfer resistance; however, there is a lack of knowledge regarding the related EET process. There are no theoretical and experimental studies in this field at present. Determining the electron transfer mechanism for graphene involved in the MES pathway can further result in a major breakthrough and boost the already high EET rate. We believe that with the development of various technical tools, such as electrostatic microscopy, electrochemical surface plasmon resonance, and the establishment of mathematical models, multidisciplinary approaches can shed fresh light on the role of graphene in the EET mechanisms.117 However, this reduces the electron mobility and conductivity of graphene. We believe that with the progress of doped and functionalized materials based on graphene, a balance between the wettability and conductivity can solve this problem.(ii) The hydrophobicity of graphene is a big challenge for the large-scale development of MES as graphene has to inevitably come in contact with the electrolyte solution. Hydrophobicity is an intrinsic property of graphene that restricts its dissolution in water, and thus it can inhibit its interaction with bacteria. In order to improve its solubility, graphene is often doped or functionalized with other functional groups such as carboxyl and hydroxy.(iii) Although graphene has an outstanding specific surface area, the sufficient utilization of its large specific surface area is another problem. As we know, due to the strong π–π stacking coupled with van der Waals forces between the layers, graphene sheets are prone to restacking and easily form irreversible agglomerates. Therefore, in graphene-based cathode fabrication, the large specific surface area of graphene can be dramatically decreased, which results in an insufficient surface for MES applications. Restraining the agglomeration of graphene sheets and making efficient use of single-layer graphene in MES cathodes should be the focus of further research. As we know, large sheets of single-layer graphene can be grown by the CVD method. Therefore, we can construct a three-dimensional single-layer graphene brush or sponge as an MES cathode. Thus, a three-dimensional graphene brush or sponge can provide a sufficient surface for MES applications.118 Its production has been substantially increased in the laboratory; however, its larger-scale production has not been exhibited at present. Its high production costs limit its commercialization. The CVD method is expensive, complicated, and energy- and time consuming. The solution-based chemical reduction method can bring about toxic chemical pollution from the GO reduction process. In contrast, mechanical exfoliation can produce graphene on a large scale at low cost and high quality. Thus, in the near future, with the advancement of graphene preparation technology, we forecast that the cost of graphene-based cathode fabrication will decrease and it will be competitive in the market.(iv) The high cost of graphene has become a significant drawback for its expansion, particularly in the competitive commercial market. Graphene and graphene-based composites have been used in MES cathode modification; however, the cost of graphene is higher than that of traditional materials such as carbon cloth and carbon felt, and its chemical production is much more expensive than other methods.55,118–120 With the development of various technologies, we expect the fabrication of large-scale graphene-based cathodes to have a bright future in CO2 microbial transformation and utilization.(v) The large-scale fabrication of graphene-based cathodes is another critical issue for its industrial-scale production. The small-scale manipulation in the lab, toxic chemicals introduced for the GO reduction process, and complex and time-consuming fabrication process have become shortcomings in their further application. Large-scale graphene-based cathode fabrication is limited by multiple factors such as its inefficient construction strategies, high cost, and low quantity of chemical production in the lab. Industrial-scale graphene-based cathode development and high-quantity chemical production should be integrated with advanced technologies such as the biologically mediated production of RGO and 3D bio-printing.5.Graphene has attracted enormous interest in the fundamental research and potential application of MES due to its suitable electrical and physical characteristics with high affinity for bacterial cells and the formation of high-density electroactive biofilms. In this report, we have briefly reviewed the recent research progress on novel cathodes of graphene and graphene-based composites/hybrids in MES systems. We have emphasized the strategies behind the design and fabrication of graphene-based cathodes with their key roles in electrosynthesis, and have included our perspective and remarks as well. This review not only provides an updated progress on the fabrication of all kinds of MES cathodes based on graphene and its composites but also demonstrates their important roles in the augmentation of the surface area, reduction in the resistance and increase in the electron transfer rate. The comprehensive discussion on the fabrication techniques and critical function of these cathodes in MES can provide fundamental insight into understanding and designing graphene-based cathodes for their further optimization and improvement. Due to the research progress on graphene and related technologies, it can be expected that graphene will play a significant role in the further development of high-efficiency MES and can speed up the commercialization process.There are no conflicts to declare.


Graphene is then produced by catalytically carbonizing this Fe-supported lignin at 1200 °C. The characteristics of both the Fe catalyst and lignin are crucial for the production of high-quality graphene. Specifically, the lignin should disperse well in water, freeze dry, and carbonize via solid-state carbonization. The obtained graphene-based material is highly resistant to electrochemical oxidation, as observed in other graphene-based materials. The direct conversion of lignin to graphene described herein is an unprecedented method for synthesizing large amounts of graphene-based material at low cost, as well as being an excellent use for lignin.Methods for effectively utilizing lignin are necessary for the realization of a sustainable society. Herein, we report a method for directly converting lignin to graphene-based materials. Fe-supported lignin is prepared by dissolving lignin in an aqueous FeCl via catalytic carbonization is expected to expand the industrial use of lignin and graphene-based materials.Directly converting lignin to graphene  Novel methods for the production of graphene using metal catalysts instead of graphene oxide have been proposed. For example, Qiu et al.15 used chemical vapor deposition (CVD) for the bottom-up deposition of graphene on the surface of a porous metal support, and examined its catalytic applicability. However, this method is not suitable for use in the industrial production of graphene-based materials because of the high price of the raw materials and the difficulty of the CVD process.In recent years, various carbon materials have been studied for use in energy storage and conversion devices.i.e., high graphene content and few defect sites).16,17 The proposed method utilizes the water solubility and ion exchange capacity of lignin to produce superior graphene directly by catalytic carbonization. The direct conversion from lignin to graphene is sustainable, inexpensive, and would provide large amounts of graphene-based material. Moreover, it is expected to greatly contribute to the industrial use of both lignin and graphene-based materials.To accelerate the industrial use of graphene-based materials, it is necessary to develop a cheap, environmentally friendly, and sustainable synthetic method that does not use graphene oxide. In this study, we report a new method for synthesizing graphene-based material directly from lignin. In recent years, several studies have been reported on methods for producing graphene-based materials using lignin as a raw material, but these methods have not yet led to the synthesis of superior graphene (2 solution (50 mL) at a predetermined concentration, and the solution was stirred for 3 h. To prevent the oxidation and precipitation of Fe2+, hydroxylammonium chloride and acetic acid were added to the FeCl2 solution at respective molar ratios of 0.36 and 0.007 (for 1 mol of Fe2+). Lignin samples supported with Fe (Fe-supported lignin) were prepared by oven drying or freeze drying the obtained aqueous lignin solutions. The Fe loading was adjusted in the range of 0.028 to 2.8 mmol g−1. The Fe-supported lignin samples were heat-treated for 1 h in an inert atmosphere at a temperature of 1200 °C to obtain carbonized samples. The sample names are distinguished by the amount of Fe (X mmol g−1) and the lignin drying method (oven dried (OD) or freeze dried (FD)), and are expressed as L-FeX(OD) and L-FeX(FD). As control samples, metal-free carbonized samples were prepared by dispersing lignin in distilled water without FeCl2 followed by drying (OD or FD) and heat treating under the conditions described above. The control samples are denoted as L(OD) and L(FD), respectively.Commercially available alkaline lignin  was used as the lignin sample. The lignin sample (1.0 g) was dispersed in FeClVmeso) were determined by Barrett–Joyner–Halenda (BJH) analysis. Raman spectra of the prepared carbon samples were obtained using Raman spectrometry  with a 532 nm Nd:YAG laser. X-ray diffraction (XRD) analysis was performed by an X-ray diffractometer  with Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) measurements were performed with a Kratos AXIS NOVA instrument (Shimadzu Co.). The charge-up shift correction was conducted by setting the C 1s binding energy level of the samples to 284.5 eV.Carbonized samples were observed using transmission electron microscopy  to investigate their carbon structures. Sample lattice fringes on the microgrid were analyzed at an accelerating voltage of 200 kV. The surface areas and pore structures were evaluated using nitrogen adsorption–desorption measurements  at −196 °C. The specific surface areas were calculated based on Brunauer–Emmett–Teller (BET) theory. The volumes of mesopores  was used to measure the concentrations of the peripheral (edge) sites of the graphene layers in the carbonized samples by measuring the molecules desorbing at those sites. The average size of the graphene sheet was also calculated from the number of edge sites.−2) and Nafion®. To evaluate the electrochemical oxidation resistance and compare the electrochemical characteristics with other graphene-based materials, cyclic voltammetry (CV) was performed in the following manner. First, a CV scan (5 mV s−1) was repeated four times in the potential range of 0 to 1.0 V vs. a reversible hydrogen electrode (RHE) in 0.5 M H2SO4 aqueous solution at room temperature. Then, triangle-wave potential cycling between 1.0 and 1.5 V at 100 mV s−1 was employed as a carbon oxidation test. After 100, 200, and 500 cycles, the CV scan was repeated another four times in the potential range of 0 to 1.0 V.Electrochemical measurements were performed using a disk electrode. The working electrode was prepared by covering a glass-like carbon disk electrode with a mixture of the prepared carbonized sample ) exhibited a similar Raman spectrum to the metal-free sample, whereas the freeze-dried Fe-supported sample (L-Fe0.56(FD)) showed a strong 2D band (2700 cm−1) unique to graphene.28 Notably, the Raman spectrum of L-Fe0.56(FD) is very similar to that of commercially available graphene . Attempts to synthesize graphene from lignin have been reported so far, but the graphene obtained in those reports showed a relatively weak 2D peak.16,17 L-Fe0.56(FD) obtained by our method shows a stronger 2D band than not only the reported graphene but also the reduced graphene oxide which is often used in the production of graphene-based materials. There has never been a report of such high-quality graphene being directly synthesized from lignin, which indicates the usefulness of our approach as a method for converting lignin to graphene.3O4, as verified by XRD measurements and selected area electron diffraction (SAED) analysis  to the G band (1590 cm−1) (ID/IG) and the 2D band to the G band (I2D/IG) were calculated. The results are summarized in ID/IG was minimized, and I2D/IG was maximized, with between 0.28 and 1.4 mmol g−1 of metal loading. When the lignin contained 2.8 mmol g−1 of Fe, the ID/IG and I2D/IG values were almost the same as those of the control sample , which indicates that the beneficial effect of the Fe support on the production of graphene is lost at a high metal loading.−1 because lignin cannot maintain its dispersed state in an aqueous solution with a large amount of metal cations.29 The presence of this precipitate causes the morphology of the freeze-dried sample to differ from that of the other freeze-dried samples; the apparent density is clearly reduced, and the state appears similar to that of the oven-dried sample. This indicates that two factors are essential for graphene production from lignin, namely, solidification of the lignin molecules in a sparse state, and appropriate Fe support for the lignin molecules.30–33 but the formation of graphene observed herein has not been reported previously. Hydrophobic polymers are generally used as carbon precursors in carbon chemistry and industry. Such polymers are difficult to freeze dry, and when used as carbon precursors, a dense solid powder must be used. It is conceivable that a dense carbon precursor would yield dense carbon, that is, carbon with laminated graphene. In addition to the effect of the Fe catalyst, the use of a sparse polymer at the molecular level—such as freeze-dried lignin—is considered the most important factor in the formation of graphene in this method.Since graphene aggregates are easily laminated by van der Waals forces, the strong Raman 2D band is unique to graphene; it cannot be observed in general carbon materials. To prevent the lamination of graphene, it is considered appropriate to use a carbon precursor in which the polymer is spatially sparse, such as freeze-dried lignin. So far, we have reported on the catalytic and noncatalytic carbonization of polymers,To clarify the principle of graphene formation from metal-supported lignin, we performed a detailed structural analysis of the carbonized samples. 18 we found that the graphene sheet sizes of carbon materials had a greater dependence on the temperature of carbonization than on the carbon structure or pore structure. By comparing previous results with the current report, it can be interpreted that the spatial distribution of lignin molecules does not affect the growth rate of graphene sheets. In contrast, the sheet size of L-Fe0.56(FD), in which graphene formation was confirmed, was clearly larger than that of the other samples. This was due to the presence of the Fe support, which causes a catalytic carbonization reaction and promotes the growth of the graphene sheet. The sheet size of L-Fe2.8(FD), which had a large Fe2+ loading, was approximately the same as that of L(FD), which was formed without Fe2+. Therefore, the effect of catalytic carbonization cannot be confirmed for L-Fe2.8(FD). L-Fe0.56(FD) and L-Fe2.8(FD) differed in whether they exhibited lignin aggregation, as shown in 2+.In our previous report,31 However, the catalytic carbonization of lignin herein proceeded at an extremely high temperature of 1200 °C, which differs from previous reports on catalytic carbonization. To understand the catalytic carbonization of lignin, it is necessary to conduct more detailed investigations in the future. The high water dispersibility and solid-state carbonization characteristics of lignin play important roles in the formation of graphene from lignin. The direct conversion of lignin to graphene by catalytic carbonization in this study was achieved owing to the polymer properties of lignin.To proceed successfully with the catalytic carbonization reaction, optimal Fe loading and spatial distribution of lignin molecules are required. When the heat treatment temperature was reduced to 1100 °C or lower (data not shown), graphene formation was not confirmed for any of the lignin samples. The catalytic carbonization reaction is promoted as the temperature increases. With Fe catalysts, the catalytic carbonization reaction should start at approximately 700 °C.via catalytic carbonization promotes the reduction of these carbon edge sites, thereby improving the electrochemical oxidation resistance. Resistance to electrochemical oxidation is a common characteristic of graphene-based materials.13 Thus, this result indicates that the carbonized sample can be regarded as a graphene-based material.To compare the characteristics of the obtained graphene-based material with those of other graphene-based materials, the sample was electrochemically evaluated by CV and oxidation cycling. In this study, we show that graphene can be synthesized from lignin biomass, which is abundant in modern society. To generate graphene from lignin, two essential conditions were identified: (i) the metal-supported lignin must remain well-dispersed in an aqueous solution, and (ii) a low-density powder is required, such as that produced by freeze-drying. The water dispersibility and solid-state carbonization characteristics of lignin play important roles in conversion to graphene. Lignin-derived graphene exhibited similar electrochemical properties to other graphene-based materials. The direct conversion of lignin to graphene is an unprecedented method for synthesizing large amounts of graphene-based material at low cost and is also an excellent method for the effective use of excessive lignin produced when extracting cellulose. With further research, this method for producing graphene-based materials could revolutionize the sustainability of the energy we use.The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.There are no conflicts to declare.RA-011-D1RA02491D-s001


Capgras and Fregoli delusions are psychotic complex conditions that integrate a larger group of Delusional Misidentification Syndromes (DMS), where the patient misidentifies people, places or objects.Review of the literature and exposure of a case report of a first psychotic episode with DMS.Case report and a nonsystematic review through databases. With the keywords: Capgras; Fregoli; DMS.This case presents a caucasian male, 34-year-old, with no personal or family history of psychiatric pathology. He was taken to the emergency service, due to behavior disorder with verbal aggressiveness to his mother. During the psychiatric emergency he was restless, with accelerated speech and presented Capgras and Fregoli delusion. He believed that his mother had been replaced by an imposter and that the doctor was actually his childhood friend disguised, all with the intention of harming him. The patient was hospitalized, analytical and brain scan show no abnormally. Improvement in symptoms was been seen when a long-term injectable antipsychotic was started. The diagnosis was Bipolar type I disorder.DSM are more frequent than previously considered, they often occur in association with psychiatric or neurological disorders. Case reports like this one helps to clarify the association between DMS and psychiatric disorders. Given the high incidence of DMS, it is essential to recognize them, carry out an early treatment and be alert to other psychopathological or neurological symptoms that may coexist.No significant relationships.