ReviewBioreactor design for enzymatic hydrolysis of biomass under the biorefinery concept
Graphical abstract
Introduction
A growing interest in research involving renewable energy resources has been observed in the last decades, due to the depletion of fossil fuels and the environmental concerns, specifically global warming [1], [2].
To overcome those issues, many alternatives have been developed, including the use of these resources through a biorefinery, which is a technology that aims to produce bioenergy and high value-added products, such as biofuels, based on the concept of today’s petroleum refinery. Biorefinery involves a sequence of environmentally friendly processes that employs biomass as raw material, representing a renewable source of energy [3], [4], [5], [6].
Biofuels have been explored as an alternative to reduce the dependence on petroleum. Biofuels are obtained from biomass through chemical, physical, or biological processes. They present several advantages such as easy availability, clean processing, biodegradability, and contribution to sustainable development. Worldwide, one of the most important liquid biofuels used in transportation is bioethanol, which consists in an alcohol obtained from sugar and starchy materials that has been considered a promising substitute or partial substitute of fossil fuels. It has a high octane number (108) and has the advantage of being able to be used in vehicles engines [1], [2], [7], [8].
However, a limiting step in the biofuels production is enzymatic hydrolysis, where the high cost of enzymes and the production of inhibitors of enzymatic activity and fermentation microorganisms limit the viability of the production of biofuels through biomass. In this context, the design of the bioreactor is an important tool to be explored in order to overcome these limitations. The optimization of the design of the bioreactor allows to control some parameters that play a fundamental role in the effectiveness of the enzymatic hydrolysis, such as mixing efficiency and, mass and heat transfer.
Thus, this review focuses on the enzymatic hydrolysis step of the bioethanol production process and on the equipment required for its performance, including the most commonly used bioreactor configurations in enzymatic saccharification, the operational conditions, and operating strategies, as well as the main aspects to be considered for their design. Firstly, an overview of the biofuel production process is given, including the pretreatment, general aspects of enzymatic hydrolysis, and fermentation, as well as some types of biomass used for the bioethanol production; in the end an approach is taken on the promising enzymatic hydrolysis bioreactors for the future.
There are different types of feedstock that can be employed in the production process of biofuels. These have been divided into three generations.
Worldwide, first-generation biofuels are the most common. The production of this type of biofuels is based on food or energetic crops, particularly corn, wheat, and sugar cane [9]. However, although these crops represent a renewable alternative source of energy to fossil fuels, they have the disadvantage of raising the agricultural lands occupancy and reducing the area for food production, leading to an increase in global food prices and creating a competition between food and energy supply [10], [11].
The development of second-generation biofuels emerged as a response to overcome the food versus energy conflict. Lately, interest in them has grown widely because they represent a promising sustainable alternative to substitute petroleum energy and diminish the negative environmental impact [4]. Second generation biofuels are produced from a diversity of lignocellulosic biomasses, including industrial, agricultural and forestry residues, as well as non-edible crops. These types of raw materials are renewable low-priced, and accessible [9], [11], [12], [13], [14].
Lignocellulosic materials are mainly composed of cellulose, hemicellulose and lignin polymers that are present in the cell wall of plants. These polymers interact with each other and provide the material a recalcitrant nature due to cellulose crystallinity and the linkage through covalent and hydrogen bonds [15], [16]. Cellulose and hemicellulose can be reduced to fermentable sugars to produce bioethanol. A typical composition of lignocellulosic biomass consists of 35–50% cellulose, 15–35% hemicellulose and 10–35% lignin. However, the concentration of the components depends mainly on the source of the plant studied [12], [17].
On the other hand, third generation biofuels are obtained from aquatic biomass such as macro and micro algae, as well as fish residues [17], [18], [19]. Third generation biofuels present certain advantages over first and second biofuels, such is the case of microalgae which harvesting cycle is shorter than traditional crops, allowing higher yields due to several harvests through the year [20], [21].
The production process of bioethanol from lignocellulosic biomass involves three main stages: pretreatment, enzymatic hydrolysis, and fermentation. Fig. 1 presents a flow diagram of the production process of second-generation bioethanol.
First, the raw material must be subjected to a pretreatment, which is an essential step in the process to overcome the recalcitrance of the lignocellulosic biomass. Pretreatment consists in the fractionation of the biomass into its main components (cellulose, hemicellulose, and lignin) to facilitate the subsequent enzymatic and microbial attack. Pretreatment facilitates the access to biomass by reducing the crystallinity of cellulose, increasing the pore size and surface area of cellulose, modifying the lignin structure, and totally or partially solubilizing hemicellulose. A diversity of pretreatment methods has been developed, including physical, physicochemical, chemical and biological processes [2], [12], [22], [23], [24].
Physical pretreatment consists in the comminution of the lignocellulosic biomass with the aim to reduce the particle size and the crystallinity of the biomass by mechanical methods such as milling, grinding or chipping [25], [26], [27]. On the other hand, chemical pretreatment implies the utilization of different chemical compounds to disrupt the lignocellulosic structure. They are classified according to their chemical nature as acid, alkaline and ionic liquid pretreatments. Also, oxidizing agents can be used to chemically pretreat the biomass, including ozonolysis and peroxide oxidative pretreatment; as well as organic solvents in an organosolv pretreatment [26], [28]. Furthermore, a diversity of physical and chemical technologies combinations have been used to reduce the recalcitrance of the lignocellulosic biomass. The most commonly used physicochemical strategies are liquid hot water (also known as autohydrolysis), steam explosion, ammonia fibre explosion and, microwave pretreatment [25], [29], [30]. Finally, biological pretreatments represent an environmentally friendly alternative that comprises the use of microorganisms, mainly fungi, that produces enzymes able to degrade lignin and hemicellulose [25], [26], [31].
Once the biomass has been subjected to a pretreatment, the second stage in the production of bioethanol is the hydrolysis of the polysaccharides recovered from the pretreatment. Hydrolysis consists in the degradation of the polysaccharides in an aqueous medium catalyzed by enzyme or acid. Therefore, the hydrolysis can be carried out by chemical or enzymatic processes [2], [17]. However, enzymatic hydrolysis is considered the most promising strategy because it presents several advantages over chemical saccharification including high efficiency, low energy consumption due to the requirement of mild operation conditions, not environmental damage because enzymes are biodegradable, not toxic compounds formation and, low deterioration and corrosion problems for the equipment. Additionally, enzymatic hydrolysis produces fewer fermentation inhibitors [12], [32], [33], [34], [35], [36].
Enzymatic hydrolysis of cellulose involves the reaction between cellulases and insoluble cellulose in an aqueous medium that aims at the degradation of polysaccharides into fermentable monomeric sugars [1], [33], [37]. So, in this process, cellulolytic enzymes are used as catalysts to cleave β-1,4 glucosidic bonds present in cellulose to generate glucose that subsequently can be transformed into bioethanol by fermentation. Cellulase enzymes are a complex system of highly specific proteins that comprise three main types: endo-1,4-β-D-glucanases, exo-1,4-β-D-glucanases (also known as cellobiohydrolases) and β-glucosidase. This group of enzymes works synergistically to degrade cellulose molecule. First of all, endoglucanases randomly break down β-1,4 glucosidic bonds in the amorphous regions in the interior of cellulose backbone releasing mainly cellodextrin by the addition of a water molecule in the β-1,4 glucosidic linkage, producing new reducing and non-reducing ends. Subsequently, cellodextrin is hydrolyzed by the action of exoglucanases. So, exoglucanases hydrolyzes cellulose and these cellodextrins from its reducing and non-reducing chain ends to produce cellobiose (dimer conformed by two units of glucose); and finally, β-glucosidases cleave cellobiose releasing soluble glucose monomers. β-Glucosidases does not have a significant effect over insoluble cellulose [2], [33], [35], [38], [39], [40], [41], [42], [43], [44]. Fig. 2 shows the described mechanism of action of cellulase enzyme. Another important factor in the enzymatic reaction development is the adsorption of cellulases onto the surface of the lignocellulosic material. Most endoglucanases and exoglucanases have a carbohydrate binding module that promotes this adsorption process. Typically, adsorption has been reported to take between 10 and 15 min, which is related to the reaction rate [45], [46].
The efficiency of cellulose enzymatic saccharification is determined by different factors. The optimal conditions for cellulolytic enzymes activity are temperature value between 45 and 55 °C and pH in the range of 4–5 [15], [25], [33]. Also, mixing conditions and solid-liquid ratio play an essential role in the hydrolysis of the polymer because suitable mass and heat transfer are required to ensure a good enzyme-substrate interaction [47].
Enzymatic saccharification has not been successful in the attempt to scale up the process to the industry due to several obstacles. Economically, enzymatic hydrolysis is considered commercially unfeasible due to the high cost of the cellulolytic enzymes [2], [17], [48]. Currently and according to Sigma Aldrich, the market price of the cellulases is approximately $9500 USD/gal of enzyme [49]. However, Novozymes reported that their second generation cellulases, Cellic CTec2®, only represents a cost of $0.50 USD/gal of ethanol produced [50]. According to Liu et al. [51], it is unfeasible to purchase cellulolytic enzymes from current market suppliers; therefore, the authors propose on-site enzyme production as an alternative to boost economically feasible industrial lignocellulosic ethanol production, which could lead to cellulase enzyme cost reduction from 30 to 70% [51], [52], [53]. The cost of cellulases is still difficult to certainly stablish, because it depends on several factors, including enzyme dosage and the bioethanol production yield, as well as, negotiation between suppliers and consumer [51], [54]. The cost contribution of enzymes has been reported to vary from $0.1 USD to $0.4 USD per gallon of bioethanol produced [54], [55], [56]. However, Klein-Marcuschamer et al. [55], in their techno-economic model for the production of bioethanol from corn stover, reported a cellulase enzyme cost $0.68 USD/gal ethanol assuming the maximum theoretical yields in the process, and an enzyme price of $1.47 USD/gal using previously reported yields. In a more recent techno economical evaluation, Liu et al. [51] found an enzyme cost of $2.71 USD/gal based on the commercial price of enzyme and conventional yields of bioethanol. Moreover, it has been reported that the enzyme cost represents about 15–28% of the bioethanol selling price, or 20% of the overall bioethanol production costs [45], [53], [57], [58]. Therefore, enzyme cost is considered one of the main barriers that hinder the commercialization of second generation bioethanol [59].
Additionally, enzymatic hydrolysis undergoes other limitations, including the slow rate of the hydrolysis reaction that is related to the decrease in cellulase activity, due to the inhibition of the enzyme by several compounds present during the saccharification process [34]. The presence of xylan (major component of hemicellulose) and lignin in the biomass reduces the efficiency of hydrolysis, because they act as a physical barrier that prevents the access of the enzyme to the cellulose surface. Particularly, lignin is an important inhibitor due to the high affinity of cellulase to it, so that the enzyme tends to adsorb into lignin’s surface generating non-productive binding that reduces the enzymes activity [47], [60], [61], [62]. Moreover, during the pretreatment, a wide variety of inhibitors are produced, such as phenolic compounds, furfural, 5-hydroxymetil furfural (HMF), weak acids, among others. However, the type and amount of inhibitors generated depend on the type of pretreatment employed [35], [40], [41].
The crystallinity of cellulose and polymerization degree are also obstacles that hinder the action of enzyme action and diminish the glucose production yield [2], [37]. Furthermore, during the enzymatic hydrolysis, oligosaccharides, disaccharides, and monosaccharides are produced which also act as inhibitors of cellulase enzyme. It has been demonstrated that these enzymes undergo feedback inhibition, as in the case of cellobiose that can inhibit cellulase. Likewise, β-glucosidase generally has its activity reduced in the presence of glucose [38], [41], [63], [64].
Consequently, a diversity of strategies has been proposed to improve the cellulose bioconversion into glucose in the enzymatic saccharification process, with the aim to make it more efficient and cost-effective. In previous studies, percentages of 40–50% of cellulases have been reported to stay absorbed onto lignin surface after saccharification processes, which represents economic losses due to decreased glucose yields [45], [59]. As a result of this difficulty, one of the strategies developed consists in the addition of surfactants or non-catalytic proteins to prevent the non-productive binding of the cellulolytic enzymes to lignin and thus decrease the deactivation of cellulases [25], [47], [65], [66]. Also, the production of more efficient cellulolytic enzymes with enhanced catalytic activity, the modification of the operational conditions, particularly the adjustment of the pH, as well as the enzymes immobilization and recycling (see Section 2.3.1) are considered promising alternatives to increase the enzymatic hydrolysis yields [47], [59], [65], [66], [67]. Enzyme recycling is an option to reduce enzymatic bioconversion costs, as it allows to reuse cellulolytic enzymes for multiple lots, decreasing new enzyme feeding [68]. It has been stated that enzyme recycling could lead to enzyme cost savings reduction of 50–60% [45]. Unfortunately, little information has been published and reported about enzyme recycling economics.
Another strategy to enhance the economics on enzymatic hydrolysis is the well-known simultaneous saccharification and fermentation (SSF), which consists in the integration of enzymatic and fermentative processes in a single step (see Section 1.2.3), as it reduces equipment, operation, and production costs. This alternative also allows to reduce feedback cellulase inhibition, as end products are promptly transformed into bioethanol making the process more efficient [69], [70], [71]. A techno-economical evaluation carried out by Wingren et al. [72] indicated that the utilization of high solid loadings in SSF processes, the recycling of the process streams, as well as the reduction of yeast dosage could lead to significant economic improvements in bioethanol production.
Regarding to the strategies to overcome the economic limitations of the enzymatic hydrolysis, one of the most studied alternative is the employment of high solid concentrations. It is well documented that a cost-effective distillation process requires a concentration of ethanol over 4% w/w, which implies that at least a concentration of 8% w/w of fermentable sugars should be achieved at the enzymatic hydrolysis stage [1], [64], [73], [74]. Hence, to ensure an increased yield of glucose production, it is necessary to perform the saccharification with a high concentration of solids, specifically solid loadings above 15% on dry basis. However, a high concentration of solids may result in a highly viscous solution that may affect the mass and heat transfer between the enzyme and the substrate and produce an increased amount of inhibitory compounds. Many researches have been developed around these limitations with satisfactory results [1], [28], [75].
The final stage in the production of bioethanol from lignocellulosic biomass is the fermentation, which consists in the conversion of monomeric sugars obtained in the enzymatic hydrolysis into bioethanol by the action of microorganisms such as yeast, bacteria, and fungi. The biological fermentation agents most commonly used for bioethanol production are yeasts, particularly Saccharomyces cerevisiae, which is capable of fermenting hexose sugars, especially glucose, in ethanol under controlled conditions [2], [40], [76]. Once the fermentation is completed, a subsequent purification step by distillation is required, where the produced bioethanol is separated from the water [40], [77].
Certain strategies have been developed to improve fermentation, including separate hydrolysis and fermentation (SHF) which consists in the execution of the enzymatic hydrolysis and fermentation in different stages. On the other hand, simultaneous saccharification and fermentation (SSF) performs the enzymatic hydrolysis and fermentation at the same time in a single reactor [15], [25], [78], [79], [80]. To meet desired overall yields during ethanol production from lignocellulosic materials, it is important to use both hexoses and pentoses. This can be achieved by co-fermentation of sugars in SSF, so called SSCF (simultaneous saccharification and co-fermentation), using genetically modified yeast strains [81], [82], [83].
Section snippets
Bioreactor design
Bioreactors are essential elements in every biological process; even more they are considered the core of bioprocesses. Bioreactors are the units in which a reaction or a biological process occurs. The bioreactor must provide the optimal conditions to satisfy the requirements of the biological system (enzyme, microorganism or cell) and to ensure a high yield of the bioprocess [84], [85].
A bioreactor for the enzymatic saccharification stage must fulfill certain requirements. As mentioned in
Future promising bioreactors for enzymatic hydrolysis
There are other types of reactors known as pneumatically agitated bioreactors, which usually employ gas to induce the mixing in the reaction vessel. The gas mixture, normally air, is introduced at the bottom of the bioreactor via nozzles, perforated plates, or a sparger ring, which promote the liquid circulation, without the need for moving mechanical parts. In pneumatically agitated bioreactors the gas-liquid contact is an important aspect. The gas is mixed with the liquid phase during the
Future perspectives
Currently, enzymatic hydrolysis still represents a bottleneck of the production process of bioethanol, due to the high cost of enzymes and the generation of inhibitory compounds that diminish the glucose production efficiency. Therefore, further study should be made to ensure an adequate conversion of cellulose to glucose by considering the lignocellulosic biomass nature, because it determines the characteristics and the behavior of inhibitory compounds as well as the entire conversion process.
Acknowledgement
This work was financial supported by the Secretary of Public Education of Mexico – Mexican Science and Technology Council (SEP-CONACYT) with the Basic Science Project-2015-01 (Ref. 254808), and the Energy Sustainability Fund 2014-05 (CONACYT-SENER), Mexican Centre for Innovation in Bioenergy (Cemie-Bio), and Cluster of Bioalcohols (Ref. 249564). The author Marcela Pino also thanks the National Council for Science and Technology (CONACYT, Mexico) for her Master Fellowship support (grant number:
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