Effect of Chemical-Hydrothermal Pretreatments on Compositional and Morphological Changes of Spruce Wood Exploited in Biogas Systems

Agricultural crops and forest residues are of valuable resources to produce biofuel due to anaerobic digestion. But, the recalcitrance nature of these lignocellulose residues limits its enzymatic degradation and decreases biogas production. Therefore, efficient pretreatments prior to anaerobic digestion are essential. In present study, hydrothermal-chemical pretreatments using Phenol (Ph), Sodium Hydroxide (SH) and Phosphoric Acid (PA), and combined pretreatments included Ph + SH and Ph + PA as chemical pretreatments were used for spruce wood. For hydrothermal pretreatments, the samples were put into the autoclave at 134 ºC for 20 min. Acid hydrolysis, FTIR and SEM analyses were carried out. The results indicated that all pretreatments were effective lignin removal having the highest value for Ph (42.362%). Adding Ph to PA, caused to increase lignin removal from 1.580– 6.112%. Mixing Ph to SH represented the same trend in changing structure of spruce wood as compared to individually use of SH. All results proposed that when Ph contributed in binary pretreatment with SH and PA, it could be more effective on the morphological changes of spruce wood. In general, Ph was more effective on changing the crystalline structure of spruce wood than the others. After that, Ph + SH was more effective compared to Ph + PA on structural changes of spruce wood. In comparison between alkali and acidic pretreatments, SH represented more structural change in spruce wood than PA one. To have an intermediate state, it is recommended to use the combination of Ph with SH as infectious pretreatment instead of individually Ph. Because, this increases the biodegradation power of SH while the toxicity of Ph decreases. The obtained results are very important in biogas production systems.


Introduction
With appearing new technological advancements particularly in developing countries, energy demands are continuously growing up. Currently, the world's largest source of energy is fossil fuels which have some disadvantages such as resource limitation, rising prices and environmental pollution. Accordingly, many attentions have been concentrated in the world to access alternative energy sources which are both economically and environmentally acceptable (Zilouei and Taherdanak, 2015). Among different types of renewable energy resources, bioenergy plays an important role as fourth largest energy resource in the world (Rajput and Visvanathan, 2018). So far, three generations of biofuels have been introduced around the world, including: edible parts of plants, non-edible parts of plants and microalgae. Among these three generations, the second one, especially the lignocellulose biomass, is of the greatest interest, due to lack of human nutritional value, low cost and abundance of resources. In addition, by converting these materials into fuel, the large amount of agricultural and forest residues produced annually, could be managed. It is noteworthy, in most developing countries, such residues are burnt by farmers inside the farm, which in turn causes environmental risks through greenhouse emissions. Lignocellulose materials such as stems and crop residues, forest residues and energy crops are common organic materials that have great potential for biogas production (Celiktas et al. 2014;Chen et al. 2014;Taherdanak et al. 2016). Biogas extracted from lignocellulose biomass provided that could be produced by anaerobic digestion and properly recovered, capable of achieving sustainable energy functions without contaminating the environment (Chojnacka et al. 2015). In the other words, anaerobic digestion is an environmentally friendly technology for the production and recovery of degradable organic wastes (Venturin et al. 2019). But, there is one major limitation in the degradation phase of such materials. The primary chemical composition of lignocellulose biomass consists of three types of polymers called cellulose, hemicellulose and lignin. Cellulose is the major component of biomass and many cellulose properties depend on the crystalline chain properties or its degree of polymerization (Monlau et al. 2013). The previous studies showed that decrease the crystallinity causes to increase the accessibility of cellulose to enzymatic attack and improve the yield of subsequent enzymatic hydrolysis (Mirahmadi et al. 2010). Hemicelluloses are present in almost all plant cell walls with cellulose (Per 1993), which binds between lignin and cellulose fibers. Short side chains containing different sugars can be easily hydrolyzed (Nimz 1984). Lignin is the most abundant polymer in nature after cellulose and hemicellulose (Liqian 2011) and creates a strong mechanical layer for plants (Dollhofer et al. 2018).
Lignin has a very strong binding to cellulose and hemicellulose and it is very difficult to break down.
Holocelluloses (cellulose and hemicellulose) can decompose to methane by anaerobic germs (Chen et al. 2014), but lignin is hydrophobic and resistant to attack by germs. Therefore, it reduces the availability of holocellulose to anaerobic microbes and so limits its rate of degradation (Rajput and Visvanathan 2018) and results lower biogas performance.
The available solution is to select and apply effective pre-treatments prior to anaerobic digestion in order to break the bond between polysaccharides and lignin. This allows more cellulose and hemicellulose access to the bacterium (He et al. 2008). Various pretreatment methods have been introduced to increase the anaerobic digestibility of lignocellulose biomass including physical, mechanical, chemical, biological and hybrid methods. Appropriate pretreatment assignment is one of the most important steps to increase digestibility and biogas production for a special substrate.
Because, the relative amounts of lignocellulose compounds are different in different types of agricultural biomass. On the other hand, due to the high variability of pretreatments results, depending on biomass type, the same pretreatment need to be tested on other lignocellulose biomass with different chemical and structural composition (Sambusiti 2013). However, many researchers have studied on optimal pretreatments for lignocellulose materials, more research is still needed to find the optimal conditions for applying different pretreatments. According to the aforementioned, the present study investigates the effect of different chemicals on the amount of delignin process and structural changes of spruce wood waste that is one of the largest forest wastes.
The main innovation of the present study is to investigate Hybrid pretreatments for lignocellulose biomass instead of individual one. In other words, the present study surveys whether the hybrid pretreatments could be effective in increasing lignin removal, structural changes or reducing crystallization.

Material And Methods Raw material and pretreatments
The raw material used as lignocellulose biomass was spruce wood from forest residues. These materials were made from milled wood waste used in the wood industry. Therefore, the first pretreatment applied to this material was mechanical grinding and size reduction, which were screened through meshes with 2 mm diameter to equalize their size.
The chemical pretreatments used in the study were Phenol (Ph), Sodium Hydroxide (SH), Phosphoric Acid (PA), and binary pretreatments included Ph + SH and Ph + PA. Each sample of milled wood (20 g) were pretreated with 100 ml (1% v/v) of these solutions. The prepared mixtures were kept at ambient temperature (26 ºC) for 30 min, then transferred to an autoclave. The samples were put into the autoclave at 134 ºC for 20 min. Afterwards, the samples were get out the autoclave and cooled to the room temperature. Then, the samples were washed with distilled water for several times to neutralize them. They were placed into an oven at temperature of 105 ºC for 24 h. The samples were stored at 4 ºC in the refrigerator for subsequent analysis.

Experimental Analysis
Wood lignin was analyzed using NREL standard method (Ehrman 1994;Mirahmadi et al. 2010), in which acid hydrolysis of cellulose and hemicellulose were used to break down the sugar polymers into sugar-forming units. Acid insoluble lignin content was determined by weight changes before and after acid hydrolysis. The percent of lignin removal was calculated according to Eq. 1 (Gao et al. 2014).
The amount of acid soluble lignin was also calculated by UV-Vis spectroscopy and its absorption intensity at 205 nm. The surface chemistry of the treated and untreated woods was determined using Fourier Transform Infrared spectrometer (FTIR) (JASCO 4700, International Co). In this method, the absorption spectra of treated and untreated woods ranging from 500 to 4000 cm − 1 were investigated. The crystallinity was determined by three methods including Cross-Linked Lignin ratio (CLL), Lateral Order Index (LOI) and Hydrogen Bond Intensity (HBI). The cross-linked structure is a characteristic feature of the concentration in guaiacyl. To evaluate the proportion of lignin with condensed and cross-linked structures (CLL), the ratio between band intensities at 1600 and 1508 cm In order to evaluate the influence of pretreatment on the morphology of wood, Scanning Electron Microscopy analysis (SEM) was performed using a high resolution SEM FEI Quanta 200. The dry material was coated a thin layer of gold.

Results And Discussion
In present study, the combined hydrothermal-chemical pretreatments were used for increasing lignin removal and accelerating the process of sugars digestion and the biogas production which are done by microorganisms. The chemical solutions used were SH, PA, Ph, Ph + SH and Ph + PA solutions that their effects on chemical structure of spruce wood were investigated.

Effect Of Pretreatments On Composition Of Spruce Wood
The amounts of lignin content for treated and untreated spruce woods are summarized in Table 1. As observed, Acid Insoluble Lignin (AIL) with 30.366% is the main part of lignin in spruce wood. The amount of AIL increased after using all of applied pretreatments, with the lowest and highest values for PA (31.756%) and Ph (48.878%) solutions, respectively. Acid Soluble Lignin (ASL) decreased by SH, Ph and SH + Ph pretreatments and increased by PA and PA + Ph pretreatments (Table 1).
Forasmuch as reduction in lignin content is a key outcome of an efficient pretreatment (Hendriks and Zeeman 2009), lignin removal was calculated for each solution in order to more clarify (Table 1).

Compositional And Morphological Analysis
Using FTIR spectroscopic technique, the changes in hemicellulose and cellulose structures before and after the pretreatments were investigated. Table 2 shows the FTIR peaks for the following samples:  The The crystallinity index is one of the key parameters to be considered during enzymatic hydrolysis.
Hence, the crystallinity index was evaluated based on CLL, LOI, and HBI. Generally, increased CLL, LOI and HBI values represent the highest degree of crystallinity and a more ordered cellulose structure.
While decreasing these values designate the amorphous structure of cellulose (Khedkar et al. 2018). Table 3 summarizes the values of different crystallinity ratios (CLL, LOI and HBI).

SEM Analysis
The morphological changes of the untreated and treated spruce woods in two magnitudes of 200 and 20 µm were evaluated using SEM analysis (Fig. 2). The SEM image of untreated sample indicated an intact surface with well-arranged structure of cellulose, hemicellulose, and lignin (Fig. 2a). After treating with Ph, SH and SH + Ph, the surface layer of spruce wood was destructed (Fig. 2b, c and d).
That's reason is that the pretreatment removes the amorphous cellulose and hemicellulose from inner part. According SEM image, SH + Ph pretreatment caused the lignin re-deposition on the biomass surface. This created a corrugated surface for the wood and could result to accelerate in the enzymatic hydrolysis process. The result of CLL concurs with SEM analysis for SH + Ph so that CLL value for this pretreatment had the lowest value (0.560). Moreover, in Fig. 2e, when using PA pretreatment, the morphology of wood did not change. But, using PA + Ph, the morphology of wood demonstrated tiny destruction (Fig. 2f). This result is also consistence with conformational analysis (Table 1) and CLL. As given in Table 1, the lignin removal for Ph and PA were 42.362 and 1.580%, respectively. When Ph is mixed with PA, caused to arise lignin removal from 1.580 to 6.112% for spruce sample. In addition, from

Conclusions
Due to the severe nature of lignocellulose biomass, their biodegradability and consequently biogas production is always difficult. In present study, hydrothermal-chemical pretreatments were applied to achieve an efficient lignin removal and structural changes of spruce wood. Alkali, acidic and oxidizer pretreatments were used individually as well as the combination of oxidizer with acidic pretreatment and oxidizer with alkali pretreatment. The results were summarized as follows: All chemical pretreatments including: Ph, SH, PA, Ph + SH and Ph + PA change the wood structure and prepare them for digestion.
Among all of the investigated pretreatments, Ph is more effective on changing the crystalline structure of spruce wood than the others. The amount of lignin removal obtained by Ph was 42.362% that is very high compared to the other chemicals not only in current study but also previous researches.
Combining Ph and PA, causes to rise their effect on lignin removal from 1.580% to 6.112%. Moreover, combining Ph and SH represents the same trend in changing structure of spruce wood as compared to individually use of SH. In other words, adding Ph improves the biodegradability power of PA and SH.
After Ph, pretreatment of Ph+SH is more effective compared to the Ph+PA on structural changes of spruce wood.
Alkali pretreatment (SH) represents more structural change in spruce wood than acidic (PA) one.
According the results of current research, it is recommended to use the combination of Ph with SH as infectious pretreatment instead of Ph. Because, this increases the biodegradation power of SH while the toxicity of Ph decreases.  untreated sample, (b1,2) treated using Ph, (c1,2) treated using SH, (d1,2) treated using SH+Ph, (e1,2) treated using PA+Ph

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