M Holzapfel

F Taraveau

Z Djelouadji
email: zoree.djelouadji@vetagro-sup.fr

Serological and molecular detection of pathogenic Leptospira in domestic and stray cats on Reunion Island, French Indies

Keywords: Cat, epidemiology, leptospirosis, public health, reservoir

Indian Ocean islands are endemic areas for human and animal leptospirosis. Maintenance host species for Leptospira spp. have still not been completely elucidated, and recently the role of cats (Felis catus) has been questioned. This cross-sectional study aims to determine whether cats are part of the maintenance community of different strains of Leptospira spp. in Reunion Island. The prevalence of Leptospira infection in an opportunistic sample of stray and domestic cats (n = 92) from Reunion Island has been studied using serological (microagglutination test) and molecular detection (polymerase chain reaction (PCR)). The results revealed a seroprevalence of 37.0% (34/92) (cut-off 1:40) without a significant difference in the living conditions of animals. The predominant serogroup was Icterohaemorrhagiae, but Ballum, Cynopteri and Australis were also detected. Using PCR, 28.6% (12/42) of stray cats were tested positive. Leptospiral DNA was detected in renal tissue, urine and blood of respectively 14.3% (6/42), 10.3% (4/39) and 11.9% (5/42) of stray cats, but 0% (0/3), 0% (0/50) and 0% (0/36) of domestic cats (P = non-applicable, P = 0,038, P = 0,058 respectively). Partial rrs gene (16S rRNA) sequencing identified Leptospira interrogans in all PCR-positive samples. Our study confirms that renal carriage and urinary shedding are possible, positioning cats, and especially stray cats as potential actors within the maintenance community of L. interrogans in Reunion Island.

Introduction

Leptospirosis is a worldwide zoonotic disease caused by spirochetes from pathogenic and intermediate Leptospira species. The human incidence is estimated at 1 million cases per year [START_REF] Costa | Global morbidity and mortality of leptospirosis: a systematic review[END_REF]. Leptospirosis is a re-emerging infectious disease, especially in tropical and subtropical regions, where environmental conditions favour the survival and transmission of leptospires [START_REF] Sykes | ACVIM small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention[END_REF].

The incidence ranges from 0.1 to 1 case/100 000 inhabitants per year in temperate areas to over 100 cases/100 000 inhabitants per year in the tropics [START_REF] Picardeau | Diagnosis and epidemiology of leptospirosis[END_REF]. In mainland France, the incidence reached 1 case/100 000 inhabitants in 2014, which represents 600 cases per year [START_REF] Bourhy | Diagnosis, surveillance, and epidemiology of leptospirosis in France[END_REF]. In Reunion Island, a French department located in the south-western Indian Ocean, the annual incidence ranged from 3 to 10 cases/100 000 inhabitants between 2004 and 2015 [START_REF] Pagès | Epidemiology of leptospirosis on Reunion Island, 2004-2015[END_REF]. Consequently, leptospirosis is regarded as an endemic disease and a crucial public health issue on Reunion Island.

More than 150 mammalian species can harbour Leptospira spp. The rat (Rattus rattus) is considered as the main maintenance host, even though studies have shown that many other mammals, such as hedgehog and mustelid species, can carry pathogenic leptospires [START_REF] Ayral | Hedgehogs and mustelid species: major carriers of pathogenic Leptospira, a survey in 28 animal species in France (2012-2015)[END_REF]. Maintenance hosts excrete leptospires and contaminate the hydric environment for long periods of time, independently of the presence of clinical signs. The maintenance hosts either develop chronic infection of the renal tubules while remaining asymptomatic, or present acute disease [START_REF] Levett | Leptospirosis[END_REF]. The infection of animals and humans occurs from direct contact with the urine of infected animals or indirectly from contaminated water [START_REF] Adler | Leptospira and leptospirosis[END_REF].

Cats are susceptible to Leptospira spp., but the prevalence of clinical leptospirosis in this species is low [START_REF] Hartmann | Leptospira species infection in cats: ABCD guidelines on prevention and management[END_REF]. Cats can present mild polyuria/polydipsia and increased body temperature after infection. Liver and kidney lesions have also been reported together with the presence of Leptospira-specific antibodies [START_REF] Arbour | Clinical leptospirosis in three cats (2001-2009)[END_REF].

However, the role of cats in the epidemiology of leptospirosis remains poorly understood. Recent studies testing the seroprevalence of Leptospira spp. in cats in various geographic regions reported from 5% to 36% seropositivity [START_REF] Alashraf | Serological detection of anti-Leptospira antibodies in shelter cats in Malaysia[END_REF][START_REF] Dybing | Leptospira species in feral cats and black rats from Western Australia and Christmas Island[END_REF][START_REF] Weis | Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany[END_REF][START_REF] Zaidi | Urinary shedding of pathogenic Leptospira in stray dogs and cats, Algiers: a prospective study[END_REF][START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF][START_REF] Žákovská | Seroprevalence of antibodies against Borrelia burgdorferi s. l. and Leptospira interrogans s. l. in cats in district of Brno and its environs, the Czech Republic[END_REF]. Moreover, in the tropical island of Christmas Island, a study found a much higher prevalence of leptospiral DNA carriage in feral cats than in rats [START_REF] Dybing | Leptospira species in feral cats and black rats from Western Australia and Christmas Island[END_REF]. On Reunion Island, a seroprevalence of 26.7% (cut-off 1:100) and renal carriage of 28.6% were registered in stray cats (n = 30) [START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF]. Yet, a recent study on feral cats reported only one renal carriage out of 172 animals tested with quantitative real-time polymerase chain reaction (PCR) (0.6%). Interestingly, this isolated strain presented high DNA sequence similarity with a strain isolated from an acute human case of leptospirosis on the island [START_REF] Gomard | Feral cats do not play a major role in leptospirosis epidemiology on Reunion Island[END_REF].

Evidence has indicated that cats can intermittently shed leptospires in their urine up to 12 weeks following infection [START_REF] Weis | Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany[END_REF]. In recent studies, leptospires from cat urine samples were successfully grown in culture, emphasising the potential role of cats in the contamination of the environment and transmission to other species [START_REF] Dorsch | Cats shedding pathogenic Leptospira spp.an underestimated zoonotic risk?[END_REF][START_REF] Alashraf | First report of pathogenic Leptospira spp. isolated from urine and kidneys of naturally infected cats[END_REF].

The first aim of the present study was to estimate the seroprevalence for different serovars of Leptospira spp. among cats and to determine the main serogroups circulating in domestic and stray cats from Reunion Island. The second purpose was to record the urine excretion, kidney carriage and presence of Leptospira spp. in the blood to consider possible long-term carriage and shedding of the bacteria from this species; and to identify causal Leptospira strains by sequencing.

Materials and methods

Ethics statement

The present study was approved by the ethical committee of VetAgro Sup (named ethical committee Jacques Bonnod) under the licence number 1970-18. This committee is registered at the French Ministry of Research and reviews all license applications for preclinical research and all clinical research projects. The use of animals for scientific purposes is covered in France, by the European Directive 2010/63/EU [START_REF] Parliament | Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes[END_REF], that was transposed into French law on 1 February 2013.

Sample collection

Experiments were designed to estimate the apparent prevalence of Leptospira spp. in stray and domestic cats from northern and western Reunion Island. The sample size of this study was calculated to ensure estimation with a precision of at least 10% (i.e. obtaining a confidence interval (CI) of less than 20% width). Theoretical seroprevalence was fixed at 27% [START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF], confidence levels to 95% and the population of cats in the Reunion Island was considered as infinite regarded to the sample size of the study. The optimal sample size (n = 87) was determined with a binomial test. Finally, we included a total of 92 cats, presenting different living conditions (domestic vs. stray cats). Domestic cats (n = 50) were selected among voluntary clients from the Veterinary Clinic of Front-de-Mer in Saint-Paul. Domestic cats included in the study were healthy and were presented for sterilisation; animals under antibiotic treatments or presenting underlying diseases were excluded. All cats lived partially outdoor. Three of the domestic cats included in the study were euthanised by the veterinarians for owners' personal reasons. Stray cats (n = 42) were captured in urban areas, by the animal shelters of Sainte-Marie and Saint-Paul. If neither identified nor adopted after 8 days, the cats were sedated using acepromazine and ketamine and euthanised by the shelter with pentobarbital after blood and urine were collected. Health check was not performed on stray animals, but at the time of necropsy, no macroscopic lesion compatible with leptospirosis was observed ( jaundice, hepatomegaly and no visible renal changes) [START_REF] Arbour | Clinical leptospirosis in three cats (2001-2009)[END_REF]. At the time of the consultation or necropsy, no domestic or stray cats respectively were excluded because all of them fit in the inclusion criteria. For each cat, 1-4 ml of blood was collected from the cephalic vein or jugular vein in a dry tube and in an ethylenediaminetetraacetic acid (EDTA) tube. During the hours following sampling, the dry tube was centrifuged for 15 min at 2000 g, and the serum was separated. Both the serum and the EDTA tube were stored at 4 °C for further analyses. Urine samples were collected by cystocentesis (1-5 ml) and kept in a dry tube neutralised with 0.5 ml of phosphate-buffered saline (PBS) at 4 °C. Kidneys were collected from euthanised animals in a sterile manner, and renal tissue was stored in sterile tubes at 4 °C.

A total of 92 cats were sampled between 1 February and 27 February 2013 (rainy season). The samples included 92 blood (92 for serological analysis and 78 for PCR), 89 urine and 45 kidney samples. All samples were kept at +4 °C (maximum 7 days) and sent progressively to the Leptospires Laboratory at VetAgro Sup (the veterinarian school of Lyon, France) for serological and molecular analyses.

Microscopic agglutination test (MAT)

Sera were tested against 24 pathogenic serovars of Leptospira spp. and two non-pathogenic serovars (Table 1), selected based on their prevalence in French overseas territories and on the reference strains available at the Leptospires Laboratory at VetAgro Sup [START_REF] Marquez | Overview of laboratory methods to diagnose leptospirosis and to identify and to type leptospires[END_REF]. Sera were centrifuged for 5 min at 3600 g. Then, each well of a sterile microtitre plate was filled with 25 μl of serum in Sorensen buffer (1:20 dilution) and 25 μl of a specific leptospiral strain (serum diluted at 1:40 in the final solution). For each leptospiral strain, controls consisted of replacing the cat's serum with 25 μl of a serum with antibodies specific for the strain tested (positive control) or with 25 μl of Sorensen buffer (negative control).

Microtitre plates were then incubated for 1 h at 37 °C followed by 1 h at 4 °C and examined using a dark-field microscope. All positive sera were tested again for the suspected serovars by using serial dilutions to get titres ranging from 1:20 to 1:320. The endpoint titre was considered the greatest dilution showing at least 50% agglutination compared to the control. Titres of 1:40 or higher were considered as indicators of exposure for the strain tested, as described in previous studies [START_REF] Luciani | Réceptivité et sensibilité du chat aux leptospires[END_REF][START_REF] Boni | Rats et chats errants: enquête épidémiologique en milieu urbain[END_REF][START_REF] Shivakumar | Diagnosis of leptospirosisrole of MAT[END_REF]. For sera reacting with more than one serovar, the seropositivity was attributed to the strains with the highest titre and serovars with lower agglutination titres were considered as cross-reactions.

DNA extraction

DNA was extracted with a KingFisher® automation instrument (Thermo Scientific, Illkirch, France) and a universal kit (LSI MagVet Universal Kit MV384, Thermofisher, Courtaboeuf, France) using 200 μl blood, pellet from centrifuged urine (1 ml of urine was centrifuged at 3000 g for 10 min at room temperature, pellet was washed with 1 ml PBS 1× and the pellet of a second centrifugation at 2000 g for 10 min was used) or 25 mg of minced renal tissue (cortico-medullary junction). PCR and real-time PCR (qPCR) were performed as described below in the Leptospires Laboratory at VetAgro Sup.

Real-time PCR

Real-time PCR (qPCR) was conducted using the LSI kit (TaqVet PathoLept kit, Lifetech, Lissieu, France), which amplifies a specific region of pathogenic Leptospira spp. The qPCR were performed using 20 μl of mix and 5 μl of extracted DNA. Positive and negative controls were included by replacing 5 μl of sample DNA with 5 μl of Leptospira interrogans serogroup Icterohaemorrhagiae DNA or 5 μl of PBS, respectively. qPCR was carried out using Rotorgene 6000 machine (Qiagen, Courtaboeuf, France), samples with a cycle threshold (Ct) over 38 were considered negative (cycle threshold corresponds to the number of qPCR cycle from which an amplified DNA is detected by fluorescence).

End-point PCR and sequencing

All positive samples analysed by qPCR were further submitted to species identification using the rrs (16S rRNA) sequencing. The extracted DNA (5 μl) was added to the amplification mix (45 μl; buffer 10×; MgCl 2 25 mM; dNTPs 10 mM each; primer F 10 μM; primer R 10 μM and TaqPolymerase 5 U/μl). The primers used targeted the 331-bp rrs gene with the following primer sequences: forward: 5 ′ -GGCGGCGCGTCTTAAACATG-3 ′ and reverse: 5 ′ -TTCCCCCCATTGAGCAAGATT-3 ′ . Positive and negative controls were included by using 5 μl of L. interrogans serogroup Icterohaemorrhagiae DNA and 5 μl of PBS instead of sample DNA, respectively. The PCR cycling programme was 15 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at °C and 1 min at 72 °C, followed by 10 min at 72 °C for final elongation. PCR was performed using Sure Cycler 8800 machine (Agilent Technologies, Les Ulis, France). The results were evaluated under UV light after migration using agarose gel electrophoresis. All PCR products were subjected to Sanger sequencing with the BigDye Terminator sequencing kit using a 3730XL DNA analyzer (Applied Biosystems, Saint Aubin, France). The Leptospira species were identified using the sequence information from NCBI nucleotide BLAST (http://blast.ncbi.nlm.nih.gov).

Statistical analysis

CIs around proportion were calculated assuming a binomial distribution and calculated with the binom.test function of R (Version 4.0.3 (2020-10-10), R: A language and environment for statistical computing, R foundation for statistical computing, Vienna, Austria). The chi-squared test was used to compare seroprevalence and a Fisher exact test to compare DNA carriage between stray and domestic animals. Differences were considered statistically significant when P < 0.05. Due to the sampling size, cats were not classified into groups by age, sex and breed. 1a). No significant difference between the two groups was revealed (chi-squared test, χ 2 (1, N = 92) = 0.77, P = 0.38).

Results

Serological results

During

The antibody titre ranged from 1:40 to 1:320 (Fig. 1b). Only one animal presented an antibody concentration high enough to be detected at a titre of 1:320, and 71% of positive results were obtained at dilutions of 1:80 or 1:40.

The positive MAT results revealed serogroup Icterohaemorrhagiae to be the main serogroup in circulation with 47% of the positive results. Considering serovars, the main serovars detected were Icterohaemorrhagiae Icterohaemorrhagiae (19 and VER) and Ballum Castellonis (BAL), with seroprevalences of 7.6% (7/92) and 4.3% (4/92), respectively, followed by serovars Cynopteri Cynopteri (CYN, 2/92), Icterohaemorrhagiae Copenhageni (COP, 2/92) and Australis Australis (AUS, 1/92) (Fig. 1c).

The results can also be considered using the serogroups from which the serovars belong. In this case, 22 cats were positive for only one serogroup (Icterohaemorrhagiae, Australis, Ballum or Cynopteri), one cat was positive for two different serogroups (Icterohaemorrhagiae and Cynopteri) with one predominant serogroup (Icterohaemorrhagiae) and 11 cats were presenting cross-reactions between two or more serogroups. Among domestic cats, specific antibodies for the serogroup Icterohaemorrhagiae were present in 12% (6/50) of the tested animals, while Ballum was detected in 8% (4/50) and Cynopteri was detected in 4% (2/50) of the tested animals. Among stray cats, two serogroups were detected: Icterohaemorrhagiae at 24% (10/42) and Australis at 2% (1/42).

A total of 53% (18/34) of the positive sera presented antibodies for several serovars. Including cross-reactions, three serogroups appeared to be more prevalent than the other serogroups: Icterohaemorrhagiae (19, COP and VER), Ballum (BAL) and Cynopteri (CYN) (Fig. 1c). Additionally, serovars Icterohaemorrhagiae and Copenhageni (12 animals out of 19 cross-reactions), as well as Copenhageni and Cynopteri (7/19) and Icterohaemorrhagiae and Cynopteri (5/19) often cross-reacted.

Molecular results

Due to technical issues (insufficient volume of blood or urine, alteration of samples during transport), some samples could not be used for PCR analyses. A total of 78 blood samples and 89 urine samples were tested. Renal tissue samples were collected from euthanised animals only, which represented 45 cats.

Regardless of the template tested, 12 out of 92 animals (13% (6.1-19.9%)) were tested positive by PCR analyses, with Ct ranging between 30 and 34. More specifically, leptospiral DNA was found in 5/78 (6.4%) blood samples, 4/89 (4.5%) urine samples and 6/45 (13%) renal samples (Table 2). All positive results were detected in 12 stray cats (Table 3), with four of them harbouring leptospiral DNA only in their blood, two shedding leptospiral DNA only in urine and four harbouring leptospiral DNA only in kidneys. One animal presented both urinary and kidney carriage, and one animal tested positive in all three templates. No leptospiral DNA was detected in domestic cats. There was weakly statistically significant difference between stray and domestic cats in the proportion of PCR-positive urine samples (Fisher's exact test, P = 0.034) and PCR-positive blood samples (Fisher's exact test, P = 0.058), with stray cats more likely to present positive results.

Based on rrs gene PCR and sequencing, L. interrogans species was identified in all positive stray cat samples.

When comparing the molecular results with the serological results, out of 92 cats, 4 (4%) were positive by both PCR and serological methods, 30 (33%) were seropositive but had negative PCR results and 8 (9%) were seronegative but had at least one positive PCR result. In total, 42/92 cats (46%) were positive by at least one of the tests performed.

Regarding the four animals with positive results via both MAT and PCR, all of them were seropositive for the serogroup Icterohaemorrhagiae. One PCR result was positive only in the blood sample, one was positive only in the renal sample, one was positive in both renal and urine samples and one was positive in the renal sample without PCR analysis of the urine sample (volume of urine too small for investigation).

Discussion

In the present study, we evaluated the prevalence of Leptospira infection among cats on Reunion Island, in order to investigate the role of feline species as maintenance hosts of different strains of Leptospira spp. Serological (MAT) and molecular (PCR) methods were used in order to determine the infectious status of 92 cats, including 42 stray cats and 50 domestic cats. Sampling of blood (n = 92), urine (n = 89) and kidneys (n = 45) were performed in 2013. At that time, our study was the most important in terms of the number of cats included and tested on Reunion Island [START_REF] Pagès | Human leptospirosis on Reunion Island: past and current burden[END_REF]. It was also the first study to investigate whether the living condition of cats (domestic vs. stray) influenced infection status or seroprevalence. We found no significant difference in seroprevalence between stray and domestic cats. However, our molecular results highlighted the presence of L. interrogans in the blood, kidneys and urine of stray cats, but not domestic cats, which should be considered in leptospirosis control policies.

In this study, we found 37% (cut-off 1:40) of seroprevalence for Leptospira infection, which is close to the only study published regarding the cat population in the island at that time that reported 26.6% (cut-off titre 1:100) [START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF]. When looking at studies performed in other countries published over the last 5 years, the mean seroprevalence observed in cats was 14%, with only one study with a prevalence over 20% in Serbia (cut-off titre 1:100) [START_REF] Alashraf | Serological detection of anti-Leptospira antibodies in shelter cats in Malaysia[END_REF][START_REF] Weis | Detection of Leptospira DNA in urine and presence of specific antibodies in outdoor cats in Germany[END_REF][START_REF] Žákovská | Seroprevalence of antibodies against Borrelia burgdorferi s. l. and Leptospira interrogans s. l. in cats in district of Brno and its environs, the Czech Republic[END_REF][START_REF] Alashraf | First report of pathogenic Leptospira spp. isolated from urine and kidneys of naturally infected cats[END_REF][START_REF] Azócar-Aedo | Leptospira spp. in domestic cats from different environments: prevalence of antibodies and risk factors associated with the seropositivity[END_REF][START_REF] Chan | Serological and PCR detection of feline Leptospira in southern Taiwan[END_REF][START_REF] Palerme | Seroprevalence of Leptospira spp., Toxoplasma gondii, and Dirofilaria immitis in free-roaming cats in Iowa[END_REF][START_REF] Rodriguez | Serologic and urinary PCR survey of leptospirosis in healthy cats and in cats with kidney disease[END_REF][START_REF] Garoussi | Seroprevalence of leptospiral infection in feline population in urban and dairy cattle herds in Mashhad, Iran[END_REF][START_REF] Sonja | Seroprevalence of cat leptospirosis in Belgrade (Serbia)[END_REF]. Our study records a high seroprevalence in cats, which could be due to a lower cut-off value (1:40). Moreover, knowing that there is currently no leptospirosis vaccine for cats due to the low morbidity of the disease, seropositivity is a true indicator of exposure and cannot be confused with postvaccination seropositivity. This high seropositivity suggests that cats from Reunion Island are in contact with the bacteria, probably through other maintenance host species such as rodents (predator-chain transmission), within a favourable environment for Leptospira spp. transmission (tropical climate) [START_REF] Gomard | Feral cats do not play a major role in leptospirosis epidemiology on Reunion Island[END_REF][START_REF]Manual of diagnostic tests and vaccines for terrestrial animals[END_REF].

None of the animals tested presented clinical signs (for domestic cats) or macroscopic lesions (for stray cats) compatible with leptospirosis (fever, weight loss, icterus, lethargy, ascites that could reflect renal failure, with hepatitis) [START_REF] Arbour | Clinical leptospirosis in three cats (2001-2009)[END_REF]. This is consistent with the idea that cats are weakly susceptible to show clinical signs of Leptospira infection. Nevertheless, clinical cases of leptospirosis have been reported in cats infected with higher titres (at least one serovar above 1:800) of serovar Pomona (more pathogenic than the serovars we found in Reunion Island) [START_REF] Arbour | Clinical leptospirosis in three cats (2001-2009)[END_REF]. In conclusion, cats do not seem to be clinically susceptible to the Leptospira serovars circulating in Reunion Island.

Cross-reactions between two or more serogroups represented 32% of positive MAT in our study. This confirms the difficulties encountered in identifying infecting serovars in patients and in maintenance hosts. However, serogroups Icterohaemorrhagiae, Ballum and Cynopteri were detected in a significant proportion of domestic cats, and serogroups Icterohaemorrhagiae and Australis were detected in a significant proportion of stray cats.

Interestingly all human epidemic surveys in Reunion Island reported L. interrogans serogroup Icterohaemorrhagiae to be the predominant infecting serogroup. Between 2004 and 2012, this serogroup represented 57.4% of the confirmed human cases [START_REF] Pagès | Human leptospirosis on Reunion Island: past and current burden[END_REF]. Serogroup Australis has also been identified occasionally 1 for abbreviations). Cross-reactions (between two or more serovars) and reactions with one predominant serovar are presented. CIs at 95% are indicated. MAT cut-off titre 1:40. [START_REF] Mailloux | Leptospirosis on the Island of Reunion. I. Human leptospiroses[END_REF]. In animals, considered potential maintenance hosts or sensitive species, such as rats, cattle and dogs, L. interrogans species was also predominantly identified, with the occasional presence of L. borgpetersenii and L. kirschneri detected in a few animals [START_REF] Guernier | Human leptospirosis on Reunion Island, Indian Ocean: are rodents the (only) ones to blame?[END_REF]. In our study, the same species and serogroups were identified in cats, suggesting important inter-species contamination. This contamination could occur through predation of murine species, especially for stray animals.

To study the potential role of cats in shedding bacteria in the environment, PCR was performed on blood, urine and kidney samples. The results revealed leptospiral DNA in 13.0% of the animals tested, but in 28.6% of the animals when considering only the stray cat population. More specifically, kidney carriage of leptospiral DNA may represent a risk of contamination of the environment. In our study, the renal carriage was estimated at 13% (6/46) among sampled cats, which is lower than what was recorded in 2012 by Desvars et al. (28.6%) [START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF]. This difference may be due to different sampled feline species population (different areas, way of life and proximity to cattle), or difference of sample conservation throughout the experiments. Moreover, the high Ct (30-34) obtained for qPCR suggested that the DNA excretion is close to the detection limit, which could also lead to false-negative results. We detected pathogenic Leptospira DNA in urine of 10% (4/39) of stray cats (4.5% of all 89 sampled cats), which is more than what was already published (0% and 0.6% [START_REF] Desvars | Endemicity of leptospirosis in domestic and wild animal species from Reunion Island (Indian Ocean)[END_REF][START_REF] Gomard | Feral cats do not play a major role in leptospirosis epidemiology on Reunion Island[END_REF]). Interestingly, in a study conducted recently, authors discovered a correlation between rrs2 and lipL32 DNA sequences from a case of L. borgpetersenii detected in a feral cat and the DNA previously sequenced in a human case of leptospirosis in Reunion Island [START_REF] Gomard | Feral cats do not play a major role in leptospirosis epidemiology on Reunion Island[END_REF]. Altogether, it suggests that contamination from cats to humans could be possible, which supports the hypothesis of cats as maintenance host population for Leptospira spp. [START_REF] Gomard | Feral cats do not play a major role in leptospirosis epidemiology on Reunion Island[END_REF]. Another hypothesis could be to consider cats as bridge hosts that link maintenance host population (mainly rats) and susceptible species of concern (mainly human, cattle and pets) [START_REF] Caron | Bridge hosts, a missing link for disease ecology in multi-host systems[END_REF]. Indeed, they present characteristics of maintenance host population but could harbour fewer bacteria in their kidney and urine than rats (since all positives samples showed a high cycle threshold (Ct) between 30 and 34, which means that small amount of DNA is detected).

As observed in previous studies [START_REF] Chan | Serological and PCR detection of feline Leptospira in southern Taiwan[END_REF][START_REF] Sant'anna R | High number of asymptomatic dogs as leptospiral carriers in an endemic area indicates a serious public health concern[END_REF], the actual bacterial DNA carriage in seropositive animals is relatively low (11.8% in our study), confirming that MAT is probably not the best method to detect active potential maintenance host species for Leptospira infection. In this case, various scenarios can be described in relation to the status of the animals tested. First, 54% of the animals presented negative results in both MAT and PCR. Regarding the sensitivity of the methods involved, those animals represent the percentage of the population that has probably not been exposed to leptospires. Similarly, 33% of the animals were seropositive with no leptospiral DNA detected by PCR. This part of the population may contain the animals that have been exposed to the bacteria but have eliminated it with the initiation of the adaptive immune response; or did not excrete Leptospira at the time of sampling. Among the 8/92 animals (9%) presenting positive results in PCR and negative results in MAT, DNA was detected only in the blood for three of them, which is consistent with a recent exposure and ongoing infection with bacteraemia between the 3rd and 10th day post-exposure [START_REF] Levett | Leptospirosis[END_REF]. During this period, leptospires can invade renal tubules, and urinary excretion is possible for several months [START_REF] Shophet | Feline Leptospiral Infection: With Particular Emphasis on Leptospira Interrogans Servor Ballum (thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Veterinary Pathology and Public Health at Massey University[END_REF]. This is observed in cat number E41 with DNA detected in the blood, urine and kidneys. Subsequently, leptospires in the blood are eliminated by the immune system, and only renal carriage persists (animals E15 and E22) with intermittent urinary shedding. Interestingly, two cats were tested positive for DNA in urine sample but not in kidney sample (E25 and E27). This can be explained by the fact that DNA extraction was performed from small pieces of kidneys where bacteria may not have been present. Indeed, rodents and other animals asymptomatically carry leptospires in the lumen of proximal renal tubules [START_REF] Sterling | Urban rats as chronic carriers of leptospirosis: an ultrastructural investigation[END_REF][START_REF] Holzapfel | Escape of TLR5 recognition by Leptospira spp.: a rationale for atypical endoflagella[END_REF]. This also suggests that renal carriage recorded by PCR could be underestimated. Another aspect to consider is the potential impact of storage conditions on the quality of the DNA that could lead to an underestimation of prevalence. Finally, four cats presented positive results by both PCR and MAT with the Icterohaemorrhagiae serogroup. The blood sample from E29 was positive via PCR and presented a titre of 1:40, which could be characteristic of the initiation of adaptive immunity in an animal with relatively recent infection. This particular case could also be the result of a new infection with serological traces of a previous infection, as leptospiraemia is known to last only a few days [START_REF] Levett | Leptospirosis[END_REF]. In E36, leptospiral DNA was detected in the kidney sample, and the MAT titre was 1:40. This is indicative of an old infection and chronic carriage. E32 exhibited the same situation, but DNA was also detected in the urine, which is indicative of intermittent urinary excretion. Finally, for E19, leptospiral DNA was detected in the kidney sample, but PCR could not be performed with the urine sample from this animal.

The PCR results showed a significant difference between the two populations, with 12/42 positive results for stray cats compared with 0/50 in domestic cats. These data must be considered carefully for several reasons. First, because few of the domestic cats (3/50) were evaluated for renal samples. Consequently, some of those animals could carry leptospires in their renal tubules which may not have been detected in urine samples. Second, even if cats rarely present clinical signs of leptospirosis, clinical examinations were only performed on domestic cats, and this could represent a bias of population recruitment. The way the recruitment was performed aimed at limiting this bias by verifying the absence of lesions characteristic for leptospirosis during the necropsy of stray cats. Finally, the small sample size in this study hindered a robust comparison of the two groups. Despite those limitations, the PCR results led us to hypothesise that the environment of the animal could influence the actual chronic kidney carriage of the bacteria. Higher rates of excretion of Leptospira among stray cats could be linked with exposition to higher doses or more frequent exposure due to their environment, contact with stray dogs and predation on small mammals. In addition, their immune system could be compromised and less efficiently control the bacteria carriage due to lower general health or co-infections, with feline immunodeficiency virus for example [START_REF] Da | Detection and modeling of anti-Leptospira IgG prevalence in cats from Lisbon area and its correlation to retroviral infections, lifestyle, clinical and hematologic changes[END_REF].

Species identification analysis performed on positive samples confirmed that the cats from our study harbour a pathogenic species (L. interrogans). As previously reported [START_REF] Gomard | Feral cats do not play a major role in leptospirosis epidemiology on Reunion Island[END_REF], leptospiral DNA (L. borgpetersenii) detected in cats can be congruent with genome sequences reported for human cases. Interestingly, we show evidence of exposure to diverse serogroups, but evidence of infection with L. interrogans only. Even, if the serological and genomic classifications of Leptospira genus do not correlate, we suggest here that several serogroups associated with several different species of Leptospira circulate in the island with probably different maintenance communities. These observations highlight the idea of leptospirosis being a multi-host-multi-pathogen system with strong variability between different ecosystems. In this situation and with the significant carriage we reported in stray cats, controlling the population of stray animals on the island is an increasingly important public health issue as they very probably are part of the maintenance community for L. interrogans. Moreover, more public awareness campaigns should be undertaken to advise people in contact with stray animals about the risks of transmission.

Future investigations could be performed to improve data collection on cat populations. Positive samples by PCR could be grown on tween-albumin EMJH medium [START_REF] Marquez | Overview of laboratory methods to diagnose leptospirosis and to identify and to type leptospires[END_REF] to determine whether the detected DNA originates from dead or living leptospires. Indeed, several recent studies reported the isolation of pathogenic Leptospira spp. from kidneys and urine of cats [START_REF] Dorsch | Cats shedding pathogenic Leptospira spp.an underestimated zoonotic risk?[END_REF][START_REF] Alashraf | First report of pathogenic Leptospira spp. isolated from urine and kidneys of naturally infected cats[END_REF]. This information could provide crucial insights into the actual risk following urinary excretion. Regarding the MAT results, it would be interesting to perform a second serological measure after a 2-weeks' time interval for the animals with bacteraemia to consider the sensitivity of MAT in asymptomatic and recently infected animals. Combining MAT and PCR analyses of blood, urine and kidney samples, our study confirms that cats are involved in the epidemiological cycle of leptospirosis, and that special care must still be taken with stray cat populations on Reunion Island. Nevertheless, we still have to determine if cats are contaminated through the predator-chain transmission with rats (being the main source of infection), if transmission withinspecies occurs between cats, if they are contaminated from the environment, or a combination of all, in various proportions.

Fig. 1 .

 1 Fig. 1. Seroprevalence of cats tested by micro-agglutination test (n = 92). (a) Percentage of animals tested negative or positive for one serogroup of leptospires or several serogroups (cross-reaction) via MAT according to their condition of living, stray cats (n = 42) or domestic cats (n = 50). (b) Higher dilution for which the MAT is positive (n = 34) for one predominant serovar and for any serovar. Cross-reactions (between two or more serovars) and reactions with one predominant serovar are presented. (c) Percentage of seropositive samples (n = 92) for each of the 26 Leptospira spp. serovars tested (*see Table1for abbreviations). Cross-reactions (between two or more serovars) and reactions with one predominant serovar are presented. CIs at 95% are indicated. MAT cut-off titre 1:40.

Table 1 .

 1 Leptospiral strains used as capture antigens for micro agglutination test

	Serogroup	Serovar	Strain	Abbr.	Species
	Australis	Australis	Ballico	AUS	L. interrogans
		Muenchen	Muenchen C90 a	MUN	L. interrogans
		Bratislava	Jez Bratislava a	BRAT	L. interrogans
	Autumnalis	Autumnalis	Akiyami A a	AKI	L. interrogans
		Bim	Strain 1051 a	BIM	L. kirschneri
	Ballum	Castellonis	Castellòn 3 a	BAL	L. borgpetersenii
	Bataviae	Bataviae	Van Tienen a	BAT	L. interrogans
	Canicola	Canicola	Hond Utrecht IV	CAN	L. interrogans
	Cynopteri	Cynopteri	Strain 3522C a	CYN	L. kirschneri
	Grippotyphosa	Grippotyphosa	Moskva V a	GRIP	L. kirschneri
		Vanderhoedoni	Kipod 179 a	VAN	L. kirschneri
	Hebdomadis	Kremastos	Kremastos a	HEB	L. interrogans
	Icterohaemorrhagiae	Icterohaemorrhagiae	Strain 19	19	L. interrogans
		Copenhageni	Strain M20	COP	L. interrogans
		Icterohaemorrhagiae	Verdun	VER	L. interrogans
	Panama	Panama	Strain CZ 214K a	PAN	L. noguchii
	Pomona	Pomona	Pomona a	POM	L. interrogans
	Pyrogenes	Pyrogenes	Salinem a	PYR	L. interrogans
	Sejroe	Sejroe	Strain M84 a	SJ	L. borgpetersenii
		Saxkoebing	Strain Mus 24 a	SAX	L. interrogans
		Hardjo	Hardjoprajitno	HJ	L. interrogans
		Wolfii	Strain 3705 a	WOLF	L. interrogans
	Tarassovi	Tarassovi	Perepelitsin a	TAR	L. borgpetersenii
	Mini	Mini	Sari a	MINI	L. borgpetersenii
	Semaranga	Patoc	Patoc 1 a,b	Bifl P	L. biflexa
		Semaranga	Veldrat a,b	Meyeri	L. meyeri

Serogroup and serovar according to antigenic classification, complete name and abbreviation (Abbr.) of the strain used, and species according to genomic classification. a Reference strain. b Non-pathogenic Leptospira strain.

Table 2 .

 2 PCR results for blood, urine and renal samples of domestic and stray cats, and comparison with seroprevalences

	Population considered	PCR on blood samples*	PCR on urine samples**	PCR on renal samples	Seroprevalence (MAT)

Table 3 .

 3 Individual status for MAT and blood, urine and kidney tissue PCR for the twelve cats presenting at least one positive result at PCR analysis

	Cat
	ID	Blood	Urine	Kidney	Serological results
	E15	-	-	+	-
	E19	-	NE	+	1:80 (19
					Icterohaemorrhagiae)
					1:80 (COP Copenhageni)
					1:40 (VER
					Icterohaemorrhagiae)
	E22	-	-	+	-
	E25	-	+	-	-
	E27	-	+	-	-
	E29	+	-	-	1:40 (19
					Icterohaemorrhagiae)
	E31	+	-	-	-
	E32	-	+	+	1:80 (19
					Icterohaemorrhagiae)
					1:40 (COP Copenhageni)
	E35	+	-	-	-
	E37	-	-	+	1:40 (19
					Icterohaemorrhagiae)
					1:40 (COP Copenhageni)
	E41	+	+	+	-
	E43	+	-	-	-

https://doi.org/10.1017/S095026882100176X Downloaded from https://www.cambridge.org/core. INRAE UMR APBV, on 06 Jan 2022 at 13:04:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.

NE, non-evaluated; +, positive result for L. interrogans via qPCR and sequencing. If the animal was seropositive, the seroprevalence titre is indicated for all serovars detected. Cat ID, identification number associated with the animal for the study.

Acknowledgements. The authors acknowledge the Veterinary Clinic of Front-de-Mer in St Paul, the 50 customers, who allowed sample collection from their animal, the shelters of Sainte-Marie and Saint-Paul, who allowed sampling on the stray cats captured, and Thibault Lurier for his precious advice on statistical analysis.

Data availability statement. The data that support the findings of this study will be available on request via e-mail from the corresponding author.

Author contributions. MH performed the experiments and analysed the data. FT analysed the data and wrote the manuscript. ZD conceived and coordinated the study, analysed the data and wrote the manuscript. All authors read and approved the final manuscript.

Financial support. This study was supported and financed by the Laboratoire des Leptospires, VetAgro Sup, Campus de Lyon, France.

Conflict of interest. None.


Dian Zhong

Kuo Zeng
email: zengkuo666@hust.edu.cn

Jun Li

Yi Qiu

Gilles Flamant

Ange Nzihou

Sergey Vasilevich

Vladimirovich

Haiping Yang

Hanping Chen

Characteristics and evolution of heavy components in bio-oil from the pyrolysis of cellulose, hemicellulose and lignin

Keywords:

Three main components of biomass were pyrolyzed individually in a closed reaction system at 500-700 • C for 60s and 90s. Then bio-oil heavy compounds were further analyzed with Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) and Kendrick mass defect (KMD) analysis. The evolution paths of heavy compounds for the different pyrolysis stages were proposed. It was found that the sugars and phenolic-like species in heavy compounds were the most active substances during secondary reactions. Moreover, the rising temperature promoted this secondary reaction of phenolic-like species as the decrease in their abundances growing from 13% to 54%, while contrarily inhibited it for hemicellulose as the decrease in their abundances changing from 44% to -2%. The lignin-derived lipids and unsaturated hydrocarbons that generated in the secondary reactions increased with rising temperature. KMD analysis showed that the heavy compounds of cellulose and hemicellulose prefer homologous evolution during pyrolysis, while those of lignin had more complex evolution paths like cracking and recombination.

Introduction

Pyrolysis technique has been widely applied for bio-oil production from biomass like microalgae, wood, crop residues and other carbonaceous materials, which can be used as biofuel and as high value-added chemical products [START_REF] Hu | Progress of the applications of bio-oil[END_REF][START_REF] Sepehri | Effect of nitrifiers community on fouling mitigation and nitrification efficiency in a membrane bioreactor[END_REF][START_REF] Sepehri | Interaction between Chlorella vulgaris and nitrifying-enriched activated sludge in the treatment of wastewater with low C/N ratio[END_REF]. However, it has been reported that the composition of liquid products of biomass pyrolysis is very complex and chemically unstable due to the large number of heavy compounds [START_REF] Oasmaa | A guide to physical property characterisation of biomassderived fast pyrolysis liquids[END_REF]. Generally, the heavy compounds results in bio-oil condensation in the downstream pipeline during the production and utilization, which seriously deteriorates the working condition of the equipment, and causes additional production costs. In addition, the formation of heavy compounds is harmful to both the quantity and the quality of pyrolysis oil. Consequently, for a better utilization of bio-oil, it is important to clarify the composition and formation mechanism of the heavy compounds.

Pyrolysis of biomass is a complex physical and chemical process, which characteristics are closely related to the feedstock composition [START_REF] Yu | Characteristics of tar formation during cellulose, hemicellulose and lignin gasification[END_REF]. Biomass can be regarded as organic matter formed by cross-linking of three main components (cellulose, hemicellulose and lignin), therefore the pyrolysis process of biomass can be simplified as the superposition of the pyrolysis behavior of its three main components [START_REF] Yang | In-depth investigation of biomass pyrolysis based on three major components: hemicellulose, cellulose and lignin[END_REF], which are different from each other [START_REF] Demirbas | Competitive liquid biofuels from biomass[END_REF]. Hence, the formation mechanism of bio oil and the heavy compounds can be derived from the evolution of the three main components of biomass during the pyrolysis process.

Cellulose has a relatively more regular structure among the three components. Therefore, the formation mechanism of cellulose bio-oil is now well developed, among which the model proposed by Bradbury and Shafizadeh (B-S model) is widely accepted [START_REF] Bradbury | A kinetic model for pyrolysis of cellulose[END_REF]. According to this model, cellulose undergoes depolymerization and dehydration at a pyrolysis temperature below 300 • C, and is transformed into an intermediate called "active cellulose". Most of the light compounds in cellulose pyrolysis are generated by the active cellulose, and several possible pathways has been proposed 9. Mettler et al. [START_REF] Mettler | Revealing pyrolysis chemistry for biofuels production: conversion of cellulose to furans and small oxygenates[END_REF] showed that oligosaccharides are more likely to produce furans, and under certain conditions the furan compounds are easily condensed to coke, which indicates a possible pathway of formation of heavy compounds. Lu [START_REF] Lu | Influence of pyrolysis temperature and time on the cellulose fast pyrolysis products: analytical Py-GC/MS study[END_REF] and Hosoya [START_REF] Hosoya | Different pyrolytic pathways of levoglucosan in vapor-and liquid/solid-phases[END_REF] found that levoglucan is thermally stable in the gas phase, while undergoes secondary reactions to generate coke and other heavy components in the liquid phase. Impressively, the pyrolysis experiments in Hosoya's works [START_REF] Hosoya | Different pyrolytic pathways of levoglucosan in vapor-and liquid/solid-phases[END_REF][START_REF] Hosoya | Solid/liquid-and vapor-phase interactions between cellulose-and lignin-derived pyrolysis products[END_REF][START_REF] Hosoya | Pyrolysis gasification reactivities of primary tar and char fractions from cellulose and lignin as studied with a closed ampoule reactor[END_REF] were conducted with a closed ampoule reactor, which allows an easy control of the reaction process and reaction time.

The separation of hemicellulose from biomass is difficult to perform and this compound is unstable during pyrolysis due to its complex structure. Therefore, the pyrolysis mechanism of hemicellulose was less studied, and to overcome this problem xylan is commonly used as a model compound. Chen et, al [START_REF] Chen | Torrefaction, Pyrolysis and Two-stage Thermodegradation of Hemicellulose, Cellulose and Lignin[END_REF]. found that the thermogravimetric behavior of xylan could be divided into the primary volatilization stage (210-340 • C) and the secondary stage (340-500 • C) corresponding to the intensive secondary reaction. Fisher et al. [START_REF] Fisher | Pyrolysis behavior and kinetics of biomass derived materials[END_REF] compared the thermogravimetric behavior of cellulose and xylan, and found that xylan has a lower thermal stability but higher coke yield than cellulose, resulting an earlier peak of oil yield at 480 • C. Ponder et al. [START_REF] Ponder | Thermal synthesis and pyrolysis of a xylan[END_REF] explained that xylose molecules cannot exist stably through intramolecular dehydration to form glycosides due to the lack of one hydroxyl group. Further study showed that hemicellulose plays an important role in the formation of solid products and can be regarded as a potential precursor for the coke formation [START_REF] Shen | The thermal performance of the polysaccharides extracted from hardwood: cellulose and hemicellulose[END_REF]. It also has a certain influence on the formation of heavy components during pyrolysis.

The pyrolysis of lignin starts at about 200 • C, and the decomposition continues as the temperature rises [START_REF] Yang | Characteristics of hemicellulose, cellulose and lignin pyrolysis[END_REF]. Kawamoto et al. compared the thermolysis behavior of 8 different lignin dimers, and found that the types of substituents on the aromatic ring and the connection ways between monomers affect the pyrolysis products [START_REF] Kawamoto | Pyrolysis reactions of various lignin model dimers[END_REF]. Other studies found that monophenols and their derivations are easy to polymerize into products with larger molecular weight [START_REF] Patwardhan | Understanding the fast pyrolysis of lignin[END_REF][START_REF] Xiao | An insight into the OPAHs and SPAHs formation mechanisms during alkaline lignin pyrolysis at different temperatures[END_REF]. It has been proved that there are a lot of molecule with molecular weight in the range 200-1300 Da existing in the pyrolysis products of lignin, which cannot be analyzed by GC-MS [START_REF] Xiao | An insight into the OPAHs and SPAHs formation mechanisms during alkaline lignin pyrolysis at different temperatures[END_REF][START_REF] Hua | The tribological behavior of diester-containing polysulfides as additives in mineral oil[END_REF]. All these data indicate that lignin is likely to generate heavy components.

Most of previous studies on bio-oil formation mechanism only focus on substances with small molecular weight using GC-MS, UV-F and FT-IR, which can not give the detail information of heavy compounds previously mentioned. Thus, the heavy compounds in bio-oil are less discussed so far because of the lack of appropriate analysis methods before Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) has been introduced. The unique element composition of each substance can be given for further characterization thanks to its V represents volatile, FC represents fixed carbon, A represents ash. a Calculated by difference. And V. ultrahigh resolving power (>200,000) and wide detectable range of molecular weight (200-1000 Da). Consequently, the global composition of bio-oil has been studied with the help of FT-ICR-MS [START_REF] Hertzog | Toward controlled ionization conditions for ESI-FT-ICR-MS analysis of bio-oils from lignocellulosic material[END_REF][START_REF] Terrell | Novel strategy to analyze fourier transform ion cyclotron resonance mass spectrometry data of biomass pyrolysis oil for oligomeric structure assignment[END_REF][START_REF] Liu | Characterization of red pine pyrolysis bio-oil by gas chromatography-mass spectrometry and negative-ion electrospray ionization fourier transform ion cyclotron resonance mass spectrometry[END_REF]. Xiong et al. [START_REF] Xiong | Formation of the heavy tar during bio-oil pyrolysis: a study based on Fourier transform ion cyclotron resonance mass spectrometry[END_REF][START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF] showed the pyrolysis behavior of bio-oil, and then assessed the chemical composition of heavy components derived from three main components of biomass (400-650 • C). They found that the oxygen-containing functional groups play an important role during the formation of heavy compounds. Xiao et al. [START_REF] Xiao | The formation mechanism for OPAHs during the cellulose thermal conversion in inert atmosphere at different temperatures based on ESI(-) FT-ICR MS measurement and density functional theory (DFT)[END_REF] explored the formation mechanism of the oxygenated polycyclic aromatic hydrocarbons during cellulose pyrolysis. The formation of phenolic oligomers in the lignin-derived heavy compounds has also been studied [START_REF] Bai | Formation of phenolic oligomers during fast pyrolysis of lignin[END_REF]. Their works suggested that the formation processes of these heavy compounds differ from the three components of biomass, and is influenced by the temperature and heating rate.

In order to gain insights into the formation mechanism of these components, the Kendrick mass defect (KMD) method was applied to mass spectrometry analysis [START_REF] Zhang | Characterization of lignin compounds at the molecular level: mass spectrometry analysis and raw data processing[END_REF]. Qi et al. used FT-ICR-MS combined with 2D KMD plots to characterize lignin depolymerization products and showed the activities of different side-chains [START_REF] Qi | Two-dimensional mass defect matrix plots for mapping genealogical links in mixtures of lignin depolymerisation products[END_REF]. Dhungana et al. introduced KMD methods to compare the bio-oil from corn stover and pine shavings pyrolysis, and their differences on aliphatic chains can be visually observed from KMD plots [START_REF] Dhungana | Characterization of slow-pyrolysis bio-oils by high-resolution mass spectrometry and ion mobility spectrometry[END_REF]. Nevertheless, the composition as well as the evolution of heavy compounds from the three components of biomass pyrolysis remained unclear, and it is helpful to understand the formation mechanism of heavy oil and guide the upgrading process of bio-oil.

In this paper, the fast pyrolysis of cellulose, hemicellulose and lignin with 500,600 and 700 • C for 60s and 90s were conducted in a closed reaction system. FT-ICR-MS and KMD analysis was used to study the chemical composition and evolution of their heavy compounds.

Material and methods

Raw material

The cellulose sample (CAS 9004-34-6), xylan (CAS 9014-63-5) and alkali lignin (CAS 8068-05-1) were dried at 105 • C for 4 h before used. Xylan was used as the alternative of the hemicellulose in this paper. The proximate and elemental analysis of the three components is shown in Table 1. N and S contents in lignin are significantly higher than in the other two components, while the oxygen content is relatively lower. This difference has a great impact not only on the subsequent data processing but also on its pyrolysis behavior.

Pyrolysis experiment

The experiment was conducted in a closed reaction system shown in Fig. 1a. The system includes a vertical tube furnace (OTF-1200X-4-VTQ) and a quartz U-tube reactor. The specific size of the reactor is shown in Fig. 1b.

Before pyrolysis, 100 mg sample was loaded in the U-tube reactor, and then the system was flushed by N 2 (99.999%, 300 ml/min) for 3 min. The thermocouple was placed at a depth of 250 mm from the entrance of the furnace to measure the temperature of reaction zone. Once the target temperature is reached (500 • C, 600 • C, 700 • C), the gas inlet and outlet valves were closed, and the reactor was quickly lowered down for pyrolysis during 60s/90s. Hosoya and Jiang [START_REF] Hosoya | Pyrolysis gasification reactivities of primary tar and char fractions from cellulose and lignin as studied with a closed ampoule reactor[END_REF][START_REF] Jiang | Modeling pyrolytic behavior of pre-oxidized lignin using four representative β-ether-type lignin-like model polymers[END_REF] proved that the secondary reactions mainly take place after 60s for pyrolysis of a small amount (100 mg) of cellulose and lignin, and that the pyrolysis is nearly completed after heating for 90s or longer. Thus, the reaction time were set as 60s and 90s, representing the primary pyrolysis and secondary pyrolysis respectively.

After pyrolysis, the reactor was taken out immediately and cooled with compressed air for 2 min and subsequently immersed in ice-water mixture for 1 min. Then the valves were open again and uncondensed gas was wiped out by N 2 (99.999%, 300 ml/min) for 3 min and gathered by gas bag. The liquid products condensed in the reactor was extracted with CH 3 OH to give a 10 ml solution. The dark-colored residue that can not be dissolved and the solid product were defined as char fraction in this paper.

Analysis and characterization of bio-oil 2.3.1. GC-MS setting

The bio-oil were analyzed using gas chromatography-mass spectrometry system (GC/MS, Agilent 7890B/5977A, America). Capillary column model is DB-5MS (30 m × 0.32 mm × 0.25 μm), The oven temperature program was as follows: initial temperature of 50 • C, increase to 100 • C at 10 • C/min, increase to 200 • C at 5 • C/min, increase to 300 • C at 10 • C/min, and hold for 3 min. The inlet temperature was 280 • C, the split flow rate was 40 ml/min, the split ratio was 40:1, the mass spectrometry ionization mode was EI (70 V). The results were compared with the NIST 14 database, and the m/z scanning range was 35-550.

FT-ICR-MS analysis

FT-ICR-MS (Bruker, Solarix 7.0 T) was used to analyze the heavy compounds in oil in negative mode using electrospray ionization (ESI) source. The ionization method of FT-ICR-MS has a preference for different target components based on their characteristics like different functional groups, aromatic content, and polarity. Among these soft ionization methods, ESI (electrospray ionization) and APPI (atmospheric pressure photoionization ionization) are mostly used in analyzing biomass pyrolysis oil for their wide range of application [22, Table 2 The rules applied in calculation. Fig. 2. The oil yields of three samples. [START_REF] Xiong | Formation of the heavy tar during bio-oil pyrolysis: a study based on Fourier transform ion cyclotron resonance mass spectrometry[END_REF][START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF][START_REF] Deng | Effects of cellulose, hemicellulose, and lignin on the structure and morphology of porous carbons[END_REF][START_REF] Xiong | Effects of vapor-/solid-phase interactions among cellulose, hemicellulose and lignin on the formation of heavy components in bio-oil during pyrolysis[END_REF][START_REF] Xiong | Effects of AAEMs on formation of heavy components in bio-oil during pyrolysis at various temperatures and heating rates[END_REF][START_REF] Xiong | Effects of the gas-/liquid-phase interactions on the evolution of bio-oil during its thermal treatment[END_REF][START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF]. Considering that the bio-oil is generally acidic and oxygen-contained, the ESI ionization source which is more sensitive to polar substances was chosen [START_REF] Yue | Unravelling the impact of lithofacies on the composition of NSO compounds in residual and expelled fluids of the Barnett, Niobrara and Posidonia formations[END_REF]. Furthermore, the negative ion mode (ESI (-) ) was chosen instead of the positive one (ESI (+) ) because the bio-oil obtained from model compounds has little nitrogen and sulfur, which are preferred by ESI (+) [START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF]. The details of basic instrument setting have been mentioned in our previous study [START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF]. Before analysis, the bio-oil was diluted with CH 3 OH (LC/MS grade) to 0.4 mg/ml for a better ionization and separation in the FT-ICR-MS [START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF][START_REF] Staš | Application of orbitrap mass spectrometry for analysis of model bio-oil compounds and fast pyrolysis bio-oils from different biomass sources[END_REF][START_REF] Miettinen | Compositional characterization of phase-separated pine wood slow pyrolysis oil by negative-ion electrospray ionization fourier transform ion cyclotron resonance mass spectrometry[END_REF], and the instrument was calibrated by NaCOOH solution [START_REF] Wang | Characterization of brominated disinfection byproducts formed during the chlorination of aquaculture seawater[END_REF]. Then the sample was infused into the MS at a rate of 120 μml/h. Each spectrum was gained after co-adding 128 scans to enhance the signal-to-noise ratio (S/N).

After testing, those signal with a S/N ≥ 3 were selected [START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF] for further calculation using MATLAB script [START_REF] Li | Characteristics and evolution of nitrogen in the heavy components of algae pyrolysis bio-oil[END_REF] and their chemical formulas were determined by several rules listed in Table 2. Besides, the solvent used to dilute the oil sample was also analyzed, and the prominent signals in solvent mass spectrum were abandoned in the experiment results.

KMD analysis

FTMS gives the relative molecular mass (RM) of each molecule, which takes the 12C (12.000000 Da) as the mass reference. Replace the 12C with a certain group F, then the relative molecular Mass RM of a molecule will be transformed into Kendrick Mass (F) (Kendrick Mass taking F group as the mass reference, KM(F)) shown as Eq (1) [START_REF] Kendrick | A mass scale based on CH 2 = 14.0000 for high resolution mass spectrometry of organic compounds[END_REF]:

KM(F) = RM × NM F RM F (1)
Fig. 3. The distributions of the heavy components of three samples produced at 500, 600 and 700 • C under 60s and 90s pyrolysis time.

Table 3

The percentages of different O-containing species in heavy compounds in cellulose-oil (C), hemicellulose-oil (H) and lignin-oil (L) produced at 500, 600 and 700 • C for 60s and 90s.

Sample Content (%) where, RM F is the relative molecular weight of the F group, and NM F is the relative molecular weight of the F group after rounding. For example, RM CH2 ≈ 14.01565, NM CH2 = 14.

ON = 0 1 < ON < 3 4 < ON < 6 7 < ON < 9 ON > 9 C-
It follows that a series of homologues with the same characteristic structure will have identical Kendrick mass defects [START_REF] Kendrick | A mass scale based on CH 2 = 14.0000 for high resolution mass spectrometry of organic compounds[END_REF]:

KMD(F) = KM(F) -NM (2)
where the KMD(F) represents the Kendrick mass defect on the basis of F group, and NM represents the rounded relative molecular weight.

The existence of molecules with the same KMD in the pyrolysis products indicates that such substances have undergone the removal or addition of F group during the pyrolysis process [START_REF] Kendrick | A mass scale based on CH 2 = 14.0000 for high resolution mass spectrometry of organic compounds[END_REF][START_REF] Dier | Enhanced mass defect filtering to simplify and classify complex mixtures of lignin degradation products[END_REF][START_REF] Roach | Higher-order mass defect analysis for mass spectra of complex organic mixtures[END_REF][START_REF] Kilgour | Appropriate degree of trust: deriving confidence metrics for automatic peak assignment in highresolution mass spectrometry[END_REF]. Therefore, the evolution of heavy components can be explored by analyzing the KMD of different groups [START_REF] Hughey | Kendrick mass defect spectrum: a compact visual analysis for ultrahigh-resolution broadband mass spectra[END_REF].

Results and discussion

Bio-oil yield and macroscopic characteristics of heavy compounds

The trend of bio-oil yield under different reaction time is basically the same as temperature rises as shown in Fig. 1. The oil yield of cellulose was generally the highest, followed by those of hemicellulose and lignin. The oil yields of cellulose and hemicellulose decreased with the increase of reaction time, indicating that the secondary reaction would reduce the yield of bio-oil [START_REF] Lanza | Gas phase chemistry in cellulose fast pyrolysis[END_REF][START_REF] Shen | The mechanism for thermal decomposition of cellulose and its main products[END_REF]. The oil yield of lignin increased with the increase of reaction time, possibly because the secondary stage of lignin pyrolysis tends to promote the formation of liquid products [START_REF] Hosoya | Pyrolysis gasification reactivities of primary tar and char fractions from cellulose and lignin as studied with a closed ampoule reactor[END_REF].

The oil yield of cellulose pyrolyzed for 60s continually increased as temperature rose from 500 • C to 700 • C, While it slightly decreased at 700 • C for longer pyrolysis time, which is consistent with the results of Shen et al. [START_REF] Shen | The mechanism for thermal decomposition of cellulose and its main products[END_REF]. With the increase in temperature, the content of levoglucan in bio-oil gradually decreased due to the secondary reactions.

The lignin-derived oil yield steadily increased at higher temperature, while the hemicellulose-derived oil yield tended to decrease. Same results can also be observed in other works [START_REF] Xin | Assessment of pyrolysis polygeneration of biomass based on major components: product characterization and elucidation of degradation pathways[END_REF].

The distributions of molecular weight of heavy compounds in cellulose and hemicellulose have the highest abundance in a range of 250Da-300Da (Fig. 3). The molecular weight of heavy compounds in lignin concentrated on the range of 300Da-400Da, and its total abundance of heavy compounds was less than in others. Fig. 3 also shows that the temperature and reaction time affect the molecular weight distribution of the heavy components. The abundance of heavy oil produced from cellulose and hemicellulose in the primary pyrolysis stage was significantly greater than that in the secondary pyrolysis stage particularly in a range of 250Da-450Da. These differences between the two stages shrunk with the increase in temperature. These could be mainly caused by the differences of the total oil yield (Fig. 2). However, the abundance of lignin-derived heavy compounds changed more slightly with the increase of pyrolysis time compared with the others. The abundance of compounds with a higher molecular weight (>600Da) increased significantly with temperature rising from 500 to 700 • C, which indicated that these larger compounds of lignin was more likely produced in the secondary reaction stage.

Xiong et al. found the oxygen-containing species participated in the formation of most heavy compounds. The changes of oxygen content in heavy oil at different reaction stages is shown in Table 3 according to Eq. (3).

C ON=x = ∑ I(O x ) ∑ I ( 3
)
where the C ON=x represents the relative content of the compounds with x oxygen atoms, I is the peak abundance of each molecular, and I(O x ) is the peak abundance of molecular with x oxygen atoms.

The increase of reaction time results in the decrease of the content of almost all substances with oxygen number (ON) greater than seven. More substances with lower oxygen content was generated. However, the substances without oxygen atoms in lignin showed a slight decrease in the second reaction stage. According to the classification based on H/ C and O/C, the substances that do not contain oxygen are mainly lipids, unsaturated hydrocarbons and condensed aromatic hydrocarbons. This indicates that the secondary reaction has an inhibitory effect on the generation of these substances, but the details still needed further studies.

Characteristics of heavy compounds

Distribution of species in heavy compounds

The van Krevelen plot shows the H/C ratio versus O/C ratio of each compound. Previous studies concluded that the distributions of species in bio-oil correspond to unique constant regions on the van Krevelen The heavy compounds were classified according to the number of oxygen atoms and presented on van Krevelen diagram, as shown in Fig. 4. The heavy compounds of cellulose and hemicellulose were mainly distributed in the regions of sugars and phenolic-like species, but less in the lipids, unsaturated hydrocarbons and condensed aromatic hydrocarbons regions. All kinds of Oxygen-containing species, especially those with 4-9 ON are found in phenolic-like region, while the sugars are mainly substances with ON greater than 7. It meant that these components were produced by the depolymerization of various polysaccharide precursors [START_REF] Wang | Initial pyrolysis mechanism and product formation of cellulose: an Experimental and Density functional theory (DFT) study[END_REF]. The distributions of lignin heavy compounds were different from those of another two samples in both elemental composition and compounds distribution. The heavy compounds of lignin were more abundant than those of cellulose and hemicellulose, and mainly distributed in the regions of lipids, unsaturated hydrocarbons, condensed aromatic hydrocarbons and phenolic-like species in Fig. 4. Almost no saccharides are generated. Although lignin is composed of various methoxy p-hydroxyphenyl propane, its main heavy components were lipids and unsaturated hydrocarbons whose maximum abundances account for 58.59% and 29.62%, rather than phenolic substances. On the contrary, a lot of "phenolic-like species" were generated in the heavy compounds of cellulose and hemicellulose whose origin structure does not contain any benzene ring. Lignin having more side chains, it generates more saturated/unsaturated free radicals during the pyrolysis process. These radicals can further recombine and form unsaturated hydrocarbons and lipid substances [START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF]. "Phenolic-like species" in the heavy compounds of cellulose and hemicellulose can only be derived from the recombination of small unsaturated radicals generated during pyrolysis or the derivatives of sugar units with large degree of unsaturation and ring structure formed through multi-stage dehydration, such as 1,4:3, 6-dihydro-α-d-glucopyranose (C 6 H 8 O 4 ) [START_REF] Scheer | Radical chemistry in the thermal decomposition of anisole and deuterated anisoles: an investigation of aromatic growth[END_REF].

With the increasing of reaction time, the sugars and phenolic-like species in heavy compounds of cellulose reduced, and so did their abundances. The dehydration of sugars happened at a low temperature (550 • C), during which "phenolic-like species" were produced [START_REF] Kawamoto | Review of reactions and molecular mechanisms in cellulose pyrolysis[END_REF]. As the reaction time goes on, those species undergo secondary reaction like ring opening and cracking. It results in the formation of more kinds of derivatives and saturated/unsaturated hydrocarbons radicals with small molecular weight [START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF][START_REF] Kawamoto | Review of reactions and molecular mechanisms in cellulose pyrolysis[END_REF]. The abundance difference between two reaction times became larger with the increase in temperature (abundance reduced by 13% at 500 • C and by 54% at 700 • C). This trend suggests that the rising temperature promote the secondary reaction of sugars and phenolic-like species. This also explained the lower abundances of these two compounds at higher temperature for longer reaction time. The contents of lipids and unsaturated hydrocarbons for 60s pyrolysis were lower than for 90s at 600 and 700 • C. This finding suggests that higher temperature promotes the formation of lipids and unsaturated hydrocarbons in secondary reaction. Meanwhile, the content of condensed aromatic hydrocarbons decreased during secondary reactions, but the abundance of these compounds from 90s pyrolysis increased with temperature. This suggests that higher temperature inhibits the decrease of condensed aromatic hydrocarbons. Xiao et al. [START_REF] Xiao | The formation mechanism for OPAHs during the cellulose thermal conversion in inert atmosphere at different temperatures based on ESI(-) FT-ICR MS measurement and density functional theory (DFT)[END_REF] proved that most of condensed aromatic hydrocarbons could be generated during secondary reaction of cellulose pyrolysis by Diels-Alder reaction, explaining the final increase of condensed aromatic species.

The heavy compounds of hemicellulose mainly distribute in the phenolic and saccharide regions that is similar to the results of cellulose. With the rise of temperature, both of them produce more phenolic-like species with O/C < 0.3, and show a trend of condensation. This suggests that the increase of temperature promotes the condensation by removing their oxygen-containing functional groups. However, hemicellulose generates more phenolic-like species and sugars in the primary stage, possibly because of its intensive depolymerization process [START_REF] Wang | Mechanism of xylan pyrolysis by Py-GC/MS[END_REF]. With the increase of temperature, the phenolic-like species generated from the 60s pyrolysis decrease and the secondary decomposition process weakened, leading to the lower difference between the two stages (abundance reduced by 44% at 500 • C and by -2% at 700 • C). Meanwhile, the weakening decomposition also provides less pyrolytic radicals needed in the formations of lipids and unsaturated hydrocarbons. This trend explains their abnormally decrease at 700 • C in secondary pyrolysis. It can be speculated that lipids and unsaturated hydrocarbons can be formed by recombination of small radicals in both primary and secondary pyrolysis, while being further decomposed in secondary reaction. Temperature indirectly affects their formation by promoting or inhibiting the radicals generated from the secondary reaction of phenolic-like species and sugars. Fig. 5 indicates that the increase of temperature inhibits the formations of lipids and unsaturated hydrocarbons in the lignin-derived heavy compounds in the initial pyrolysis. However, it promotes their formation Fig. 10. KMD of oxygen-containing compounds in cellulose pyrolysis oil.

during the secondary reactions and even overcame the secondary decomposition of unsaturated hydrocarbons leading to the increase of its abundance after 90s pyrolysis. The side chains on the lignin units begins to be remove at 400 • C [START_REF] Hosoya | Role of methoxyl group in char formation from lignin-related compounds[END_REF], and the removal reaction tends to be completed with the increase of temperature. Therefore, it is speculated that the formation of lipids and unsaturated hydrocarbons are in competition at 700 • C leading to the weakening of the promoting effect of the secondary reaction of lipids. In the secondary reaction stage, phenolic substances will be further decomposed and transformed, making their content slightly lower. Meanwhile, those phenolic substances from lignin pyrolysis also acted as the precursors of the condensed aromatic hydrocarbons, which produced more condensed aromatic hydrocarbons compared to cellulose and hemicellulose.

Characterization of DBE of heavy compounds

In the DBE/CN diagram (Fig. 7) of cellulose and hemicellulose, the substances with different ON show obvious "layered" distribution, especially for the 60s pyrolysis. It shows that substances with high ON generally have low DBE/CN, indicating that oxygen-containing functional groups play an important role in the formation of heavy components [START_REF] Xiong | Assessing the chemical composition of heavy components in bio-oils from the pyrolysis of cellulose, hemicellulose and lignin at slow and fast heating rates[END_REF]. Moreover, a "special island" can be observed in the 60s pyrolysis diagram, which is consisting of condensed aromatic hydrocarbons and unsaturated hydrocarbons with DBE >15, CN > 20 and ON<6. This clearly separated distribution indicates that two reaction path ways for the formation of heavy compounds in primary pyrolysis stage of cellulose are possible. One corresponds to the production of unsaturated macromolecules with low oxygen content such as condensed aromatic hydrocarbons, and the other one is related to the generation of pyrolytic derivatives of sugars and phenolic-like species with higher oxygen content.

With the increase of time, the DBE distribution tends to "collapse": many species in the "special island" (unsaturated hydrocarbon and condensed aromatic hydrocarbon) disappear, while some substances with DBE less than 15 and CN more than 20 are generated. It is worth noticing that less abundance and fewer species of condensed aromatic hydrocarbons (also shown in Figs. 4 and5) may not be due to their decomposition in the second reaction. It could be the result of further polycondensation and transfer into secondary coke with longer pyrolysis time, which cannot be dissolved and detected. This can be supported by comparing the coke formed on the inner wall of the reactor after pyrolysis, as shown in Fig. 6. In addition, the substances with ON > 9 and DBE ≈5 gradually disappear with an increase of reaction time, which corresponds to the results shown in Fig. 4. Generally, the unsaturation of heavy components tends to reduce as the reaction proceeds. Considering the decrease of high ON species in longer reaction time, it is speculated that more decarbonylation reactions happen in the secondary reaction of cellulose-derived heavy compounds. Individually, hemicellulose produces less substances of the "island" in the primary stage than cellulose indicating that the condensation of hemicellulose is by far inferior to cellulose. With the rise of pyrolysis temperature, the change between the two stages gradually decreases, which indicates that the pyrolysis of hemicellulose is accelerated by temperature and the secondary reactions are promoted. As the reaction time goes on, more long-chain hydrocarbons with smaller DBE appear in the lignin pyrolysis heavy oil (Fig. 7) that are the corresponding lipids. For lignin-derived compounds, phenolic-like species with ON > 6 and CN of 15-20 in the primary stage are further replaced by the same species with lower ON accompanying with deoxidization in the secondary reaction.

Changes of O/C with molecular weight of the heavy components for different reaction times

As shown in Fig. 8, the compounds with O/C < 0.3 or >0.8 have the largest molecular weight for 60s pyrolysis of cellulose and hemicellulose. For 90s pyrolysis, the O/C ration of the compounds with large molecular weight are generally less than 0.2. It is more obvious in the lignin-derived heavy compounds that these large molecular are produced by the growth of lipids, unsaturated hydrocarbons and condensed aromatic hydrocarbons in the secondary reaction consistent with the distributions of DBE of the three samples. Supported by previous results, one can assume that the heavy components undergo deoxidation reactions such as decarboxyl, decarbonyl, dehydration and other reactions [START_REF] Xiong | Formation of the heavy tar during bio-oil pyrolysis: a study based on Fourier transform ion cyclotron resonance mass spectrometry[END_REF] during their formation.

KMD analysis of heavy compounds

Distributions of heavy compounds in KMD diagram

The formation and evolution of three kinds of heavy compounds were different because of their unique pyrolysis behavior, in which the oxygen-contained functional groups played an important role. The results of pyrolysis at characteristic temperature of each samples are selected for KMD analysis based on CH 2 , H 2 O, CO 2 and CO, etc, to reveal the evolution of heavy compounds. Considering the heavy oil yield and the intensity of secondary reaction, 500, 600 and 700 • C are chosen as the characteristic temperatures of hemicellulose, cellulose and lignin respectively.

Before discussing the evolution route based on the reaction time by KMD analysis, it is necessary to investigate the characteristic KMD distributions of the five kinds of species in three heavy oil samples based on CH 2 group (Fig. S1). It can be found that most of heavy components of both cellulose and hemicellulose are concentrated around a linear region ("main line" for short). This region contains saccharides, phenoliclike species, condensed aromatic hydrocarbons and parts of unsaturated hydrocarbons, while other parts of unsaturated hydrocarbons and lipids are found outside the region. Although the distribution of lignin-derived heavy compounds in the KMD diagram is quite different from that of cellulose and hemicellulose, the "main line" can still be distinguished.

Evolution of cellulose-derived heavy compounds

Fig. 9 shows the distribution of KMD with molecular weight based on CH 2 , H 2 O, CO and CO 2 groups. The two pyrolysis stages are distinguished by circles with different colors, and the homologues of the new products generated in the second stage are represented by solid points. Combined with the species distribution in Fig. S1, it appears that the heavy components of cellulose mainly undergo the transformation between sugars, phenolic-like species and condensed aromatic hydrocarbons in the primary reaction stage. Some products in the primary reaction stage disappear with the increase of reaction time, while many new substances are produced in the secondary reaction stage.

The KMD(CH2) diagram indicates that most of the new substances (lipids and unsaturated hydrocarbons) outside the "main line" are generated in the secondary reaction stage. These substances are homologues of CH 2 group including some high molecular weight substances such as C 37 H 74 O 6 . This indicates that in the secondary reaction stage, the newly generated lipids and unsaturated hydrocarbons are produced through the growth of alkane chains. The KMD(H2O) diagram reveals that the multi-stage dehydration reactions happen in some substances existing only in the primary stage which was also can be deduced from the van Krevelen diagram based on Stankovikj's work [START_REF] Stankovikj | Quantification of bio-oil functional groups and evidences of the presence of pyrolytic humins[END_REF]. For example, the trisaccharide units C 18 H 30 O 15 dehydrated into C 18 H 28 O 14 detected by FTMS as shown in Fig. 9. More sugar units as well as some oxygen-containing lipids and unsaturated hydrocarbons ) detected by GCMS and FTMS as an example, its carbonyl group is removed to form C 8 H 14 O 7 with lower DBE, whose path is similar with Liu's work [START_REF] Liu | Theoretical studies on formation mechanisms of CO and CO2in cellulose pyrolysis[END_REF]. The distribution of KMD(CO2) is similar to that of KMD(CO), but it shows that the removal of carboxyl groups in the secondary reaction only take place in sugars and phenolic-like species.

Many solid points appeared in different KMD diagrams, indicating that these substances might act as intermediates with different reaction paths for different substances production in the evolution of heavy compounds. At the same time, some primary pyrolysis products are not collinear with any products of secondary reaction, which means these products undergo more complex reactions in the secondary reaction step, such as ring opening, carbon chain cleavage, etc. Fig. 10 illustrates the evolution of oxygen atoms replacing the molecular weight with the number of oxygen atoms in KMD diagram. The dehydration and decarbonylation reactions mainly take place for substances with ON < 10 in the secondary reaction stage, while only few substances with ON > 10 undergo deoxidation, indicating that the compounds with large ON mainly experience more complex reactions during the secondary reaction.

Evolution of hemicellulose-derived heavy compounds

Compared with cellulose, the hemicellulose-derived heavy compounds in two-stage pyrolysis widely overlap in the "main line" region, which indicates that the pyrolysis process of hemicellulose is dominated by homologue evolution, assisted with some cleavage of carbon chain or other complex reactions.

In the KMD(CH2) diagram, just like cellulose, a part of lipids and unsaturated hydrocarbons generated by the growth of alkane chains also appear outside the "main line", like C 31 H 40 O 3 and C 35 H 48 O 3 shown in Fig. 11. In KMD(H2O), it is observed that the dehydration reaction occurs mainly in sugars and phenolic-like species but less in lipids and unsaturated hydrocarbons, which is different from those of cellulose. This result indicated that nearly no oxygen atoms existed in the lipids and unsaturated hydrocarbons in the form of hydroxyl groups. The KMD (CO) and KMD(CO2) figures show that the removal of carbonyl and carboxyl groups also occur in sugars, phenolic-like species and condensed aromatic hydrocarbons, while these reactions hardly take place in unsaturated hydrocarbons and lipid. Meanwhile, the removal reaction of CO 2 is much weaker than that of CO in the secondary reaction of hemicellulose. It was indicated in previous study that many carboxylic groups were intermolecularly dehydrated while lots of C--O bond were retained at the ends of small pyrolytic fragments [START_REF] Yang | Unveiling the pyrolysis mechanisms of hemicellulose: experimental and theoretical studies[END_REF], which explained the stronger removal of CO in the secondary reaction. The variation of KMD with the ON in Fig. 12 shows that the hemicellulose-derived substances with large ON in the heavy compounds transform into lower ON compounds mainly through dehydration and decarbonylation during the secondary reactions.

Evolution of lignin-derived heavy compounds

Due to the abundant of methyl or methoxy side chains in the lignin units [START_REF] Shen | A study on the pyrolysis mechanism of a β-O-4 lignin dimer model compound using DFT combined with Py-GC/MS[END_REF], the changes of OCH 2 groups are also considered in addition to CH 2 , H 2 O, CO and CO 2 groups introduced above. As shown in Fig. 13, lignin generates varieties of products during the primary stage of pyrolysis. As the reaction time increase, many non-collinear products appear. The homologous evolution of lignin-derived compounds in the "main line" region is not as strong as that of cellulose and hemicellulose, but obviously more active in the unsaturated hydrocarbons and lipids in outside region. This finding suggests that the heavy components of lignin tended to be cracked or condensed in the secondary reaction.

The KMD(CH2) diagram in Fig. 13 indicates that the alkane chain growth mainly takes place in the unsaturated hydrocarbons, lipids and phenolic substances. Some phenylpropanes detected by GCMS and FTMS showed the evolution caused by multi-stage demethylation (KMD (CH2) = 0.12243). In addition, the phenolic substances produced during lignin pyrolysis experience the removal of OCH 2 groups, CO groups and a few H 2 O groups attributed to the abundant sidechains. However, the removal of CO is more intensive than that of H 2 O, and both of them take place on the propylene or propanol sidechains [START_REF] Shen | A study on the pyrolysis mechanism of a β-O-4 lignin dimer model compound using DFT combined with Py-GC/MS[END_REF][START_REF] Kawamoto | Lignin pyrolysis reactions[END_REF]. This indicates that the oxygen-contained groups on the propyl sidechains are mainly present in the form of C--O. The dehydration reaction takes place in the lipids, which probably due to the involvement of fragments containing hydroxyl side chains during the recombination. At the same time, some lipids and unsaturated hydrocarbons also undergo the removal of OCH 2 groups (seen in KMD(OCH2)). This could be attributed to the presence of methoxy benzene side chains of these substances or the involvement of hydroxymethyl fragments during the formation of lipids and unsaturated hydrocarbons. The KMD(CO) figure shows that the CO removal only occurs in lipids and some phenolic substances. It should be pointed out that there is almost no compounds collinear in the KMD(CO2) figure, consequently, the KMD(CO2) figure of lignin is omitted here. Moreover, this result also proves that C--O in lignin heavy compounds mainly exist in the form of aldehyde or ketone.

Fig. 14 shows that the evolution of oxygen-contained heavy compounds of lignin is far more complicated than these homologous transformations, and there are more unstable structures with large molecular weight resulting from a lot of complex cleavage and condensation.

Evolution routes of heavy bio-oil compounds

The heavy components derived from pyrolysis of the three components at 500, 600 and 700 • C with different reaction times have been analyzed in the previous sections. From these results, the possible evolution routes of the heavy components are shown in Fig. 15. All the five kinds of species (classified in section 3.2.1) are already generated during the primary pyrolysis. The lipids and phenolic-like species are dominant in lignin-derived and cellulose/hemicellulose-derived heavy oil, respectively. Then during the secondary reaction stage, the phenolic-like species and sugars are the most active components that undergo cracking, condensation and homologous evolution (-CH 2 -, -CO, -CO 2 , -H 2 O, -OCH 2 ). Most of lipids and unsaturated hydrocarbons (UHs) are produced by the recombination of radicals generated by the secondary reactions of phenolic-like species and sugars. These possible routes of evolution, especially the formation of lipids and UHs, are differently affected by temperature (500,600 and 700 • C). Generally, high temperatures promote the formation of these two species in secondary reactions of cellulose and hemicellulose while inhibiting those of lignin.

Conclusion

The species and abundance of heavy components change greatly with temperature in different pyrolysis stages. The increase in temperature promotes the pyrolysis of phenolic-like species and sugars, and facilitates the formation of other species in cellulose pyrolysis oil. For hemicellulose, a temperature increase accelerates the pyrolysis process and inhibits the secondary reactions of its phenolic-like species. The formation of lipids, unsaturated hydrocarbons and condensed aromatic hydrocarbons during the primary pyrolysis of lignin are inhibited by the temperature increase. However, it tends to promote their formation during the secondary reaction step. KMD results show that there are two pathways for the evolution of heavy components: homologous evolution and complex cracking or condensation. With the increase of reaction time, heavy components of cellulose and hemicellulose mainly undergo homologous evolution, which is achieved through dehydration, decarbonylation, and mildly decarboxylation. The lipids and some unsaturated hydrocarbons produced during the secondary pyrolysis mainly form by growth of alkane chains. Many other complex reactions like cracking and condensation happen during secondary pyrolysis of ligninderived heavy compounds because of its unstable structure. This work is intended to bring new insights in the conversion mechanisms of cellulose, hemicellulose and lignin in pyrolysis.

Credit author statement

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .

 1 Fig. 1. The closed pyrolysis system (a) and the U-tube reactor (b).

Fig. 4 .

 4 Fig. 4. The van Krevelen plots of heavy components in cellulose-oil (C), hemicellulose-oil (H) and lignin-oil (L) where first letter represents feedstock, first number represents temperature ( • C) and second number represents reaction time (s).

Fig. 5 .

 5 Fig. 5. The abundances of different species in the heavy compounds derived from three components.

Fig. 6 .

 6 Fig. 6. The coke formation of cellulose pyrolysis if different reaction stages: A is 600 • C, 60s pyrolysis; B is 600 • C, 90s pyrolysis.

Fig. 7 .

 7 Fig. 7. The DBE distribution with CN of cellulose-oil (C), hemicellulose-oil (H) and lignin-oil (L) pyrolyzed at 500, 600 and 700 • C for 60s and 90s.

Fig. 8 .

 8 Fig. 8. The O/C distribution with molecular weight of cellulose-oil (C), hemicellulose-oil (H) and lignin-oil (L) produced at 500, 600 and 700 • C for 60s and 90s.

Fig. 9 .

 9 Fig. 9. KMD based on different groups of heavy compounds in cellulose pyrolysis oil.

Fig. 11 .

 11 Fig. 11. KMD based on different groups of heavy compounds in hemicellulose pyrolysis oil.

Fig. 12 .

 12 Fig. 12. KMD of oxygen-containing compounds in hemicellulose pyrolysis oil.

Fig. 13 .

 13 Fig. 13. KMD based on different groups of heavy compounds in lignin pyrolysis oil.

Fig. 14 .

 14 Fig. 14. KMD of oxygen-containing compounds in lignin-oil.

  Dian Zhong: Writingoriginal draft, Software, Visualization, Kuo Zeng: Writingreview & editing, Methodology, Formal analysis, Jun Li: Investigation, Formal analysis, Visualization, Yi Qiu: Resources, Gilles Flamant: Methodology, Formal analysis, Ange Nzihou: Investigation, Formal analysis, Visualization, Vasilevich Sergey Vladimirovich: Formal analysis, Polishing language, Haiping Yang: Conceptualization, Resources, Validation, Hanping Chen: Validation.

Fig. 15 .

 15 Fig. 15. The evolution of heavy compounds derived from three main components of biomass:(a) Cellulose, (b) Hemicellulose and (c) Lignin. UHs represents unsaturated hydrocarbons and CAHs represents condensed aromatic hydrocarbons.

Table 1

 1 The proximate and ultimate analysis (dry basis, wt.%).

	Proximate analysis	Ultimate analysis

Acknowledgement

Authors acknowledge funding from the National Key R&D Program of China (2017YFE0124200), the National Natural Science Foundation of China (52076098), the International Cooperation Project of Shenzhen (GJHZ20190820102607238) and the Young Top-notch Talent Cultivation Program of Hubei Province. There are no conflicts to declare.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.rser.2021.111989.