Biorefinery process for production of bioactive compounds and bio-oil from Camellia oleifera shell

A biorefinery process was developed in this study to obtain bioactive compounds and bio-oil from Camellia oleifera shells. Four different extraction techniques (water, ethanol, ultrasound-assisted deionized water, and ultrasound-assisted ethanol) were utilized to extract tea saponin and tannin from C. oleifera shells. Results showed that ethanol had better extraction capacity than did deionized water, and ultrasound could promote the dissolution of tannin and tea saponin in solution. The thermogravimetric curves of the samples treated under the four conditions moved toward high temperatures. This phenomenon indicated the thermal stability of the residue was significantly improved. The pretreatment showed a slight effect on the chemical compositions of bio-oil. Specifically, the samples treated with ethanol and ultrasound-assisted deionized water contained higher phenol contents (81.07% and 81.52%, respectively) than the other samples. The content of organic acid decreased with an increase in the phenol content.


Introduction 
Researchers are focusing on sources of clean renewable energy because of the dramatic depletion of nonrenewable energy, such as oil, and the environmental pollution caused by the use of fossil energy.Biomass resources, which are abundant, renewable, and eco-friendly, play an important role in energy supply [1] .The conversion of biomass resources to renewable fuels or chemicals by various techniques is thus a hot research topic.
Camellia oleifera shells are regarded as agricultural waste and cannot be treated economically and effectively in most cases.Each ton of C. oleifera is processed to produce tea oil along with 0.54 t of C. oleifera shells.The main components of C. oleifera shells are cellulose, hemicellulose, lignin, and bioactive substances.According to Luo et al. [2] , lignocellulose in C. oleifera shells is a common green carbon-neutral material used for producing new energy sources.Apart from green energy [2] , saponins, tannins, xylose, xylitol, furfural, and activated charcoal are produced with use of the lignocellulose in C. oleifera shells [3,4] .
Tannins are natural extracts derived from plants and used as processing aids or flavoring substances in the food industry.Chemically, tannins are polyphenolic compounds with molecular weights ranging from 500 Da to 20 000 Da and can be classified as condensed and hydrolyzed tannins.Concentrated tannins are mainly obtained from grapes and tea.Meanwhile, the main sources of hydrolyzed tannins are oak and chestnuts [5] .C. oleifera is a good source of tea saponin and has not been utilized in the production and processing of tea oil.Tea saponin has good properties, such as powerful foaming, emulsifying, dispersing, and wetting performance [6] and anticancer [7] , anti-inflammatory, and antibacterial characteristics.Thus, tea saponin has been widely used in the food, medicine, pesticide, and other fields [82] .
In recent years, solvent and supercritical fluids assisted extraction techniques were the most common methods of extracting compounds from plants.Downey et al. [9] investigated the effective range of the extraction of concentrated tannin-containing aqueous ethanol mixtures from Syrah grape skins.They observed the tannin extraction rate of the extracted procyanidins and the solvent type or volume in aqueous solution [9] .In the present study, we speculated that the alcohol extraction method may be better than the water extraction.Ultrasound-assisted extraction plays an important role in extracting phenolic compounds from solid materials.Compared with the traditional solvent extraction method, ultrasound-assisted extraction can significantly reduce extraction time, energy consumption, and solvent usage.Moreover, the operation is simple and easy [10] .The adoption of ultrasound-assisted extraction for extracting pectin from peels and phenolic substances from lees has been investigated [11,12] .However, the feasibility of using ultrasound-assisted and solvent extraction to obtain phenolic substances from C. oleifera shells has not yet been explored.
C. oleifera shells can be used reasonably and economically.The lignocellulose in the residue is fully utilized and pyrolyzed to produce bio-oil after tannin and tea saponin are extracted.Bio-oil can be used to produce high-value-added chemicals after upgrading [13] .Pyrolysis is the main method of converting biomass into energy and chemical products [14] .
In an oxygen-isolated environment, the biomass organic matrix is broken down at high temperatures and used to produce three substances, namely, solid biochar, bio-oil, and non-condensable gases [15] .Fast pyrolysis can increase the yield of bio-oil, whereas slow pyrolysis is used to obtain maximum solid biochar [16] .Bio-oil can be used directly as biofuel for power generation, and solid biochar can be further processed to become a soil improver and activated carbon.The adoption of biomass pyrolysis for producing new energy materials has become a frontier research field.
In this study, the effects of different extraction techniques on the extraction yields of tea saponin and tannin were investigated.Thermogravimetric (TG) and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS) analysis techniques were used to analyze the pyrolysis behaviors of residue.This study aims to lay a foundation for the efficient and systematic processing of C. oleifera shells.

Material and method 2.1 Materials
C. oleifera shells were purchased from a farm near Nanchang City, Jiangxi Province, China.The samples were sifted using a 60-mesh sieve and then dried in vacuum at 105°C overnight.Proximate and ultimate analyses of the samples are shown in Table 1.An eighty percent ethanol solution was made by mixing anhydrous ethanol with deionized water at a ratio of 4:1.Analytical-grade anhydrous ethanol was obtained from Xilong Scientific Co., Ltd., Shenzhen, China.Deionized water was processed by a laboratory water purification system produced by Hetai Instruments Co., Ltd., Shanghai, China.The ash, fixed carbon, and volatile matter contents of the raw samples were measured according to ASTM e870-82 (2013).The contents of C, H, N, and O in the biomass samples were determined using an element analyzer (Vario EL III, Elementar, Hanau, Germany).All experiments were carried out thrice to reduce the experimental error.The high heating value (HHV) was measured by a calorimeter (DYZDHW-6, Hebi Daewoo Instrument Co., Ltd., China).

Extraction of tannin and tea saponin
C. oleifera shells were smashed and sifted using an 80-mesh sieve to obtain the C. oleifera shell samples.Then, 20 g C. oleifera shell samples were weighted and placed in 500 mL round-bottom flasks.Deionized water solution and 80% ethanol solution were added to each flask.The mixture was heated at 70°C for 1.5 h and then filtered to separate the extract and residue under vacuum.Finally, for evaluating the role of ultrasonic assistance, the residue and the extract were collected for all sample groups.
The tea saponin and tannin contents of the extracts of the four groups were measured and recorded.The detailed measurement method has been described in the litereture [17] .Meanwhile, residues were dried for subsequent use.

TG analysis of C. oleifera shell and residue
TG analysis of C. oleifera shell and residue was conducted using a TG analyzer (TGA 4000, PerkinElmer, Waltham, MA, USA) to explore the thermal behavior.Prior to TG analysis, the sample was sifted using a 60-mesh sieve and then dried to constant weight in vacuum at 105°C.
For a typical experiment, approximately 4 mg of each sample was placed in a ceramic sample pan and high-purity nitrogen was used to provide a pyrolytic experimental inert atmosphere.The nitrogen flow rate was 100 mL/min.The samples were heated from 30°C to 800°C at a constant rate of 10°C/min.

Py-GC/MS analysis of C. oleifera shell and residue
Py-GC/MS analysis was conducted using a double-shot pyrolyzer (PY-2020iD, Binder, Tuttlingen, Germany) coupled with a gas chromatograph/mass spectrometer (GC/MS; Agilent Technologies, Santa Clara, CA, USA) with a chromatographic column (HP-5ms; 30 m × 0.25 mm × 0.25 µm).Approximately 0.5 mg of the sample was introduced into a quartz reactor.High-purity helium was used as the carrier gas to create an inert atmosphere for the Py-GC/MS experiment at a flow rate of 1 mL/min.
The sample was rapidly heated from room temperature to 600°C at 20°C/ms.The resulting volatiles were analyzed by GC/MS.The helium gas transported the steam generated by the pyrolysis to the injection portion of the GC.The syringe temperature was 250°C, and a 20:1 split ratio was used.The GC column was heated to 60°C for 2 min.Then, the temperature was increased to 280°C at 10°C/min for 10 min.MS was operated at an ionization energy of 70 eV, and the mass spectrum was obtained at m/z 12 to 500.
The chemical composition of the pyrolysis vapor was identified by a comparison of its spectrogram with the MS data of the National Institute of Standards and Technology.
The relative content of each component of the pyrolysis oil was determined by calculating the peak area percentage.

Extraction of bioactive substances under different conditions
Ethanol solutions are commonly used to extract bioactive substances, such as tannin, tea saponin, and active polysaccharides, from biomass.According to the previous study by Tao et al. [12] , ultrasound can facilitate the extraction process of bioactive substances.
Figure 1 shows that the 80% ethanol solution had better extraction capacity than the deionized water for the bioactive substances, regardless of ultrasound-assisted treatment.This finding indicated that the solubility of bioactive substances in organic solvents is higher than that in deionized water.The experimental results also showed that ultrasound-assisted extraction helps extract bioactive substances in two kinds of solutions, and this finding is consistent with those in the previous research [18] .In the ultrasound-assisted treatment process, the local pressure in the sample suddenly decreased to less than the saturation vapor pressure and formed a cavity.The bubbles burst at high frequencies of vibration, producing shear waves and turbulence of high energy, which caused damage to the cell walls of the samples and facilitated the dissolution of protected bioactive substances [19,20] .
Figure 1 Extraction of bioactive substances under four conditions 3.2 Pyrolysis of residue 3.2.1 TG analysis TG and derivative TG (DTG) curves are used to show the relationship between the weight change of a sample and temperature, which allows for an understanding of the behaviors in pyrolysis [21] .Figure 2 shows that the pyrolysis behavior could be divided into three stages.In the first stage (from 30°C to ~210°C), the sample was decomposed slowly.The volatilization of water and light compounds occurs within this temperature range.In the second stage (from 210°C to ~380°C), the remarkable weight loss (~70%) was observed due to hemicellulose and cellulose decomposition, which is also the main pyrolysis stage of the samples.Finally, in the third stage (from 400°C to 800°C), the residues, such as lignin, were again pyrolyzed slowly [22,23] , forming relatively stable char.
In the DTG curve, two characteristic peaks could be observed.The shoulder at 200°C to 300°C was mainly ascribed to the decomposition of hemicellulose, and the pointed characteristic peak at 300°C to 380°C was attributed to the decomposition of cellulose.Compared with the pyrolysis of the raw C. oleifera shells, the TG and DTG curves of the pretreated samples was removed to the high-temperature zone.This result indicated that the process of extracting tannin and tea saponin from the C. oleifera shells through the four methods contributed to the thermal stability of the cellulose and hemicellulose in the C. oleifera shells.Furthermore, different solvents used in the pretreatment considerably changed the residual weight loss.The samples that was treated with deionized water showed a sharp peak at approximately 350°C in the DTG curve.By contrast, the samples that was treated with the ethanol solution exhibited only a gentle shoulder at approximately 350°C, which indicated that the content of cellulose in the samples treated with deionized water was higher than those from other treatments [24] .This phenomenon could be attributed to the difference in extraction content in the samples treated with deionized water, which resulted in prominent peaks [25] .Carbon is produced by the dehydration and decomposition of sugar [26] .Thus, the char yields (Y char ) of the samples treated with deionized water were lower than those of the samples treated with the ethanol solution.Differences in the pyrolysis behaviors of the various pretreatment solvents were mainly observed in the range of 330°C to 400°C, indicating that the decomposition temperature of the pretreated samples is nearly in the same range as that of the main components of the sample [27] .  2 for a further understanding of the pyrolysis behavior of hydrolysate [28] .The table also shows that the pretreatment process increased the T i (temperature at which the weight loss was 5 wt%) and T m (temperature at which the maximum weight loss rate was obtained) of the samples.This finding also indicated the thermal stability was improved after extraction [29] .Compared with the DTG max (maximum weight loss rate) of the raw samples, that of the samples with pretreatment was substantially better.Thus, the extraction process could promote the pyrolysis of the C. oleifera shell samples.A comparison between Samples 1 and 3 or between Samples 2 and 4 showed that ultrasound-assisted extraction could reduce the T i of the samples.The thermal stability of cellulose and hemicellulose was lowered because of the damage caused by ultrasound to the samples.

Py-GC/MS analysis
Py-GC/MS is an efficient tool for analyzing organic matter and an important method of studying the evlution of biomass during pyrolysis [30] .On the basis of the different types of chemicals, pyrolysis products are mainly divided into nine categories, including acids, ethers, hydrocarbons, phenols, aldehydes, esters, ketones, amines, and inorganic substances.Information about different chemical compositions is summarized in Table 3.Among these compounds, phenols and acids are the two main organic compounds, accounting for approximately 90% of all.Organic acids contained nearly only acetic acid, which was generated by the removal of acetyl from hemicellulose [28] .In the early stages of hemicellulose pyrolysis, with the cracking of the carboxyl group, the formation of acetic acid was caused by the fracture of the 4-O-methyl-D-glucuronic acid base [31] .
We speculated from the change in the organic acid content produced by five groups of samples in bio-oil that ethanol processing and ultrasound-assisted extraction could reduce the acetyl group in cellulose and hemicellulose, thus reducing the acetic acid in bio-oil.However, in the group of samples treated with ultrasound-assisted ethanol, the content of acetic acid did not reflect this prediction potentially because the methoxy and vinyl groups in the sample were transformed into the precursor organic compounds of acetic acid, such as acetyl, in the ultrasound-assisted ethanol treatment process.This finding could also be confirmed by the reduction in the contents of phenol, 2-methoxy-and phenol, 4,4′-(1-methylethylidene)bis-.Phenolic compounds in bio-oils are mainly composed of derivatives of phenol, whose content is one of the indices used to evaluate the quality of bio-oil [32] .Figure 3 shows that in the two groups of samples treated with different extraction solutions in the pretreatment stage, the effect of ultrasound on the content of phenolic substances in the bio-oils did not show the same rule.The data from the samples after deionized water leaching showed that the samples with ultrasonic assistance during extraction contained a large amount of phenolic compounds.The content of methoxy phenol decreased, and that of alkyl phenol increased significantly.This finding could be attributed to the fact that ultrasound promoted the breaking of methoxy in the sample.For the sample extracted with 80% ethanol solution, ultrasound-assisted extraction adversely affected the phenol content of the bio-oil produced by the samples possibly because the ultrasound-assisted ethanol extraction reduced the content of hemicellulose and the destruction of alkyl led to a decrease in the content of alkyl phenol.The alkyl phenol content of the bio-oil produced by pyrolysis was higher in the treated samples.Moreover, the content of alkoxyl phenol was low, thereby leading to good bio-oil quality.The low content of aldehydes and ketones may be caused by the dissolution of some bioactive substances in the samples by pretreatment.Meanwhile, the content of inorganic substances in the bio-oils was also subjected to the same change during filtration as the extraction content of biological active substances.The more the organic matter was dissolved, the higher the content of inorganic matter was.A certain amount of organic acid can reduce the high calorific value of bio-oils [28] .According to the above analysis, bio-oil with enhanced quality was obtained from the pyrolysis of samples extracted from ethanol or ultrasonic-assisted deionized water.

Conclusions
The experimental results showed that the 80% ethanol solution had better extraction capability for tannins and tea saponin in C. oleifera shells than the deionized water.Ultrasound-assisted extraction can facilitate positively the extraction of bioactive substances from the biomass.TG analysis indicated that the process of extracting tannin and tea saponin from the C. oleifera shells through the four methods contributed to the increase of thermal stability of the cellulose and hemicellulose in the C. oleifera shells.The content of alkyl phenol produced from pyrolysis increased significantly after treatment with mild ethanol.Ultrasound-assisted deionized water extraction of the C. oleifera shells produced the same effect.However, ultrasound-assisted ethanol extraction had no regular effect on the C. oleifera shells.Moreover, the content of alkyl phenol decreased, whereas the content of acid increased.

Figure 2
Figure 2 TG and DTG curves of raw sample and residueThe characteristic parameters of the TG and DTG curves are summarized in Table2for a further understanding of the pyrolysis behavior of hydrolysate[28] .The table also shows that the pretreatment process increased the T i (temperature at which the weight loss was 5 wt%) and T m (temperature at which the maximum weight loss rate was obtained) of the samples.This finding also indicated the thermal stability was improved after extraction[29] .Compared with the DTG max (maximum weight loss rate) of the raw samples, that of the samples with pretreatment was substantially better.Thus, the extraction process could promote the pyrolysis of the C. oleifera shell samples.A comparison between Samples 1 and 3 or between Samples 2 and 4 showed that ultrasound-assisted extraction could reduce the T i of the samples.The thermal stability of cellulose and hemicellulose was lowered because of the damage caused by ultrasound to the samples.

Figure 3
Figure 3 Chemical composition distribution of pyrolysis vapors produced from raw sample and residue

Table 1
Characteristics of raw sample a Dry basis.

Table 2
Thermal degradation characteristic of samples a

Table 3
Relative contents of main chemical compositions in bio-oil obtained from pyrolysis of each sample a a ND = Not Detected.Run CS represented untreated.Run 1-4 represented ultrasound-ethanol, water, ethanol, ultrasound-water.