Annually, large amounts of agricultural residues are produced in Chile, which can be turned into a good opportunity to diversify the energy matrix. These residues have a slow hydrolysis stage during anaerobic digestion; therefore, the application of a pretreatment seems to be an alternative to improve the process. This work focused on applying a thermochemical pretreatment with NaOH on two lignocellulosic residues. The experiments were performed according to a 24 factorial design. The factors studied in a 24 factorial design were: temperature (60 and 120 °C), pretreatment time (10 and 30 minutes), NaOH dose (2 and 4%), and residue size (<1 and 1–3 mm for wheat straw; 1–5 and 5–10 mm for corn stover). The analyzed response variables were the solubilization of organic matter, and the biodegradability of the lignocellulose hydrolysate. The statistical analysis of the data allowed the identification of the experimental conditions that maximized solubilization of organic matter and biodegradability. The main results showed that more aggressive experimental conditions could increase the breaking down of the structure; in addition, the time of pretreatment was not significant. Conversely, the less aggressive experimental conditions, regarding regent dosage and downsizing, favored the release of biodegradable organic matter. The main conclusion of this study was the identification of the operational conditions of the thermochemical pretreatment that promote maximum biogas production, which was caused due to the solubilization of a large amount of organic matter, but not because of the increase in biodegradability of the released organic matter.

INTRODUCTION

Nowadays, a significant issue in Chile, and in most other countries, is energy production, whose consumption has been steadily increasing due to economic growth. Specifically in Chile, the main consumption of energy is carried out by the mining sector; therefore, by 2020, an increase in power consumption of around 100,000 GWh of total electricity demand is projected, which represents the challenge of incorporating 8,000 MW in to the system (Ministerio de Energía 2012). To address this challenge, it will be necessary to expand the energy matrix, which is mostly composed of thermal generation (63%) (Ministerio de Energía 2012).

In the case of Chile, it has shown a significant potential to produce second generation solid, liquid and gaseous biofuels from biomass generated by the agriculture and forestry sectors. It is estimated that the annual generation of residues by these sectors is about 10 million tons, which could be utilized for the production of biogas (methane) using an anaerobic digestion system. As an illustration, the potential for biogas production in Chile was estimated to be at least 1.2 billion m3/year, equivalent to 2.4% of primary energy consumption in the country. Eighty-four percent of the potential biogas and methane in Chile could be obtained from agroforestry sector biomass (Chamy & Vivanco 2007). The production of methane via anaerobic digestion of lignocellulose residues would be beneficial by providing a clean fuel from renewable feedstock; in addition, methane is considered to be a competitive fuel in terms of efficiencies and costs when compared to other forms of biomass energy.

Anaerobic digestion (AD) is a bioprocess carried out by a microorganism consortium in the absence of oxygen, which produces the breakdown of organic matter and its later conversion to methane and carbon dioxide. This process can be divided into four main stages of substrate transformation: hydrolysis, acidogenesis, acetogenesis and methanogenesis. The microorganisms involved in the process have a symbiotic relationship, due to the compounds released by one stage being used as a substrate in the next stage. If the balance is altered, some product accumulation occurs, such as the volatile fatty acids, reducing the pH value and inhibiting bacterial metabolism (Siegert & Banks 2005).

In recent decades, actions have been taken to consolidate Chile as an agri-food power and also to promote the sustainable use of renewable resources. Consequently, the activity of the agroforestry sector has grown by 4.7% during 2013, expansion that was led by the agricultural subsector due to of the increased production of grains such as corn and wheat (Traub 2014). In the period 2012/2013, the production of each of these cereals in Chile reached approximately 1.5 million tons (INE 2012). In addition, the emission factor of crop residues is around 1 ton and 1.3 tons per ton of grain for corn and wheat, respectively (Kim & Dale 2004); each year 3.5 million tons of residues are generated. The agricultural residues are defined as lignocellulosic matter (LM) because of their chemical composition. The cellular wall and the global structure are composed of lignin, cellulose and hemicellulose: all high molecular weight polymers of carbohydrates. According to bibliographic data, the corn stover has 78.5% of dry matter, of which 58.3% are carbohydrates, while the wheat straw has a higher content of dry matter (90%) and the carbohydrates only reach 54% (Kim & Dale 2004). In both cases, the lignin is lower than 20%.

The production of biogas from solid substrates is limited by the AD stage of hydrolysis; therefore, pretreating the LM seems to be a good alternative for breaking down the waterproof lignin and so hydrolyze the cellulose and hemicellulose into simple sugars (Mao et al. 2015). Several studies have shown different pretreatments that affect the surface area and porosity; they could also reduce or increase the crystallinity of cellulose and hemicellulose, as well as decrease the degree of polymerization (Emmel et al. 2003; Taherzadeh & Karimi 2008; Hendriks & Zeeman 2009; Galbe & Zacchi 2012). The comparison of different pretreatments showed that alkaline thermochemical hydrolysis appears to be the most effective method for breaking the linkage between lignin, hemicellulose and cellulose (Chandra et al. 2007; Hendriks & Zeeman 2009). Besides, an alkaline pretreatment is compatible with a subsequent anaerobic digestion process so it would not be necessary to adjust the pH after pretreatment (Pavlostathis & Gossett 1985). There are studies that have shown the effectiveness of using NaOH as a chemical pretreatment, increasing the anaerobic biodegradability of substrate and the biogas production from different residues such as corn stover (Chen et al. 2009) and wheat straw (Pavlostathis & Gossett 1985). Sodium hydroxide is used widely for the fractionation of various complex materials such as agricultural residues due to its relatively high alkalinity. The main effect of pretreatment with NaOH on LM is to reduce or modify the lignin by breaking the ester bonds that form the cross-links between lignin and xylan (Tarkow & Feist 1969). High concentrations of alkali generally cause an increase of biomass delignification grade (Silverstein et al. 2007). The application of an alkali pretreatment using high NaOH concentrations (between 6–20% w/w) resulted in cellulose dissolution however, the removal of lignin was also reduced (Mirahmadi et. al. 2010).

Despite this, some studies have shown an effective removal of lignin and hemicellulose using alkaline pretreatments (Taherdanak & Zilouei 2014). The best pretreatment condition associated with a combination of different operational conditions such as temperature, NaOH dose in addition to residue size and time of pretreatment must be established.

The main objective of this study is to clarify the effect of an alkaline pretreatment on the solubilized organic matter of two lignocellulosic residues: wheat straw and corn stover. The study focuses on the combined effect caused by the residue size, NaOH dose, temperature and time of pretreatment.

MATERIALS AND METHODS

The experiments were performed according to a 24 factorial design. The factors studied were: temperature (T: 60 °C and 120 °C), pretreatment time (t: 10 and 30 minutes), NaOH dose (: 2 and 4%), and residue size (S: smaller than 1 mm and between 1 and 3 mm for wheat straw; between 1 and 5 mm and between 5 and 10 mm for corn stover).

The difference in size of the residues reported was caused by the dissimilar aspect ratio. Both residues were chopped manually/mechanically, to obtain a similar value for their characteristic length, being the long wheat straw cylinder and the long side of the corn stover bucket (diameter and thickness were uncontrolled and similar). However, characterization using a sieve gave a different characteristic diameter, due to the assumption that both particles were spheres.

The study into the variables effect and its interactions was carried out by the quantification of solubilization and biodegradability of the leachable fraction of organic matter.

Materials

Residues

Two residues were used in this study, wheat straw (WS) and corn stover (CS), which were collected from a small farm located in the Town of Paine, Santiago (Chile). The residues were dried and separated into two size ranges, using a sieve shaker (Gilson Company INC., SS-15 model).

Anaerobic inoculum

The inoculum came from an anaerobic digester operated for the treatment of waste yeast production by Lefersa Company in Santiago (Chile).

Experimental procedure

Sampling and chemical characterization

Samples of raw residue were characterized before experimentation. The analyses performed were chemical oxygen demand (COD), total solids (TS) and volatile solids (VS) using Standard Methods (APHA, AWWA & WEF 2005). The leachate fraction was prepared by adding 50 mL of water to 5 g of residue; this solid-liquid mixture was agitated for 30 minutes in an orbital shaker and centrifuged at 5,000 rpm for 10 minutes. Finally, the supernatant, a soluble sample of the residue (SSR), was recovered and characterized. The chemical analyses performed on SSRs were COD by Standard Methods (APHA, AWWA 2005), as well as the content of carbohydrates (CHs) by the Dubois method (Dubois et al. 1956), and protein content (Prots) by Bradford's method (Bradford 1976).

Chemical pretreatment

The pretreatment was carried out in 250 mL Pyrex bottles with 5 g of residue and 18 mL of water with NaOH (concentration according to experimental design). Closed bottles were introduced into a thermostatic bath at 60 °C, which corresponds to pretreatment at low temperature; the pretreatment at 120 °C (high level) was carried out using an autoclave. Both cases were experimentally designed to determine the dynamics of heating the sample, establishing the time required to achieve the desired temperature (5 minutes for the thermostatic bath and 30 minutes for the autoclave), which were added to the value defined in the experimental design.

Anaerobic digestion

The biomethane potential (anaerobic biodegradability assay) was performed in 100 mL serum bottles (50 mL digestion volume). The soluble samples obtained in the pretreatment (SSR) were used as substrates. The experimental conditions used were: 1 g/L of NaHCO3 as buffer and 1 mL/L of macro and micro nutrients (Angelidaki et al. 2009). The pH of the assays was previously adjusted to 7, the VS content of inoculum used was 3 g/L; and the substrate/inoculum ratio was 0.5 (S0/X0).

The biogas production was measured daily using a digital transducer IFM. The biodegradability (BID) (Equation (1)) was calculated as the cumulative production of biogas divided by the theoretical production 395 [mLCH4/gCOD] (Speece 2008) (658 [mLBiogas/gCOD] assuming 60% methane). 
formula

Statistical analysis

The response variables CODS, CHS, ProtS and BID were analyzed using the software STATGRAPHICS® Centurion XVII. The analyses were performed with a 95% confidence level.

RESULTS AND DISCUSSION

Lignocellulosic residues characterization

The comparison of COD and CODs of the residues before pretreatment showed that the amount of leachable organic matter was lower than 1% (Table 1). This behavior was expected due to the residues having a non-permeable structure, provided by the lignin. In addition, the content of CHs and Prots were lower than 20 mg/g, even being non-detectable. In contrast, the solid content was high for all sizes, being higher than 93% for TS and higher than 86% for the ratio VS/TS (Table 1).

Table 1

Residue characterization before pretreatment

 WS CS 
Size [mm] < 1 1–3.35 1–5 5–10 
COD [g/g] 440.9 ± 4.9 469.1 ± 2.1 533.3 ± 22.1 526.7 ± 17.7 
TS [%] 94 ± 0.07 94 ± 0.07 93 ± 0.20 93 ± 0.59 
VS [% ST] 88 ± 0.2 92 ± 0.2 86 ± 0.3 89 ± 0.6 
CODs [mg/g] 35 ± 0.3 15 ± 0.6 30 ± 1.6 17 ± 1.7 
CHs [mg/g] 8 ± 0.1 2 ± 0.1 17 ± 0.8 10 ± 0.4 
Prots [mg/L] ND ND ND ND 
 WS CS 
Size [mm] < 1 1–3.35 1–5 5–10 
COD [g/g] 440.9 ± 4.9 469.1 ± 2.1 533.3 ± 22.1 526.7 ± 17.7 
TS [%] 94 ± 0.07 94 ± 0.07 93 ± 0.20 93 ± 0.59 
VS [% ST] 88 ± 0.2 92 ± 0.2 86 ± 0.3 89 ± 0.6 
CODs [mg/g] 35 ± 0.3 15 ± 0.6 30 ± 1.6 17 ± 1.7 
CHs [mg/g] 8 ± 0.1 2 ± 0.1 17 ± 0.8 10 ± 0.4 
Prots [mg/L] ND ND ND ND 

ND: Non-detectable.

Table 1 shows that the COD levels were similar in order of magnitude for both residues and for both sizes. In fact, the COD content decreased slightly more than 6% when the size of WS was reduced from the range 1–3.35 mm to less than 1 mm. Additionally, a 1.2% increase was observed when the size of CS was reduced from the range 5–10 mm to 1–5 mm. By contrast, the comparison of the CODs showed a significant difference between sizes, showing an increase of 2.3-fold and 1.8-fold for WS and CS, respectively.

Selection of alkaline reagent

Alkaline pretreatment of lignocellulose is a complex chemical process that is applied with the purpose of removing the lignin and solubilizing the hemicellulose polysaccharides; thereby, it increases accessibility to the cellulose. This process involves not only reactive, but also some of the non-reactive phenomena, such as dissolution of non-degraded polysaccharides. The reactive phenomena included the peeling reaction, which refers to the removing (one at time) of the terminal sugars from the reducing end of the molecule, the hydrolysis of glycosidic bonds and acetyl groups and the decomposition of dissolved polysaccharides (Mirahmadi et al. 2010). Mechanistically, it has been postulated that the alkali cleaves the hydrolysable linkages such as α- and β-aryl ethers of the lignin and the glycosidic bonds of the carbohydrates, which are the primary reactions that lead to the dissolution of each biopolymer (Chen et al. 2013).

NaOH pretreatment is classified into processes of low-concentration and high-concentration. The first, typically 0.5–4% NaOH, is carried out at high temperature and pressure; therefore, it is called thermochemical treatment. The mechanism involved the reactive destruction of the lignocellulose, while the NaOH at high temperature disintegrates the lignin and hemicellulose, being both polymers removed from the solid phase. Conversely, the high-concentration process, usually 6 to 20% of NaOH, is used at ambient pressure and low temperature. The mechanism of this process is dissolution of cellulose; but lignin is not significantly removed for the latter.

The efficiency of NaOH pretreatment depends greatly on process conditions, e.g., temperature, NaOH concentrations and pretreatment time as well as the inherent characteristics of lignocellulose used (Zhao et al. 2008).

The slurry obtained from the alkaline treatment of lignocellulose has two phases, the solid fraction which is mainly composed of cellulose and the liquid fraction containing dissolved hemicellulose, lignin, in addition to some unreacted inorganic chemicals (Bensah & Mensah 2013).

In particular, the use of a thermochemical pretreatment increased biogas production between 5 and 20%, regarding the application of alone thermal pretreatment (121 °C for 30 minutes), or adding the alkali reactive (121 °C for 30 minutes and 7 g/L of NaOH) (Kim et al. 2003).

Solubilization process (SP)

An alkaline thermochemical pretreatment process was used for dissolving complex substrates before anaerobic digestion. The data obtained from all the experimental conditions showed the increase in the content of organic matter of SSR samples after pretreatment (Figure 1). This effect indicated that alkaline hydrolysis can disrupt the impermeable layer of the lignocellulosic residue caused by the breakdown of structural linkages between lignin and carbohydrates, which will facilitate the mass transfer phenomena occurring in the subsequent digestion stage of the hydrolyzed organic matter.
Figure 1

Solubilization reached after thermochemical pretreatment of WS: (a) < 1 mm and (b) 1–2.35 mm; and CS: (c) < 1 mm and (d) 1–2.35 mm ( 60 °C/2% NaOH, 60 °C/4% NaOH, 120 °C/2% NaOH, 120 °C/4% NaOH).

Figure 1

Solubilization reached after thermochemical pretreatment of WS: (a) < 1 mm and (b) 1–2.35 mm; and CS: (c) < 1 mm and (d) 1–2.35 mm ( 60 °C/2% NaOH, 60 °C/4% NaOH, 120 °C/2% NaOH, 120 °C/4% NaOH).

The observation of the results obtained from solubilization of organic matter shows that the time variable, in the interval of study, has not been a significant effect. The pretreatment at 60 °C of WS whose residue size was lower than 1 mm, caused an increase of the CODs of 80 to 90 mg/g when the alkali concentration doubled from 2 to 4% of NaOH. Conversely, at 120°C, the COD was modified from 100 to 160 mg/g for the same increment of the alkali dose (Figure 1). An analogous behavior was observed for the WS whose residue size was in the range 1–3.35 mm, except that the solubilization achieved for each condition was lower. The operational variables have an equivalent effect in the solubilization of CS (Figure 1(c) and (d)).

In order to determine the effect of the variables and how much they affected the responses in the study, the results were statistically analyzed. Figure 2 shows a graphical representation of the identification of significant effects, which corresponded with those that were located beyond the limit. The analysis of variance (ANOVA, P < 0.05) indicated that the individual factors that significantly affect the solubilization of the organic matter for both residues were: temperature, size and dose of NaOH; thus, the time of pretreatment was not significant for this type of pretreatment and residues (Figure 2(a)). In addition, some combined factors were identified as significant, such as temperature and NaOH dose, temperature and time, and time and size. Although the significance of the effects was different for both residues, most of the identified effects were coincidental. These results were obtained for all the responses studied (CODs, CHs and Prots), and for both residues (data of CHs and Prots not shown).
Figure 2

Standardized Pareto diagram for the effects on (a) solubilization (CODs) and (b) biodegradability (BID) ( WS & CS).

Figure 2

Standardized Pareto diagram for the effects on (a) solubilization (CODs) and (b) biodegradability (BID) ( WS & CS).

The experimental conditions of the pretreatment that maximized the solubilization were: high temperature (120°C), high NaOH dose (4%) and low particle size (<1 mm WS and < 5 mm CS), showing that more aggressive experimental conditions could increase the disruption of the structure of biopolymers. However, these operational conditions are be associated with more difficult or expensive processes, due to it being necessary to add further reagent and significantly reduce the original size of the residue. Furthermore, the R-squared statistic indicates that the fitted model explains the 91.3% and the 94.4% of the variability of the level of solubilization of WS (Equation (3)) and CS (Equation (4)) respectively (Table 2).

Table 2

Statistical models obtained for CODS and BID of WS (Equation (2) and (4)) and CS (Equation (3) and (5))

 
formula
 
Equation (2) 
 
formula
 
Equation (3) 
 
formula
 
Equation (4) 
 
formula
 
Equation (5) 
 
formula
 
Equation (2) 
 
formula
 
Equation (3) 
 
formula
 
Equation (4) 
 
formula
 
Equation (5) 

Anaerobic digestion (AD)

The biomethane potential assays of the leachate (SSR) showed different biogas production for the samples studied (Figure 3); therefore, for most of the experiments, a wide range of biodegradability was obtained (between 35 and 80%). These results indicated that the organic matter released due to the thermochemical pretreatment was not completely assimilated by microorganisms. This effect can be expected due to the lignin not commonly being biodegradable (Chandra et al. 2007); thus, the products from the structural decomposition of this biopolymer might not be completely accessible to the bacterial metabolism responsible for the anaerobic digestion process (Chen et al. 2009).
Figure 3

Accumulated biogas during anaerobic digestion test of (a) WS pretreated at 120 °C and 30 minutes, (b) CS pretreated 120 °C and 10 minutes ( small size/2% NaOH, small size/4% NaOH, large size/2% NaOH, large size/4% NaOH).

Figure 3

Accumulated biogas during anaerobic digestion test of (a) WS pretreated at 120 °C and 30 minutes, (b) CS pretreated 120 °C and 10 minutes ( small size/2% NaOH, small size/4% NaOH, large size/2% NaOH, large size/4% NaOH).

As in the previous section, the biodegradability of the samples were statistically analyzed (ANOVA, P < 0.05), where the factors that significantly affect the biogas production were identified (Figure 2(b)). For the CS, only individual factors were identified as significant, where the most significant was the residue size and, the less significant was the time of pretreatment. Conversely, the analysis for the WS showed that almost all the factors were significant (individuals and combined), where the most significant was the combined factor temperature–time and the less significant was temperature–NaOH dose. This difference would be produced due to the different shape of both residues (aspect ratio), due to WS being a needle shape with constant diameter and CS a fixed blade thickness; then, the exposed surface to the alkali attack of both residues were different.

The experimental conditions that maximized the biodegradability of WS were: high temperature (120 °C), low NaOH dose (2%) and large particle size (1–3.35 mm). For CS: high temperature (120 °C), low NaOH dose (2%) and large particle size (5–10 mm). Those results showed that the utilization of less aggressive experimental conditions, regarding regent dosage and downsizing, could increase the release of highly biodegradable organic matter.

Global analysis

The experimental conditions of the thermochemical pretreatment that maximized the solubilization of organic matter (solubilization process – SP) and the biodegradability (anaerobic digestion process – AD process) did not match for any of both residues; thus, the operational condition that it should be selected for the global process was not obtained explicitly, since some conditions will improve the solubilization but not increase the biodegradability.

In summary, the optimal operational conditions obtained for both studied variables showed that (Table 3):

  1. The amount of organic matter released (as COD), using the operational conditions of the SP was higher than that obtained in the AD process; being 3.2 times for WS and 2.3 times for CS.

  2. The biodegradability (BID) obtained by the operational conditions of the SP was lower than that obtained for the AD process, being 36% lower for WS and 65% lower for CS.

  3. The total biogas production for each residue will always be higher, if the optimal operational conditions selected are those of the SP. This result is caused due to the decrease in the biodegradability, which will be compensated by the increase in solubilization. Therefore, for WS, the total amount of biogas production will be 91.5 NL/kg versus 38.9 NL/kg, when the operational conditions selected are the optimal for the SP. In addition, the total amount of biogas production for CS will be 68.1 NL/kg versus 49.0 NL/kg, when the operational conditions selected are the optimal for the SP.

Table 3

Experimental conditions that maximize the studied responses

  WS
 
CS
 
 SP AD SP AD 
Operational conditions 
 T [ °C] 120 120 120 120 
 t [min] 30 30 30 10 
 CNaOH [%] 
 S [mm] <1 1–3.35 1–5 5–10 
Results 
 CODs [mg/g] 320 99 217 94 
 CHs [mg/g] 92 22 86 40 
 Prots [mg/g] 35 27 16 
 V/CODs [mL/g] 286 393 314 522 
BID [%] 44 60 48 79 
  WS
 
CS
 
 SP AD SP AD 
Operational conditions 
 T [ °C] 120 120 120 120 
 t [min] 30 30 30 10 
 CNaOH [%] 
 S [mm] <1 1–3.35 1–5 5–10 
Results 
 CODs [mg/g] 320 99 217 94 
 CHs [mg/g] 92 22 86 40 
 Prots [mg/g] 35 27 16 
 V/CODs [mL/g] 286 393 314 522 
BID [%] 44 60 48 79 

All the results presented above were consistent with most of the literature, where the increase in biogas production, as the main goal of an AD process, will be maximized for the more aggressive experimental conditions studied. Nevertheless, this increase should be associated with a large amount of organic matter released, not with selective production for biodegradable compounds.

CONCLUSIONS

The study of the pretreatment effect on the soluble fraction obtained from pretreated lignocellulosic residues showed that the operational conditions that increased the solubilization, and the biodegradability of the released organic matter did not match. However, it was possible to determine the experimental conditions that would increase the production of biogas in the process: high temperature, large dosage of NaOH, lengthy period of pretreatment, and small residue size.

ACKNOWLEDGEMENTS

The authors are grateful for financial support from the ‘Proyecto FONDECYT no 11130138’ from the Chilean Government, and ‘Dirección General de Investigación y Postgrado’ DGIP (General Direction of Research) at UTFSM.

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