Bioethanol production from autohydrolyzed Eucalyptus globulus by Simultaneous Saccharification and Fermentation operating at high solids loading

Abstract

Eucalyptus globulus wood samples were subjected to non-isothermal autohydrolysis in order to solubilize hemicelluloses, leading to treated solids of increased cellulose content and enzyme digestibility. Autohydrolyzed solids obtained under a variety of operational conditions were assayed as substrates for bioethanol production by Simultaneous Saccharification and Fermentation (SSF). SSF was optimized using the Response Surface Methodology. The experimental plan included as independent variables the autohydrolysis severity (S_o defined as the logarithm of the severity factor), the liquor to solid ratio and the enzyme to substrate ratio. The dependent variables considered were the maximum ethanol concentration achieved in individual experiments, the volumetric productivity, the maximum ethanol conversion, the yield of solids after SSF and the cellulose recovery in solids coming from SSF. Operating at high solids loading (S_o = 4.67, LSR = 4 g/g, ESR = 16 FPU/g), media containing up to 67.4 g ethanol/L and corresponding to 91% of the stoichiometric amount calculated from the cellulose content of wood were obtained. Wood processing resulted in the generation of soluble products from hemicelluloses, in the generation of up to 291 L ethanol/1000 kg oven-dry wood from cellulose, and in a solid material mainly made up of lignin.

Introduction

The increasing world population and the growing per capita energy demand boost the interest in producing energy from renewable feedstocks, alternative to fossil resources [1]. Lignocellulosic materials (LCMs) are clean and cheap raw materials for obtaining energy and chemicals. Other advantages of LCM as potential energy sources include their large availability, independence of geographic location, improvement of local economy derived from cultivation, neutral carbon balance and renewable character [2]. Biomass-derived biofuels are considered as the only suitable alternative to oil-derived fuels [3]. Second generation bioethanol, produced from LCM, could replace up to 30% of gasoline [4], [5].

The heterogeneous and complex structure of LCM make their fractionation and further benefit difficult. LCM contain non-structural components (extractives, moisture, etc.) and structural components (cellulose, a polysaccharide made up of glucose units, with high crystallinity and polymerization degree; hemicelluloses, made up of various sugar units, which can be substituted; and lignin, a polymer of phenolic nature).

LCM utilization may be based on the biorefinery concept: the feedstocks can be fractionated (by reactions involving the depolymerization of at least one of the structural components), and the resulting fractions can be employed for specific applications, including bioethanol production from polysaccharide-derived sugars. The manufacture of second generation bioethanol from LCM can be carried out by processes involving three major steps [6]:

• pretreating the raw material (for example, by a fractionation treatment) to increase its susceptibility to further processing (and, in some cases, to obtain valuable byproducts),

• enzymatic hydrolysis of cellulose to sugars, and

• biological conversion of sugars to ethanol.

Pretreatment is one of the most influential stages on the production of second-generation bioethanol, owing to its high incidence in the operational costs [5], [7]. An optimal pretreatment should fulfill was many as possible of the following requirements [7], [8], [9], [10], [11], [12], [13]:

• simple and economic operation;

• particle size reduction, if necessary, should be achieved at low cost;

• reduced consumption of energy, water and chemicals;

• limited corrosion effects;

• ability for reaching favorable effects on the LCM structure;

• reduced polysaccharide losses;

• recovery of valuable products from hemicelluloses;

• limited generation of unwanted products from polysaccharides (for example, dehydration products such as furfural or 5-hydroxymethylfurfural) or lignin;

• production of pretreated solids with enhanced cellulose contents and high susceptibility toward enzymatic hydrolysis;

• recovery of high-quality lignin or lignin-derived compounds; and

• limited waste generation.

There is not a general agreement on which pretreatment can be considered as the best one [14]. Pretreatments intending hemicellulose solubilization have been considered, either operating with water [15], [16], [17] or with acidic solutions [18], [19], [20].

Autohydrolysis (also called hot water processing or hydrothermal pretreatment) is one of the approaches that meet several of the above conditions. This method is carried out by heating an aqueous suspension of the LCM, and results in the partial hydrolysis of hemicellulose into soluble fragments (by means of the catalytic action of hydronium ions from water ionization and from in situ generated acids). Operating under suitable conditions, hemicelluloses can be extensively converted into soluble saccharides [21], whereas the treated solids (with increased proportions of cellulose and lignin) show an increased susceptibility to cellulolytic enzymes [6], [22], [23], [24], enabling their utilization as substrates for bioethanol production.

The fermentative production of ethanol from pretreated LCM can be carried out operating either in two sequential steps of hydrolysis and fermentation (Separate Hydrolysis and Fermentation, SHF) or in a single stage (Simultaneous Saccharification and Fermentation, SSF). SSF presents advantages derived from the decreased product inhibition, limited operational costs and decreased contamination risks [25]. SSF also compare favorable with SHF in terms of experimental results [26].

From a general perspective, the loadings of solids and enzymes are major variables affecting the economic features of bioethanol manufacture. The importance of the type of pretreatment and its repercussion on the overall process has been emphasized in literature [27], as well as the fact that a correct choice of pretreatment conditions increases the bioconversion of Eucalyptus globulus, from about 40% up to near 100% [12].

High solids loading (HSL) is a key operational strategy for both economic and technical reasons [28], [29]. Increased concentrations of solids enable higher potential bioethanol concentrations, reducing the size equipment, the consumption of energy in heating and distillation, and the downstream processing duties. According to literature, the threshold for economic profitability corresponds to bioethanol concentrations in the fermentation broth in the range 4–5 volume percent [28]. Achieving this threshold entails the utilization of media containing 15–20% solids (on dry basis). However, high solid loadings may result in limited cellulose conversions in enzymatic hydrolysis [29] or in SSF stages, owing to mass transfer limitation.

In the manufacture of bioethanol by technologies involving enzymatic hydrolysis, the cost of enzymes has been identified as the second contributor to the operational costs, just after the raw material [30], and can account for 20% of total costs [31]. Because of this, the optimization of enzyme spending has been one of the topics considered in literature [12], [25], [28], [32], [33]. However, it can be noted that enzyme loadings below a given threshold may result in increased SSF duration [33] and limited cellulose conversion.

Eucalyptus is a fast growing species with favorable features as a raw material for bioethanol production. E. globulus wood has been considered by Romaní et al. [12] as a substrate for bioethanol production by SSF by a method based on hydrothermal pretreatment and bioconversion at low-intermediate solids loading, reaching a maximum ethanol concentration of 27 g/L. Similar concentrations (up to 29 g/L) have been achieved using acid-catalyzed pretreatments [34], whereas higher concentrations (up to 35 g/L) were achieved with organosolv-processed substrates [35].

To our knowledge, no information has been reported dealing with the simultaneous assessment of variables measuring the pretreatment conditions and two variables (loadings of solids and enzymes) of major economic importance. The experimental plan developed in this work provides relevant information for preliminary technical and economic analysis.

This work deals with the optimization of bioethanol production from autohydrolyzed E. globulus wood, operating at high substrate loadings in SSF mode. The effects of substrate pretreatment conditions, liquor to solid ratio employed in SSF, and enzyme loading on selected operational variables (ethanol concentration, ethanol yield and exhausted solid composition) were assessed in selected experiments.