Pyrolysis of lemon peel waste in a fixed-bed reactor and characterization of innovative pyrolytic products

Lemon peel waste (LPW) were pyrolysed in a laboratory fixed-bed reactor at final temperature of 300 °C, 400 °C and 500 °C with an incremental heating rate of 10 °C/min, under N2 atmosphere to produce biochar, bio-oil and gas. The maximum yields of bio-oil, biochar and gas were 16.66 wt%, 66.89 wt% and 54.6 wt%, respectively, at 400 °C, 300 °C and 500 °C. The produced gas has a maximum calorific value around 12 MJ/N m3 with a composition up to 68.82 vol.% of CO, 5.25 vol.% of CH4, 1.48 vol.% of CnHm. The recovered biochar is a promising applicant for the manufacturing of carbon materials and for solid fuel applications. The bio-oil chemical characterization using GC–MS and FTIR spectroscopy shows its richness with bioactive compounds such as squalene, d-limonene, ß-Sitosterol and phenol. Biochar and bio-oil showed bactericidal activity against Bacillus cereus, Micrococcus luteus, Salmonella enterica, Listeria monocytogenes and Staphylococcus aureus. Pyrolytic products of LPW show large potential applications in agriculture and agri-food industry and allow a sustainable biomass-waste management with promising economic benefits.


Introduction
The exploitation of new renewable resources to produce biofuels as an alternative to the use of conventional petroleum derivate is gaining an increasing research interest. Global climate change associated with the massive release of greenhouse gases (GHG) has raised concerns about the use of fossilized hydrocarbons as a primary energy source [1].
The use of vegetable biomass as a renewable energy source has some advantages in comparison to non-renewable fuels: (1) solid, liquid or gaseous fuels are obtained depending on the transformation method used; (2) reductions in greenhouse gases (e.g., CO 2 , CH 4 , and N 2 O) due to biological decomposition or waste burning; and (3) the use of lignocellulosic biomass as an energy resource does not increase CO 2 levels in the atmosphere [2].
In this context, the transformation of agro-industrial waste into renewable biofuels can reduce fossil fuel dependence, help reduce GHG emissions, and significantly reduce the pollution associated with the unsafe disposal of agroindustrial residues [3]. Currently, the citrus processing industry generates a substantial waste stream in areas of significant citrus production [4]. Amongst agro-industrial wastes, citrus waste is abundant, low-cost and readily available feedstock for renewable energy production [3]. About 92,088 billion tons of citrus are produced, through the world [5]. In Tunisia, the citrus production is estimated to be 560,000 tons in 2018 [6]. It is estimated that 33% of the total global citrus harvest is used for juice and essential oil production [7]. As most agro-industrial activities, the citrus Aïda Ben Hassen Trabelsi and Nourhene Boudhrioua contributed equally.
conversion sector leads to the generation of huge quantities of organic waste. It is estimated that 40-60% of citrus is converted to organic waste: citrus peel waste (CPW), pulp and seeds [8]. Therefore, the suitable management of these organic residues constitutes a permanent concern for the citrus processing industry. Current management practices of CPW include first generation recycling methods, such as animal feed after drying, composting since they are very rich in sugar fibers [9], disposal in landfills and anaerobic digestion [10] and for recovery of biologically active composites such as phenolic acids and flavonoids pectin, fibers [11]. But these conventional methods of CPW processing are insufficient and induce many problems in terms of energy productivity and environmental consequences [12]. The chemical composition of CPW reveals a significant content of pectin in addition to cellulose, hemicellulose and lignin as major components [13]. These lignocellulosic wastes could be considered as attractive feedstock to produce renewable fuels and innovative products (biofertilizers, bioactive molecules). Regarding the significant calorific value of CPWs (around 17 MJ/kg dry basis), their direct use for energy conversion through combustion [14] or to provide energy carriers through thermochemical processes such as gasification [15] could be a solution to provide energy to the citrus product processing industry [15,16].
Pyrolysis is the thermochemical process that, in the lack of oxygen, converts organic wastes into valuable products including solid char, liquids (hydrocarbons and water) and gaseous products. Canché-Escamilla et al. [2] showed that pyrolysis of trunk and flower stalk leads to the recovery of bio-oil at 600 °C with a high elemental carbon content and good heating value. Sahoo et al. [17] carried out slow pyrolysis of agricultural residues, such as rice straw, wheat straw, and sugarcane bagasse at a temperature range of 350-650 °C with two heating rates (20 °C/min or at 100 °C/min) and reported that a maximum bio-oil yield was obtained at 550 °C for rice straw and wheat straw and at 450 °C for sugarcane bagasse. Bera et al. [18] prepared biochar by pyrolysis of rice straw, wheat straw, maize stover and pearl millet stover by only variation of pyrolysis temperature. Temperature and biomass residue modified significantly biochar properties which improved soil nutrients. Similar results were reported by Tan et al. [19]. Few studies have been conducted on the pyrolysis of CPW; most of them were performed on orange peel waste [13,[20][21][22][23][24] and mandarin peel waste [25]. Aguiar et al. [21] showed that the temperature of the pyrolysis is the parameter which exerts the most important influence on the products yields from orange peel. Miranda et al. [22] showed that dry orange peel have high potential to produce liquid fuel (around 53.1% of bio-oil) due to their volatile content (78.9% w/w) and their low ash content (2.94% w/w) with average yields of biochar and gas around 21.1% and 25.8% w/w, respectively. Morales et al. [23] reported that a high bio-oil yield (around 77.64%), a moderate biochar yield (20.93%) and only a low percentage of gas (around 1.43%) were produced from solar pyrolysis of orange peel. Volpe et al. [20] reported that bio-oil yield from pyrolysis of dried lemon and orange peel varies between 36 and 39% w/w for a temperature range of 400-650 °C; whereas the biochar yield was between 37.2 and 40.8% w/w. Alvarez et al. [13] reported that bio-oil yield of orange peel is close to 55 wt% in the interval of temperature between 425 and 500 °C and the yield of biochar varies from 27 to 33 wt%. Few studies reported the pyrolysis of Lemon Peel Waste (LPW) with a focus on all pyrolysis products (bio-oil, biochar and gas) and on their adequate and specific uses especially in nonconventional applications using active biomolecules. Adeniyi et al. [25] developed a steady state thermodynamic model for the pyrolysis of lemon peel tested at 500 °C and atmospheric pressure. The authors reported that the lemon peels were the best for bio-oil production compared to other biomasses.
Moreover, recent studies reported that biomass pyrolysis generates pyrolytic products with antibacterial activities [27][28][29][30][31][32]. Patra et al. [26] studied the antibacterial activity of the bio-oil obtained from P. densiflora in Gram-positive bacteria (Bacillus cereus and L. monocytogenes) and Gramnegative bacteria (S. Typhimurium and E. coli). The authors observed that bio-oil interfered in cell membrane integrity of both Gram positive and Gram-negative bacteria. Another study showed that the concentrated pyroligneous acid of the solid pineapple biomass showed antibacterial activity against E. coli and Corynebacterium agropyri, exhibiting zones of inhibition of 13-20 mm and 20 mm, respectively [24]. Bio-oils obtained from pyrolysis of M. ferrea and P. glabra, also showed efficacy against S. aureus and E. coli. Mesua ferrea bio-oil showed the lowest MIC, with 1.56 μg/ mL for E. coli and 3.12 μg/mL for S. aureus, while P. glabra exhibited a MIC of 6.25 μg/mL for both bacteria [25]. Kaetzl et al. [27] studied the filtration of rice husk biochar and nonpyrolyzed rice husk as low-cost filter materials for wastewater and evaluated their potential and limitation. In general, the performance of the biochar filter was superior or equal to the rice husk and standard sand filters. Results showed that the contamination with fecal indicator bacteria was more than 2.5 log units lower than the control group irrigated with untreated wastewater. Similarly, Perez-Mercado et al. [28] showed that using biochar as a filter medium, more than1 log10 CFU Saccharomyces cerevisiae was successfully removed from diluted wastewater under the condition of on-farm irrigation.
Thus, the principal objective of this paper is to explore, firstly, the pyrolysis of LPW into innovative pyrolytic products having potential applications not only in energy, but also in agriculture and food industries. It attempts to explore the consequence of pyrolysis final temperature on pyrolytic products yields and qualities.
Another aim was to extract organics compounds from the raw, bio-oil and biochar and to quantify their antimicrobial activity against Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus and Listeria monocytogenes) and Gram-negative bacteria (Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa). To the best of our knowledge, this is the first report publishing the antimicrobial activities of bio-oils and biochars produced from LPW.

Material and methods
Preparation of the samples LPW used for this work were collected from a juice citrus factory in Cap Bon (Northern Tunisia). ~ 20 kg of fresh material was collected immediately after processing. The freshly processed LPW was oven dried for 48 h at 60 °C to reduce the moisture content until ~ 10% [32]. The dried LPW was ground in a mill and then sieved to obtain homogenous products with 2 to 4 mm particle size.
In the present study, as recommended by several previous works [4,19,32,33], a pre-dehydration step was performed before the LPW pyrolysis because of its initial high moisture content (~ 78%).

Experimental setup and procedure
Raw LPW was dehydrated by air-drying until reaching a moisture content value around 10%. A dried sample of ~ 3 kg LPW was divided to perform 3 pyrolysis batches. Each experiment was conducted in duplicates, to confirm reproducibility and the obtained products yields mentioned are the average value of two equivalent runs. The pyrolysis experiments of dried LPW were carried out using a laboratory scale fixed-bed reactor at final temperatures of 300 °C, 400 °C and 500 °C, with a heating rate of 10 °C/min, a retention time of 1 h and under atmospheric pressure. The pyrolysis setup used in this work was reported in detail in [34] (Fig. 1). The pyrolysis experiments were conducted on a batch fixed-bed reactor heated with an electric furnace. The stainless-steel reactor (L: 30 cm, Ɵ: 15 cm) is flushed with nitrogen (N 2 Flow rate is 50 L/h) to prevent oxygen introduction into the reactor and to guarantee an inert medium for pyrolysis reactions and the reactor axial temperature is followed using a K-type thermocouple. The pyrolysis gases pass through a condensation system to collect bio-oil and non-condensable gases, separately.
The end products were put in dark vials and stored at 4 °C for further analyses. Pyrolysis products yields are determined by weighing the biochar and the bio-oil [35] and using the following Eqs. (1), (2) and (3):

Proximate and ultimate analysis of LPW and biochar samples
The moisture content of LPW samples was determined by the weight loss after drying at 105 °C for 24 h [36]. The volatile matter (VM) content of LPW samples was determined based on the mass loss after samples heated at 950 °C (1) Bio − oil yield wt% = bio − oil collected weight initial feedstock weight × 100 (2) Biochar yield wt% = biochar collected weight initial feedstock weight × 100 (3) Gas yield wt% = 100 − (biochar yield wt% + bio − oil yield wt%)  for 7 min without oxidation agent [36]. Ash content was calculated using the standard methods [36]. Ultimate analysis CHN-O was fulfilled using a CHN elemental analyzer (Perkin Elmer 2400, country). The oxygen content was determined by difference.
The High Heating Value (HHV) calculation was established based on the ultimate analysis data (CHN-O) using the following formula [35]: The determination of proximate and ultimate analyses of LPW and produced biochar allows the evaluation of LPW suitability as feedstock for the pyrolysis and the estimation of their energetic potential.

FTIR spectroscopy of LPW, biochar and bio-oil samples
Fourier Transform Infrared (FTIR) spectroscopy analyses were performed to find out the functional groups composition of raw material (LPW), bio-oil and biochar. FTIR analysis will be useful for further determination of potential applications for molecules having bioactive functional groups. The FTIR spectra were registered on KBr pellets using a FTIR spectrometer (Perkin Elmer, FTIR 2000), in the spectral range of 400-4000 cm −1 .

Thermogravimetric analyses of LPW
Thermogravimetric (TGA-DTG) analyses were conducted to investigate the thermal degradation behavior of LPW by following the mass loss with the temperature increase, using a TGA Thermogravimetric analyzer (SETSYS-1750) under Argon atmosphere in the temperature range between 30 and 700 °C and the heating rate of 10 °C/min. The thermogravimetric analyses (TG-DTG) of LPW are useful for the comprehension of the thermal behavior of LPW under heating in an inert medium and for the selection of end pyrolysis temperature.

Gas chromatography-mass spectrometry of bio-oil
Bio-oil samples were derivatized using bis (trimethylsilyl) tri fluoroacetamide (BSTFA) and pyridine, to produce trimethylsilylester derivatives. Around 10 mg of the bio-oil sample was derivatized with 100 µL of pyridine and 100 µL of BSTFA. The solution was jumbled in a vortex and left to stand for 30 min at 70 °C. The GC-MS analyses of the produced bio-oil were performed using an Agilent 7890A GC equipped with an Agilent 5975C mass-selective detector (MSD). The capillary column was HP-5MS 5% Phenyl Methyl Siloxof 30 m long, 0.25 mm internal diameter, and 0.25 μm film thickness. The oven was programmed to hold (4) at 70 °C for 2 min, then a ramp at 7 °C/min to 300 °C and hold there for 10 min. The injector temperature was set to 250 °C. The injector split ratio was set to 10:1 ratio. The carrier gas was helium (1 ml/min). The identification of bio-oil compounds was performed according to the NIST database and by comparing to previously published mass spectra data. GC-MS analyses of studied bio-oils were conducted to explore the molecules group content recognized in literature as active or bioactive.

Pyrolytic gas chemical composition determination
The gas chemical composition and the calculated calorific content (Low heating value LHV) of the produced pyrolytic gas were determined by a gas analyzer (GEIT 3160 model, Belgium). Before analyses, the gaseous mixture was purified to reduce tars and water [34].

Determination of minimum inhibitory and minimum bactericidal concentrations of pyrolysis products
Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of biochars and biooil freeze-dried methanolic extracts prepared from different pyrolysis products were determined according to Ben Hsouna et al. [37] against 8 foodborne and spoiling bacteria. Methanolic extracts of bio-oil and biochars (m/v, 1/10) were prepared. Freeze-dried extracts were obtained after methanol removal by rotary evaporation under vacuum and freeze drying of the extracts. A stock solution of each extract (100 mg/mL) was prepared in dimethyl sulfoxide/ water. The 8 foodborne bacteria provided from the Center of Biotechnology of Sfax belong to Gram-positive bacteria (Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 1880 and Listeria monocytogenes ATCC 19117) and to Gram-negative bacteria (Salmonella enterica ATCC 43972, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 9027). The MIC test was performed in sterile 96-well microplates with a final volume in each microplate well of 100 μl. The extracts transferred to each well obtaining a twofold serial dilution of the original sample and to produce the concentration range of 1.5-100 mg/mL. To each test well 10 μl of cell suspension were added to final inoculums concentrations of 10 6 CFU/mL for bacteria. Dimethyl sulfoxide/water was used as negative control. The plates were then aseptically covered and incubated at 37 °C for 24 h. To determine the minimum bactericidal concentration (MBC), 10 μL of each well with no visible growth were removed and inoculated in Muller-Hinton Agar (MH) (Oxoid Ltd, UK) plates in strings. After 24 h of incubation at 37 °C, the number of surviving organisms was determined. The results were expressed in mg/mL of the re-suspended freeze-dried extracts.

Proximate results
The proximate results of the LPW are set in Table 1. As it is shown, a pre-dried LPW sample has moisture content around 10.86%. The drying pre-treatment (using open air and oven) was a fundamental step before pyrolysis, since the fresh LPW samples are very humid (moisture content around 78% by weight) and the pyrolysis process requires feedstock with low water content, around 10%, [38]. The pre-drying process is usually recommended before pyrolysis experiments to avoid additional heat to remove the moisture from biomass and to reduce bio-oil moisture content.
The VM content of LPW was about 80%. This VM value was in the same range of those obtained for other LPW (Table 1) (77.22% [20]; 87.16% [39] and for other CPW (74.3% [24]; 57.5% [15]). The high VM content of the studied LPW sample reveals its suitability as raw material for pyrolysis process since biomass with high VM is easily devolatilized and also produces less biochar. The LPW ash content is around 5.37%. The low LPW ash content shows its suitability for thermochemical process and explains the low amount of remaining solid residue.

Ultimate results
Besides, the studied LPW showed high amounts of carbon (40.1%), of hydrogen (7.36%), of nitrogen (1.02%) and of oxygen (51.52%) ( Table 1). The high content of organic matter (as carbon and hydrogen) makes LPW suitable for biofuels production [40]. The results shown in Table 1 [43]. The good energetic value of LPW is a key parameter for the selection of these wastes as feedstock for energy carrier's production. LPW thermal behavior Figure 2 shows the thermal behavior of LPW by simultaneous TG (mass loss) and DTG (derivative thermos-gravimetric) evolution profiles as a function of temperature. Three stages were distinguished for the principal thermal processes up to 700 °C. The first stage starts at 26 °C and ends at 133 °C with a mass loss about 12.38% on account of the removal of moisture and very light volatile components from biomass [22,44,45]. The second stage starts at 134 °C and finishes at 407 °C with a global mass loss of 50.8%. It should be noted that this stage is mainly characterized by the degradation of pectin, hemicelluloses and cellulose [46]. In this stage, the maximum degradation temperature of pectin is at 162 °C with a mass loss of 2.18%, that of hemicelluloses is at 230 °C with a mass loss of 26.63%, and that for cellulose is at 330 °C with a mass loss of 19.08%. This interval was considered as an active pyrolysis stage [44]. Aguilar et al. [35] reported that the decomposition peaks of lemon peel are 160 °C (9%), 235 °C (22%), and 328 °C (8.3%) and correspond to pectin, hemicellulose, and cellulose, respectively. The third stage, from 408 to 700 °C, is assigned to the presence of lignins with a mass loss of 4% at final temperature of 462 °C. The last stage is referred to as passive pyrolysis [15,22,44]. LPW thermal decomposition goes down slowly under a wide range of temperature from ambient to 700 °C [47]. The thermal decomposition of hemicelluloses, cellulose and lignins occurs in the temperature ranges of 210-325, 310-400 and 160-900 °C, respectively [48,49].

LPW Fourier transform infra-red (FTIR) results
LPW FTIR spectrum is presented in Fig. 3 [46,50]. The band shown at around 622 cm −1 is attributed to CH aromatic stretching vibrations and CH 2 deformation vibration [45,51]. The aliphatic and oxygenated functional groups contents of raw LPW emphasize their potential use as feedstock for the pyrolysis process.

Pyrolysis products yields
LPW pyrolysis products distribution is given in Fig. 4. Biochar yield decreased from 66.9 to 31.5 wt% when the temperature increased from 300 to 500 °C while the pyrolytic gas yield increased from 26.9 to 54.6% when temperature rose from 300 to 500 °C. The bio-oil yield showed a maximum value (around 16.6%) at 400 °C. The bio-oil yields    Fig. 4 Yields of pyrolysis products obtained at 300, 400 and 500 °C from lemon peel waste at heating rate of 10 °C/min reduction at high temperature is mainly due to secondary reactions (rupture and rearrangement) of the pyrolysis vapors, which participate to the increase of the gaseous product [52]. At 500 °C, the yield of condensable and noncondensable products increased to reach 68.53 wt%. This reaction is compatible with TGA (Fig. 2), where weight loss at 500 °C was observed. The yield of bio-oil acquired in this work is slightly lower than that indicated for the pyrolysis of LPW or similar citrus waste, which ranges from 35 to 53% by weight [20,22,24,53]. This could be explained by the difference in the used pyrolysis reactor and namely in the condensation operational conditions (condensation temperature, number of refrigerators, cooling water circulation, etc.) of pyrolysis vapors. The decrease of biochar yield with temperature increase could be due either to the primary decomposition of the main components of the studied biomass at higher temperatures or through secondary decomposition of the char residue in the form of dehydrogenation and dehydration of hydroxyl groups reaction [48].
This result is comparative to those reported by Volpe et al. [20], who investigated the slow pyrolysis of the lemon peel juice industries in a fixed-bed pyrolysis reactor after drying, where the biochar yield decreased from 55.3 to 38.2%, when temperature increased from 300 to 500 °C. These authors reported similar trend of the LPW solid residue with temperature increase (from 300 to 500 °C) and explained the LPW char yield drop at high temperature by the presence of a larger amount of lignin in the raw lemon peel waste. For non-condensable products, it is believed that the increase of pyrolytic gas proportion is mainly due to the secondary cracking of the pyrolysis vapors at higher temperatures and to the occurrence of some secondary charring reactions. In their comparative study of several types of citrus waste, Volpe et al. [20], reported that the char produced from lemon peel is more reactive at the higher temperatures which explains the increase of unrecovered products (noncondensable gases) from 20.7% to 23.0% when temperature rose from 300 to 500 °C. However, as reported by Volpe et al. [20], secondary decomposition of char at high temperatures contributes to the release of non-condensable gaseous products.

Biochar characterization
Proximate and ultimate analyses of biochar presented in Table 2 show that by increasing the severity of the treatment, the solid residues have a moisture content below equilibrium and that the volatile mass decreases rapidly (from 75 to 58%). The obtained biochar fixed carbon content is about 16.37% (at 300 °C) and 30.41% (at 500 °C). By increasing pyrolysis temperature, most of the oxygenated compounds in the biological reservoir are lost (O% = 41.68% at 300 °C and O% = 21.36% at 500° C). Besides, carbon content increases from 51.41% at 300 °C to 72.29% at 500 °C, which positively affects the char calorific values, increasing from 19.88 at 300 °C to 24.42 MJkg −1 at 500 °C. Similar HHV's were obtained for other biochars from the pyrolysis of orange peel or citrus wastes at temperatures between 300 and 600 °C [13]. The hydrogen content of the studied biochars decreases with the temperature increase of the pyrolysis.
The biochar produced at different temperatures contain higher amounts of fixed carbon than raw LPW which indicates lower liberation of carbon during pyrolysis [54]. Moreover, the fixed carbon content increases from 16.37 to 30.41% while pyrolysis temperature increases from 300 to 500 °C. A similar trend was observed by different authors [55,56] for several feedstock and different pyrolysis temperatures. The high fixed carbon contents of biochars obtained from LPW could be associated with the high lignin content of LPW (Volpe et al. [20]). In fact, according to Sun et al. [57], for lignocellulosic biomasses, the cellulose component is responsible for the volatile releases whereas the lignin forms fixed carbon. Generally, biochar with high fixed carbon are more economically attractive if they are to be purchased for use in long-term carbon sequestration in soil [58].
FTIR spectra of biochar obtained from LPW pyrolysis are shown in Fig. 5a. The peak assignments description is provided in Table 3. Remarkably, the intensity of the hydroxyl groups of the C-H stretching associated with aliphatic compounds and the carbonyl band C=O decreased with rising temperature. The O-H stretching vibration at 3600-3100 cm −1 in the FTIR spectra of the biochar sharply decreased after pyrolysis, probably due to the further dehydration of the LPW (release of residual water) [48]. Compared to raw LPW, pyrolysis at 300 °C, 400 °C and 500 °C resulted in a decrease of the absorption intensity of O-H (3394-3309 cm −1 ) and of aliphatic C-H (2922 cm −1 ) functional groups, mainly due to dehydration of residual cellulose and/or hemicellulose and also to the cracking of aliphatic functions in LPW sample. Thus this result is similar to previous studies conducted by Volpe [59] and Wang [53] on residual biochars produced from citrus waste and rice husk, respectively. Compared to raw LPW, a strong signal related to aromatics in the FTIR spectra of biochar obtained at 300 °C reveals the occurrence of condensation reactions and forming more rigid structures [59]. At 400 °C, the distinctive absorption peaks at 3400 cm −1 , 2920 cm −1 and 1602 cm −1 representing O-H, aliphatic C-H and C=O, respectively, vanished, which indicates that at modest pyrolysis temperatures hydroxyl and CO 2 are liberated mainly by direct dehydration and decarbonylation reactions, respectively [53]. It can be spotted that signals at around 1000 cm −1 arranged in the spectra of hemicellulose and cellulose originate from the hydroxyl and either groups of polysaccharides. This strong band at around 1000 cm −1 is well distinguishable in the spectrum of the biochar produced at 300 °C but it disappears for biochars produced at high temperatures (400 and 500 °C). This could be explained by the complete degradation of cellulose and hemicellulose at high temperature.

Bio-oil characterization
FTIR analyses have also designed that the functional groups of bio-oils at different pyrolysis temperatures are quite similar (Fig. 5b). Table 3 shows the functional groups and corresponding classification of compounds. The stretching vibration of OH (3300-3600 cm −1 ) designates the presence of alcohols and phenols. The absorbance of peaks between 1578 and 1686 cm −1 representing C=C stretching vibrations is indicative of alkenes and aromatics. The absorption of aliphatic CH 2 at 1420 cm −1 indicates the presence of alkanes or aliphatic hydrocarbon chains in heteroatomic compounds. The absorption bands between 1672 and 1607 cm −1 indicate the presence of carboxylic acids and aldehydes; the three bio-oils obtained at different temperatures present the same  The possible presence of aromatic esters is indicated by the absorbance peaks between 657 and 670 cm −1 as well as C-O stretch indicate the presence of aromatic [43]. The FTIR biooil results are confirmed by the molecular identification of the predominant molecules within several obtained bio-oil using GC-MS technique. GC-MS analysis was performed to determine the organic compounds of the bio-oils presented in Fig. 6. The bio-oil was mainly composed of alcohols, carboxylic acids, phenols, fatty acids, aldehydes, nitrogenous compounds (indoles and pyridines) which are consequences of the features of the components in the pectin, hemicellulose, cellulose and lignin contained in LPW.
The major detected compounds in the bio-oil sample were n-hexadecanoic acid (palmitic acid) (19.98%) and (10.74%); 9-octadecenoic acid (oleic acid) (12.7%) and (5.97%); octadecanoic acid (10%) and (stearic acid) (8.89%), respectively, at 500 °C and 300 °C pyrolytic temperatures in Table 4. The components of the bio-oil at 500 °C were similar to the liquid product obtained by Primaz et al., [52]. The other useful compounds that were found on the bio-oil at 500 °C were: squalene, d-limonene and β-sitosterol. The components of the bio-oil were similar to the liquid product obtained by other researchers [24,60]. The most abundant phenols accounted for 16.3% at the temperature of 400 °C. The existence of phenolic and aromatic compounds in pyrolysis oil was due to the thermal degradation of lignin and was also seen in the FTIR spectral regions of 3300-3600 cm −1 [54].
The major compound found in the bio-oil fraction obtained during pyrolysis at 400 °C is glycerol (23.5%). It is a foremost renewable raw material used essentially in the chemical industry. The prevailing compounds obtained are hydroquinone and 4-coumaric acid. The oxygenated compounds correspond to saccharides. The pyrolysis mechanism of hemicelluloses is very similar to that of cellulose, which also starts with the depolymerization of polysaccharide chains to form oligosaccharides, following the cleavage of the xylan chain in the glycosidic linkage and rearrangement of the produced molecules [60]. The nitrogen-containing heterocyclic compounds in bio-oils, such as pyridines, are supposed to be derived from protein degradation [61].

Gas characterization
The composition of produced synthesis gas and their corresponding heating value (LHV) are presented in Table 5. Carbon monoxide (CO) was the largest amount of the chemicals in the pyrolytic gas which contributed to about 68.82 wt% at 400 °C but at 500 °C the concentration of CO decreased about 59.79 wt%. The contents of carbon dioxide increased from 0.12 wt% at 300 °C to 0.72 wt% at 400 °C. The CO and CO 2 contents of the gaseous fraction are indicators of the presence of oxygen in the sample. This oxygen derives from the pyrolytic decomposition of partially oxygenated organic compounds (cellulose, lignin, lipids and carbohydrates). Yang et al. [62] highlighted that the hemicellulose component gave high amounts of CO and CO 2 whereas lignins degradation release high CH 4 yield. With a higher presence of aromatic ring and methoxyl, the cracking and deformation of lignin released much more H 2 and CH 4 . In this study, the high amounts of CO (around 68.82%) obtained at 400 °C is correlated to hemicellulose complete degradation at 400 °C. Bensidhom et al. [35] reported for the gas produced from date palm waste pyrolysis a yield of CO ranging from 30 to 55 wt% and yield of CO 2 ranging from 0.33 to 0.55 wt%. The presence of CH 4 in the gaseous mixture in significant proportions gives it good fuel properties. The release of CH 4 can be caused by the cracking of methoxyl-O-CH 3 and was mainly focused at low temperatures (< 600 °C). Hemicellulose, cellulose and lignin all contributed to the releasing of CH 4 from biomass pyrolysis, at respectively low, middle and high temperature ranges. The yield of H 2 was also very low (≤ 0.46 wt%). In the present experiments, CO, CO 2 , CH 4 and LHV decreased when temperature increases from 400 to 500 °C. Furthermore, the lower heating value (LHV) of gas reaches 12 MJ/Nm 3 at 400 °C and then decreased up to 10 MJ/Nm 3 at 500 °C. This may be associated with the reduction in methane CH 4 amount which leads to a decrease in the pyrolytic gas LHV of the synthesis gas because methane has the highest calorific value than the other gases [63].  could be analyzed. For MBC/MIC ≤ 4, the effect was considered as bactericidal but for MBC/MIC > 4, the effect was defined as bacteriostatic [64]. The MBC/MIC ratio of biochar obtained at 300 °C against selected bacteria was equal to 2.0. This ratio is inferior to 4 suggested that biochar had a bactericidal effect against both Gram-negative and Gram-positive bacteria [65]. Escherichia coli, Pseudomonas aeroginosa and Enterococcus fecalis seem more resistant to the biochar produced at 300 °C. This might be due to the difference in the composition of the cell envelopes of the organisms. Yuhao et al. [66] evaluate the antibacterial efficiency of Maize straw biochar produced at 500 °C and they contribute to elucidate their antibacterial mechanism against Escherichia coli, a Gram negative bacteria and Staphylococcus aureus, a Gram-positive bacteria. The authors reported significant disruption of membrane which led to the inactivation of bacteria, resulting from the loss of cell membrane integrity and permeability. The induced oxidative damage inhibited the essential cell metabolism. Penetration of bioactive compounds through the cell wall and membrane into the cytoplasm enhanced the bactericidal effect of the extract. The authors reported that different effects of bioactive compounds on microorganisms might be due to the difference in the composition of their cell envelopes. The outer phospholipid membrane present in Gram-negative bacteria allows more resistance cells against bioactive compounds. Table 7 shows MBC, MIC and MBC/MIC ratio values of bio-oil produced at different temperatures from LPW on the tested bacteria. The bio-oil produced at 500 °C was the most active bio-oil against the eight microbial strains investigated with MICs ranging from 12-25 to 100 mg/mL. These results are in agreement with CG-MS-analysis of biooils. In fact bio-oil produced at 500 °C showed the presence of D-limonene (Table 4), suggesting this component as the main responsible for the antimicrobial activity, although the antimicrobial activity can also be attributed to a synergistic effect of biomolecules [67]. GC-MS measurement showed the presence of numerous fatty acids and their related esters. Fatty acids also have antifungal and antibacterial activities [68]. Similar results are reported by Patra et al. [26] who studied the antibacterial activity of the bio-oil obtained from P. densiflora against Bacillus cereus and L. monocytogenes andS. Typhimurium and E. coli. MIC and MBC values of P.   -----PS  50  100  2  ---25  50  2  ---Mic  50  100  2  25  50  2  ------Sa  50  100  2  50  100  2  ------List  50  100  2  25  50  2  ------Sal  50  100  2  50  100  2  ------EF  50  100  2  --------- densiflora bio-oil were 250-500 μg/mL and 500-1000 μg/ mL, respectively. The concentrated pyroligneous acid of the solid pineapple biomass showed antibacterial activity against E. coli and Corynebacterium agropyri, exhibiting a zone of inhibition of 13-20 mm and 20 mm, respectively [27]. Bio-oils obtained from pyrolysis of M. ferrea and P. glabra, also showed efficacy against S. aureus and E. coli.

Antimicrobial activities of bio-oils and biochars produced from lemon peel waste
To the best of our knowledge this is the first report proving antibacterial activities of biochars and bio-oils of LPW. Biochar and bio-oil of LPW presenting effective, recyclable and long-acting antibacterial properties, has a promising application in bacterial decontamination.

Conclusion
Dried LPW were converted into biochar, bio-oil and pyrolytic gas using a fixed-bed pyrolysis reactor. The maximum yields of biochar (66.89 wt%), bio-oil (16.66 wt%) and gas (54.6 wt. %) were obtained at 300 °C, 400 °C and 500 °C, respectively. The increase of the final temperature was followed by an increase of the production of non-condensable compounds but resulted in a decrease in biochar yield. Biochar with high fixed carbon content and high calorific value could be applied as a green biofertilizer. The produced biooil is composed namely of phenols, acids, terpenes, alcohols and nitrogen compounds. LPW could be processed to obtain bio-oil and biochar, containing active biomolecules with high added-value as antimicrobial agents. Biochar obtained at 300 °C and bio-oil obtained at 500 °C showed the most interesting bactericidal activity against selected foodborne and spoiling bacteria. This interesting finding further promotes the use of LPW pyrolysis bio-oils and biochar extracts as eco-friendly alternatives to replace conventional antibacterial agents. However, further investigations are needed to acquire detailed information on the transformations and the isolation of the bioactive molecules of biochar and biooil and to identify pathways of these specific and complex chemical of pyrolysis end products. The outcomes of this work could be completed to ensure a sustainable citrus byproducts waste management and to provide economic benefits for citrus farmers and agri-food industry.