Biomass Conv. Bioref. DOI 10.1007/s13399-017-0277-3
REVIEW ARTICLE
Alkaline hydrogen peroxide pretreatment of lignocellulosic biomass: status and perspectives Emmanuel Damilano Dutra 1 & Fernando Almeida Santos 2 & Bárbara Ribeiro Alves Alencar 3 & Alexandre Libanio Silva Reis 1,4 & Raquel de Fatima Rodrigues de Souza 5 & Katia Aparecida da Silva Aquino 6 & Marcos Antônio Morais Jr 3 & Rômulo Simões Cezar Menezes 1
Received: 3 December 2016 / Revised: 20 June 2017 / Accepted: 22 June 2017 # Springer-Verlag GmbH Germany 2017
Abstract Lignocellulosic biomass is a renewable and abundant resource that is suitable for the production of bio-based materials such as biofuels and chemical products. However, owing to its complex chemical composition, it requires a process that enhances the release of sugars. Pretreatment is an essential stage in increasing the efficiency of enzymatic hydrolysis of lignocellulosic biomass. The most widely used pretreatment methods operate at high temperatures (160– 290 °C) and pressures (0.69 to 4.9 MPa) and generate biological growth inhibitors such as furfural and hydroxymethylfurfural (HMF). Thus, there has been a growing need to adopt new approaches for an effective pretreatment that operates at ambient temperature and pressure and reduces the generation of inhibitors. Among these methods, alkaline hydrogen peroxide (AHP) is notable because it is effective for a wide range of lignocellulosic biomass * Alexandre Libanio Silva Reis
[email protected]
1
Biomass Energy Research Group, Department of Nuclear Energy, Federal University of Pernambuco, Recife, PE 50740-540, Brazil
2
Department of Engineering, State University of Rio Grande do Sul, Porto Alegre, RS 91540-000, Brazil
3
Interdepartmental Metabolic Engineering Research Group, Department of Genetics, Federal University of Pernambuco, Recife, PE 50670-901, Brazil
4
Departamento de Energia Nuclear, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, Cidade Universitária, Recife, PE 50740-540, Brazil
5
Laboratory of Bioprocessing, Northeastern Center of Strategic Technologies, Av Prof. Luiz Freire, 01, Recife, PE 50740-540, Brazil
6
Laboratory of polymers and nanotechnology, Department of Nuclear Energy, Federal University of Pernambuco, Av. Moraes Rego, 1235, Recife, PE 50670-901, Brazil
concentrations, and can provide a high degree of enzymatic hydrolysis efficiency. However, few results have been discussed in the literature. Given this, the aim of this study was to investigate the use of alkaline hydrogen peroxide (AHP) as an oxidative pretreatment agent to improve the efficiency of enzymatic hydrolysis for different types of biomass and examine the key areas of the pretreatment. Finally, there is a discussion of the challenges facing a large-scale application of this method. Keywords Bioenergy . Lignocellulosic biomass . Pretreatment . Cellulosic ethanol . Biorefinery
1 Introduction The diminishing supply of fossil fuels resulting from environmental problems caused by the burning of these materials underlines the urgency of generating new forms of energy based on renewable sources. These include lignocellulosic biomass which has a high potential, since it is part of the carbon cycle in nature, and has been used by humans ever since the dawn of civilization [1, 2]. In general, the use of biomass can be defined as a renewable natural energy resource, which can be processed to provide more complex and suitable forms for bioenergetic purposes. It includes every kind of energy linked to accumulated chemical activity resulting from photosynthetic processes. Biomass can be obtained from non-woody plants, woody plants, organic waste, and biofluids [3]. Until recently, lignocellulosic biomass was often regarded as a worthless raw material that should be disposed of as waste. However, several research groups around the world are currently seeking to make it economically feasible to make use of lignocellulosic biomass for the production of new Bvalue-added products^
Biomass Conv. Bioref.
such as biofuels, biochemicals, and biomaterials through biochemical, chemical, and thermochemical processes [4–6]. The biochemical pathway is one of the technologies most widely used for the production of biofuels, with an emphasis on the production of bioethanol and biogas. However, the chemical composition of lignocellulosic biomass makes it highly resistant to biological degradation, and it requires a pretreatment stage to enable it to be turned into energy products [7]. The main purpose of pretreatment is to disrupt the recalcitrance of the lignocellulosic matrix and facilitate the separation of polysaccharides and lignin, which leads to greater accessibility to the enzymatic hydrolysis [8]. There are several types of pretreatment reported in the literature, which can be divided into chemical, physical, and biological methods or a combination of them [8–11]. In general, the pretreatment has benefits and drawbacks. It is important to establish criteria for an effective pretreatment since this stage is one of the most costly for the production of biofuels from biomass [12, 13]. The choice of a suitable pretreatment method includes the following requirements: the production of reactive cellulose fibers, low degradation of cellulose and hemicellulose, the absence of possible inhibitors that might be formed as a result of the hydrolysis, and fermentation process and a minimum energy consumption. Moreover, there is also a need to achieve cost-effectiveness regarding the reduction of the particle size, the materials required for the construction of pretreatment reactors, waste production, and low chemical consumption [14]. Most of the various pretreatment alternatives available require a large amount of energy and/or high pressure, which results in high process costing. Steam explosion is a thermomechano-chemical pretreatment using high-pressure saturated steam with a temperature of (160–290 °C, p = 0.69–4.85 MPa) lasting from several seconds to a few minutes [11, 15]. Acid treatment is another alternative where dilute acids at a high temperature (160–200 °C) are used as catalysts [16]. Ammonia fiber expansion (AFEX) is a physicochemical pretreatment that is conducted by using liquid ammonia in contact with biomass at a relatively moderate temperature (60– 120 °C) and high pressure (1.72–2.06 MPa) for 5–30 min, after which the pressure is suddenly reduced [17]. Liquid hot water is a hydrothermal pretreatment that operates at temperatures above 200 °C at various pressure conditions for a few minutes [18]. These are examples of effective pretreatments requiring high-energy consumption and high pressures. Effective pretreatments that can be conducted at room temperature and pressure have been investigated in recent years, and these can be assessed by the increasing interest in the use of alkaline hydrogen peroxide (AHP) for several pretreatments of biomass (Table 1). AHP is an oxidative pretreatment process. It acts in the delignification of the lignocellulosic biomass which allows a greater efficiency to be achieved in the recovery of sugars in
the liquid phase of enzymatic hydrolysis [31] since the presence of lignin makes it difficult for the enzymes to attack the substrate [32]. This type of pretreatment has low-energy consumption and does not generate inhibitors like hydroxymethylfurfural and furfural [21]. However, most of the review papers in the literature [8, 10, 11, 14, 33], used for the pretreatment of lignocellulosic biomass, fail to report the use of AHP as an effective pretreatment. Thus, this review discusses the use of a pretreatment with alkaline H2O2, with the aim of altering the lignocellulosic biomass structure to make the cellulose more accessible to the enzymatic hydrolysis stage.
2 Structural composition of lignocellulosic biomass Lignocellulosic biomass is a renewable and abundant resource suitable for the production of bio-based materials such as biofuels and chemicals and is mainly composed of cellulose, hemicellulose and lignin, and small extractive fractions and ash [34]. Cellulose is a homogeneous long-chain polysaccharide that is abundant in nature and represents approximately 40% of the dry weight biomass measurement formed by Dglucose units linked by β-1,4-glycosidic bonds. It is found in crystalline form with amorphous regions and is more susceptible to enzymatic hydrolysis. The hydrolysis of cellulose leads to the production of glucose, which is easily converted by most microorganisms, like Saccharomyces cerevisiae and Dekkera bruxellensis [35]. Hemicellulose, the second most important polysaccharide after cellulose, is a complex heterogeneous polymer composed of sugar units of five and six carbon molecules. Its main chain consists of xylose, and its branches may contain L-arabinose, galactose, and mannose, as well as uronic acid and acetyl groups [36]. The hydrolysis of hemicellulose mainly produces xylose and pentose which only a few microorganisms can metabolize to ethanol at high yields, like Spathaspora sp. [37], Pichia stipitis [38], and Candida shehatae [39]. After cellulose, lignin is the main cell wall component of plants. It is a complex and heterogeneous structure that is mainly formed of phenolic compounds and their derivatives. It has low biodegradability, and only certain specialized microorganisms can degrade it, such as white rot fungi and brown rot fungi [40, 41]. These three components of the biomass are linked together through chemical bonds that form a heterogeneous composite. Hydrogen interactions are responsible for binding the polysaccharide fractions to one another, just as they do lignin. Ethertype bonds also carry out this function. These hydrogen interactions are not considered strong because the majority of hemicellulose monomers does not have an external primary alcohol bound to the pyranoside ring [42], except, e.g., by
Biomass Conv. Bioref. Table 1
Efficiencies of enzymatic hydrolysis reported for different types of biomass pretreated with alkaline hydrogen peroxide pretreatment
References Lignocellulosic biomass
Cellulose Hemicellulose Enzymes (%) (%)
Enzyme loading
Enzymatic Cellulose Hemicellulose hydrolysis conversion conversion (%) (%) time (h)
[19]
Wheat straw
35.9
44.8
Cellulase T. reesei
[20]
Wheat straw
44.24
25.23
95 961
NR
Rice husk
35.62
11.96
120
67.38
81.10
[22]
Sugar cane bagasse
39.6
21.4
24
69.4
NR
[23]
Wheat straw
40.98
36.96
Unspecified cellulases
72
95.71
NR
[24]
Bamboo
45.5
22.8
Cellulases Micelase®
48
78.92
NR
[25]
Sweet sorghum bagasse
49.78
27.72
Celluclast 1.5 L Novozyme 188
24
62.46
NR
[26]
Corn stover
34.4
22.4
48
75
71
[27]
Cashew apple 20.52 bagasse Nopalea 31.6 cochenillifera Seaweed Ulva 19.4 prolifera
10.17
Accellerase 1000, Multifect-Xylanase, Multifect-Pec, and Novozyme 188 Novozymes NS 22074
5 mg/mL 16 mL of each for 100 g of biomass 4 mL/100 g of biomass for each 3.42 FPU and 1 UI β-glycosidase/g of pretreated biomass 50 FPU/g of pretreated biomass 20 FPU/g of pretreated biomass 20 FPU/g dry biomass and 40 IU/g dry biomass 30 mg/g glucan
72 120
[21]
Celluclast Novozyme 188 Viscostar 150 L Celluclast Novozyme 188 Viscostar 150 L T. reesei cellulase and A. niger β-glucosidase
87
NR
23.4
NR
84
54.4
8.09 Microalgae biomass (Scenedesmus obliquus, Scenedesmus quadricauda, and Nitzschia sp.)
7.25
30 FPU/g 24 cellulose ACCELLERASE® 1500 20 FPU/g dry 48 biomass 48 Commercial cellulase (Jienuo 7.5 FPU and 4.5 U/g Enzyme Co., China.) and pretreated UPR cellobiase (Sigma Chemical Co., USA) 48 Celluclast 1.5 L and 10 FPU/g Novozyme 188 cellulose 20 CBU/g cellulose
78.8
NR
[28] [29]
[30]
17.1 14.4
NR
NR not reported a
Saccharification calculated with total carbohydrate conversion
glucomannans. Ester bonds are responsible for maintaining the hemicellulosic fraction bound to lignin. The fact that this structure is highly organized hampers the removal of the fermentable monosaccharides from the polysaccharide fractions of the biomass. Given this, pretreatments have been adopted as an alternative method for carrying out this procedure. The purpose of this type of process is the delignification of the biomass, as well as the solubilization of the hemicellulose and swelling of the crystalline cellulose, to assist in the removal of the cell wall and the dissociation of
the biomacromolecules [43, 44]. With the partial or total removal of lignin and hemicellulose, the cellulose portion is more exposed and can interact more effectively with the enzymes, and increase the efficiency of the enzymatic hydrolysis. This also causes a reduction in the unproductive binding of cellulases to lignin and is thus a means of positively interfering with this stage [45]. For example, the lignin content in the sugarcane bagasse and its crystallinity index (% CrI) are 23.06 and 51.7%, respectively, for in natura bagasse [46]. Added to this, the lignin
Biomass Conv. Bioref.
content is relatively high. In these conditions, it is increasingly necessary to rely on pretreatment, to expose this cellulose, and thus ensure the release of glucose, for the hydrolyzate. This is corroborated by experiments that have been carried out in which an increase of about 200% was observed in the monosaccharide concentration when the delignified bagasse was used to the detriment of the lignified bagasse [47]. It can thus be stated that the absence of the pretreatment is one of the factors that can lead to a reduction in the release rates of glucose in the cellulosic matrix. The fractions from the enzymatic hydrolysis of the polysaccharides, cellulose, and hemicellulose, as well as the macromolecule lignin obtained from the delignification of the lignocellulosic biomass, can be used in the concept of a biochemical platform in biorefineries. A biorefinery is a physical structure that is capable of processing biomass for the production of biofuels, which are chemical compounds with high added value and energy [48]. In addition to the use of glucose for ethanol production, other advanced biofuels can be produced from the hydrolysis of cellulose, such as acetone, butanol, and ethanol [49], as well as organic acids, glycerol, sorbitol, mannitol, enzymes, and biopolymers [50]. The hemicellulose fraction also has applications in the biorefinery concept of lignocellulosic biomass. A wide range of products can be produced from sugars derived from this fraction, such as xylitol, organic acids (succinic, propionic, lactic, and butyric), solvents such as butanol and acetone, and other biofuels like 1,3-propanediol [51, 52]. Lignin can be used for the production of energy through burning in the industrial sector. However, depending on the functionality of the lignin obtained in the pretreatment, several high value-added compounds can be produced, like carbon fibers, emulsifiers, dispersants, sequestrants, surfactants, and aromatic compounds [53].
Its highly oxidizing properties led Gould [55] to investigate the use of H2O2 to improve the enzymatic digestibility of agricultural residues, as well as the mechanism of the action of H2O2. Pretreatment with H2O2 has been successfully applied to a range of lignocellulosic biomasses with the main intention of improving the enzymatic hydrolysis yields through delignification. The enzymatic hydrolysis yields for different biomasses can be seen in Table 1, and depending on the conditions evaluated, values ranging from 23.4 to 95% have been reported for cellulosic conversion. It should be noted that the enzymatic hydrolysis yields largely depend on the following: the enzyme source, enzymatic loading, use of auxiliary enzymes, solids loading on enzymatic hydrolysis, and reaction time [32, 56, 57]. This means that the comparison of efficiencies for different biomasses should be made with caution. The use of H2O2 for the pretreatment of lignocellulosic biomass is based on the chemical reactions that this oxidizing agent undergoes in the alkaline liquid medium. Its dissociation generates the hydroperoxide anion (HOO−) through Eq. (1) [55]. H2 O2 þ H2 O←→HOO− þ H3 Oþ
ð1Þ
In the alkaline medium, the hydroperoxide anion can react with H2O2, leading to the formation of superoxide and hydroxyl radical, as expressed in Eq. (2). H2 O2 þ HOO− ←→OH: þ O2 −: þ H2 O
ð2Þ
In the absence of other reagents, superoxide and hydroxyl radicals can combine, and generate oxygen and water, as in Eq. (3). OH: þ O2 −: þ Hþ →O2 þ H2 O
ð3Þ
Therefore, the general equation of H2O2 decomposition in alkaline medium can be summarized in Eq. (4).
3 Chemical reactions involved in pretreatment with alkaline hydrogen peroxide in lignocellulosic biomass H2O2 is a natural metabolite present in many organisms and participates in various biological routes, especially those involved in the decomposition of lignin by fungi within complex enzyme systems [40]. Hydrogen peroxide is a highly reactive and an extremely powerful oxidizer. It is not flammable, is miscible with water capable of being mixed in all proportions, and is generally sold as an aqueous solution with concentrations ranging between 20 and 60% (w/v). For example, a 35% solution (w/v) has 35% H2O2 and 65% H2O by weight. It has also been used for many years in the cellulose and paper industry, as a bleaching agent and the effluent treatment area as a reagent in advanced oxidation processes (AOP) [54].
H2 O2 þ HOO− þ Hþ →O2 þ 2H2 O
ð4Þ
For each mole of H2O2 added, 0.5 mol of O2 is generated. However, when there are other compounds that react with radicals formed in Eq. (2), a smaller amount of O2 is produced. Furthermore, H2O2 is unstable under alkaline conditions and readily decomposes, particularly in the presence of certain transition metals such as manganese, iron, and copper, and this leads to the generation of hydroxyl radicals (OH−) and superoxides (O−2) which play a role in delignification reactions [55] through Eqs. (5), (6), and (7) H2 O2 þ M→Mþ þ OH: þ OH− þ
−
−
H2 O2 þ M þ 2OH →M þ O
ð5Þ 2
þ 2H2 O
ð6Þ
General equation 2H2 O2 þ OH− →OH: þ O− 2 þ 2H2 O
ð7Þ
Biomass Conv. Bioref.
Although there are two possible routes for H2O2 decomposition, experimental data showed the predominance of Eq. (2) in the delignification of lignocellulosic materials [55]. In a study by Karagoz et al. [31], the authors used the addition of metals in pretreatment to minimize cellulose degradation and maximize lignin solubilization. In this context, rape straw biomass was submitted to pretreatment with H2O2 under alkaline conditions (2.5% v/v H2O2 pH 11.5, 50 °C for 1 h) with six different MgSO4 concentrations (0; 0.25, 0.5, 1, 1.25, and 1.5% w/v). The effect was observed in the lignin solubilization, with results ranging from 22.83 to 65.66% when the MgSO 4 concentration increased from 0 to 0.25% w/v. However, MgSO4 concentrations above 0.25% resulted in a reduction in lignin solubilization. Recent studies demonstrated an increase in the conversion of cellulose and delignification, with the addition of transition metals, such as Cu, Fe, and Mn, when pretreatment with alkaline hydrogen peroxide was carried out. Bansal et al. [58] reported that the presence of Cu increases the delignification of the biomass, and leads to an increase of 10–100% in the conversion of cellulose. Also, the image formed from the transmission electron microscopy showed that the concentration of polymeric and oligomeric lignin soluble in alkaline medium was significantly higher when Cu was used as a catalyst in the oxidative pretreatment [59]. Lignin is probably the most important site for a chemical attack by radicals generated by H2O2 decomposition in alkaline medium [60]. Although electron micrographs indicate changes in the physical and morphological characteristics of cellulose fibers with small glucose losses 15%) to make the process economically viable. However, some problems arise from high solid concentration in pretreatment and enzymatic hydrolysis, like an increase in viscosity caused by a reduction in the supply of free water,
Biomass Conv. Bioref. Table 2 Solid concentrations, H2O2 concentration, and pretreatment time mostly used in alkaline hydrogen peroxide pretreatment of different lignocellulosic biomasses Lignocellulosic biomass Solids concentration H2O2 concentration Pretreatment time (h) Effect of pretreatment
References
Wheat straw
8.6% (w/v)
2.15% v/v
3
High total sugar and individual sugar
[20]
Rice hulls Barley straw
15% (w/v) 10% (w/v)
7.5% v/v 2.5% v/v
24 24
High sugar yields Sugar release
[21] [65]
Corn stover
2–10% (w/w)
0.5 g/g biomass
24
Increasing in glucose yields
[66]
Rapeseed straw
5% (w/v)
5% v/v
1
[31]
Sugarcane bagasse
4–15% (w/w)
7.4% v/v
1
Optimal solid concentration with respect to overall ethanol production High glucose yields
Cashew apple bagasse
2–10% (w/v)
4.3% v/v
6
Sweet sorghum bagasse
10% (w/v)
5% v/v
24
Increasing solid concentration had minor [27] effect in cellulosic digestibility High sugar yields [64]
Seaweed Ulva prolifera
10% (w/v)
0.2% v/v
12
High sugar yields
which hinders the mixing procedure and increases the energy required for agitation [69]. 4.2 Pretreatment time The pretreatment time, which can be defined as the contact time of lignocellulosic biomass with the pretreatment agent, varies widely depending on the type of pretreatment used— from several minutes (steam explosion) to hours (alkaline hydrolysis) and days (biological pretreatment). In the case of pretreatment with alkaline H2O2, several reaction times have been reported (1–24 h), depending on the operating conditions and the pretreated biomass. In the case of the wheat straw biomass pretreated with 2.15% H2O2 (v/v) and total solid content of 8.6% (w/v), 24 °C and pH 11.5, the increase from 3 to 24 h in the pretreatment reaction resulted in a greater release of total sugars (glucose + xylose + arabinose) after enzymatic hydrolysis [20]. The pretreatment reaction time was not significant in the release of sugars (glucose) at high H2O2 concentrations for sugarcane bagasse biomass. The optimum pretreatment conditions for sugarcane bagasse were 7.35% v/v H2O2, 25 °C for 1 h, with enzymatic hydrolysis performed with cellulases derived from Trichoderma reesei (3.5 FPU/g dry pretreated biomass) and β-glucosidase obtained from Aspergillus niger (1.0 CBU/g dry pretreated biomass) under conditions of 50 °C and 100 rpm [70]. 4.3 H2O2 concentration in the pretreatment The H2O2 concentration has a significant influence on improving the biomass accessibility to enzymatic hydrolysis. It can vary widely between different types of biomass, depending mainly on the recalcitrance of the raw material. Studies show variations from 0 to 10% (v/v) of H2O2 in the solution (Table 2). One of the main difficulties in using AHP pretreatment can be attributed to the concentration that has to be used
[67]
[29]
to pretreat the biomass because the best pretreatment efficiency is obtained from high concentrations of H2O2 at reduced reaction times [70]. Since the cost of hydrogen peroxide is considerably more than the value of the bioproducts, like ethanol, these processes may not be economically viable. In studies using rice husk, Saha and Cotta [21] investigated the influence of the H2O2 concentration in solution, as a means of maximizing the release of sugars after enzymatic hydrolysis with cellulase, β-glucosidase, and xylanase. The increase in H2O2 concentration (to 7.5% v/v) resulted in a greater release of sugars from rice husk biomass. However, when the concentration was set at 10% (v/v), there was a reduction in the release of sugars. Biomass with initial sugar concentration, like microalgae biomass, with an increase in H2O2 concentration, might lead to the degradation of sugars and formation of by-products composts and inhibitors [30]. Although most studies involve high concentrations of H2O2, it is necessary to establish the optimum conditions for each type of biomass at low concentrations of H2O2, since this parameter has a direct bearing on the cost of the pretreatment.
4.4 Temperature of pretreatment The pretreatment temperature is a variable that is closely linked to the energy requirements of the pretreatment method, which influences the cost of the whole process. The pretreatment methods, used at the pilot plant and for demonstration purposes, consumed large amounts of energy because they operated at high temperatures (180–250 °C). Studies with alkaline H2O2 are performed at a mild temperature 25–70 °C and in standard conditions for ambient pressure (Table 3). The effect of increasing temperature in pretreatment with H2O2 varies according to the lignocellulosic biomass that is evaluated. When rye straw biomass was pretreated with 2% (w/v) H2O2, pH 11.5 and 12 h of reaction, the temperature increase
Biomass Conv. Bioref. Table 3 Effect of the temperature and pH in delignification and solubilization of hemicellulose in alkaline peroxide pretreatment of rye straw and kenaf
Biomass fraction
Effect of the temperature (°C) on alkaline peroxide pretreatment of rye strawa
Effect of the pH on alkaline peroxide pretreatment of kenaf biomassb
20
10.5
30
40
50
60
70
11
11.5
13
Lignin soluble (%)
52.7
75.7
81.8
83.1
85.8
87.8
25
38
50
25
Hemicellulose soluble (%)
44.2
52.5
70.0
70.0
71.3
71.9
ND
ND
ND
ND
ND not determined a
Rye straw biomass pretreated with 2% (v/v) H2O2, pH 11.5 and 12 h of reaction [61]
b
Kenaf biomass pretreated with 1% (v/v) H2O2, 2% (w/v) total solids, 25 °C and 24 h of reaction [55]
led to higher lignin and hemicellulose solubilization to the liquid fraction (Table 3) [61]. About sugarcane bagasse biomass, increasing the temperature did not assist in the release of total reducing sugars and glucose in the enzymatic hydrolysis. The best results in the conversion of cellulose into glucose (62.4%) were obtained under conditions of 20 °C, 5% H2O2, and 4% total solids (w/v) for 6 h [22].
4.5 pH of pretreatment The effectiveness of pretreatment with alkaline H2O2 is largely dependent on pH, as it is combined with the generation of the hydroperoxide anion, as described in Eq. (1). Studies by Gould [55] have shown that the optimal pH value is 11.5. Studies have reported that the use of H2O2, without pH correction, results in less efficiency in the release of sugars during enzymatic hydrolysis. In a comparative study of the chemical pretreatment of aquatic plants used in water purification, pretreatment with H2O2 was not effective in improving enzymatic hydrolysis (10% w/v total solids; 1% (v/v) H2O2; room temperature for 2 h). However, the combination with NaOH solution achieved the best enzymatic hydrolysis results (1% NaOH w/v, room temperature for 12 h followed by the addition of 1% (w/v) H2O2, at room temperature for 12 h) [71]. Li et al. [29] employed alkaline hydrogen peroxide for pretreatment seaweed biomass with pH 4, 6, 8, and 10. The results showed that the yields obtained, from reducing sugar and glucose in pretreated groups, were higher than those of the control group. The optimum pH was 4.0 with a maximum rate of reducing sugar. The lowest yield of glucose and sugar reduction was obtained at pH 6.0. A smaller increase was observed at pH 8.0 and 10.0. The authors suggested that, when compared with another lignocellulosic biomass, the differences with the best condition are pH 11.5. The lower content of lignin might be the reason why the yield of sugar reduction was sensitive to the pretreatment of pH. The combined analysis of variables that affect pretreatment with alkaline H2O2 is a very important means of describing the optimal conditions for each type of lignocellulosic biomass
evaluated and, depending on the intended use of the released sugars, these parameters can be adjusted and optimized [12].
5 A comparative study of pretreatment with alkaline hydrogen peroxide in lignocellulosic biomass The evaluation of comparative studies between pretreatment with alkaline hydrogen peroxide and other types of pretreatments is fundamental to establish the conditions of the process and the types of biomass in which H2O2 demonstrates greater efficiency. Some types of biomass were evaluated in comparative studies, such as corn stover [66], cotton stalks [72], sweet sorghum bagasse [25], and barley straw [65]. It should be noted that these studies only evaluated the technical aspects, such as delignification efficiency or conversion efficiency of cellulose and hemicellulose into glucose and xylose, respectively. In addition to the technical aspects, there is a need for performing an economic analysis to express the comparative cost of the different types of pretreatment. The hydrolysis efficiency varied between the pretreatment options evaluated. In the treatment of corn straw and barley straw, pretreatment with alkaline hydrogen peroxide increased cellulose conversion efficiency by 54.47 and 6.81%, compared to ammonia explosion and H2SO4 (0.75% w/v), respectively. When pretreating barley straw with lime, this increase was only 3.30%, while cotton stalks, pretreated with H2SO4 (2% w/v), presented the greatest difference of this efficiency, relative to H2O2: 108.89%. On the other hand, cotton stalks and sweet sorghum bagasse, when submitted to pretreatment with NaOH (2% w/v) and NaOH-soaked in H2O2 (2% w/v), respectively, presented higher efficiency than the H2O2 solution. Reduction in cellulose conversion, in the comparison between the treatments, averaged 18.05 and 26.5%, respectively. That is, pretreatment, using alkaline H2O2 solution, was more efficient than treatments with ammonia explosion, H2SO4, and lime, for the evaluated biomass types, because it removes higher lignin content, while the others act mainly in the removal of hemicellulose. The efficiency of the enzymatic hydrolysis is
Biomass Conv. Bioref.
dependent of biomass delignification and hemicellulose solubilization [32]. In contrast, oxidative pretreatment is less efficient than those with NaOH, but, when associated with it, increases the efficiency of hydrolysis.
6 Concluding remarks The use of hydrogen peroxide in the alkaline medium has benefits and drawbacks, like any other pretreatment method. Among the main advantages are low-energy consumption, the fact that there is no generation of inhibitors (hydroxymethylfurfural and furfural), the availability and easy acquisition of H2O2 and NaOH, with no need for special reactors for the pretreatment reaction, and its compatibility with high solids loadings. Furthermore, the use of alkaline H2O2 leads to sterility conditions for the pretreatment and enzymatic hydrolysis, and thus eliminates the need for antibiotics [26]. Although it is unable to form inhibitors from acid reactions, such as HMF and furfural, this type of pretreatment adds a high pH to the biomass. An alternative method that involves separating the solid-liquid fractions, with the washing of the solid fraction, to a neutral pH, has been employed as a means of overcoming this kind of obstacle [62]. However, this method entails using large volumes of water to achieve the required pH. If one decides not to separate these fractions, one can either Bdestroy^ H2O2, through the action of the enzyme catalase, or correct it through the HCl, until the pH value of the normal condition of most hydrolyses is 4.8 [73]. However, the latter alternative causes the formation of salts, which is a drawback in employing this method. Also, this type of pretreatment generates other inhibitors from the decomposition of lignin, like sodium acetate and pcoumaric (pCA) and ferulic (FA) acids. This problem can be overcome by adopting genetic strategies with the aim of making microorganisms resistant to the fermentative inhibitors that are present in the hydrolysates of biomasses pretreated with hydrogen peroxide. Sato et al. [73] have evaluated the resistance of S. cerevisiae strains to sodium acetate and pcoumaric (pCA) and ferulic (FA) acids. They observed that the adaptive changes (which include LOH in PDR1 and increased copy numbers from PDR16 to PDR18) in the GLBRCY87 strain of S. cerevisiae led to an improved PDR gene response. This response, subsequently, allowed for increased cell survival at high concentrations of pCA and FA, in addition to increasing the number of functional cells capable of fermenting xylose. A further drawback when using H2O2 is the price of this reagent in conjunction with NaOH. It is estimated that to process 1 ton of corn straw biomass, the cost involved would be US$102–125 for pretreatment reaction with low H2O2 concentration (0.125 g H2O2/g biomass), with an addition of US$ 36 for NaOH (US$300/t) [66]. One means of offsetting the
high cost of H2O2 is to use it at low concentrations in the pretreatment stage, which usually results in a need to control the reaction of pH. The use of in situ production techniques, new alkalinizing agents for pH correction and a recycling of the liquid fraction in pretreatment with AHP, can reduce the use of inputs and procedural costs [74]. Another important challenge is the need for an initial grinding of the biomass material so that it can be pretreated with H2O2. It is a fact that particle size reduction increases the surface area and improves the pretreatment and efficiency of enzymatic hydrolysis. Most of the studies cited in this review used reduced particle diameters (0.5 to 3 mm), which leads to an increase in procedural costs. However, there have been cases reported in the literature of how the use of alkali H2O2 for the pretreatment of sugarcane bagasse biomass without the grinding process has achieved satisfactory results for enzymatic hydrolysis [22]. Furthermore, experimental studies are usually conducted with amounts of 1–10 g, which requires a high degree of pretreatment and is efficient for enzymatic hydrolysis. However, when the scale of experimental protocols is increased, the values reported in the laboratory stage are not always achieved. This is mainly due to difficulties such as mass transfer and stirring for solid reagent contact. However, in a recent study conducted with corn straw biomass, the increase in the scale was carried out successfully. In the laboratory scale, 1 g corn straw was pretreated with 0.125 g H2O2/(g solids) with pH control and glucose release efficiency of 73.8 ± 2.2%; and in the demonstration scale, 1 kg of corn straw was pretreated under similar conditions with 75% glucose release efficiency [26, 66]. Finally, alkaline hydrogen peroxide has proved to be efficient in the pretreatment of a variety of lignocellulosic biomass with high rates of efficiency for enzymatic hydrolysis when used alone or in combination with another pretreatment method. Furthermore, high lignin and hemicellulose solubilization values for the liquid fraction strongly validate this pretreatment method in the biorefinery concept. Acknowledgements This work was sponsored by the Bioethanol Research Network of the State of Pernambuco (CNPq-FACEPE/ PRONEM program, Grant No. APQ-1452-2.01/10), the CNPqUniversal program (Grant No. 472106/2012-0), and the Ministry of Science and Technology, Brazil (SIGTEC number PRJ03.33).
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