Appl Biochem Biotechnol DOI 10.1007/s12010-014-0873-7

Continuous Enzymatic Hydrolysis of Lignocellulosic Biomass with Simultaneous Detoxification and Enzyme Recovery Raghu N. Gurram & Todd J. Menkhaus

Received: 28 January 2014 / Accepted: 23 March 2014 # Springer Science+Business Media New York 2014

Abstract Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products. Keywords Biorenewable products . Enzyme recycle . Bioseparations . Enzymatic hydrolysis . Biorefining . High-value coproducts

Introduction Driven by the rapid depletion of petroleum reserves, as well as environmental and political concerns associated with petrol feedstocks, there has recently been extensive research on the production of biorenewable fuels such as ethanol, butanol, biodiesel, and biogasoline from non-food biorenewable lignocellulosic feedstocks [1–3]. Biochemical conversion of R. N. Gurram : T. J. Menkhaus (*) Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, 501 East Saint Joseph Street, Rapid City, SD 57701, USA e-mail: [email protected]

Appl Biochem Biotechnol

lignocellulosic biomass into a liquid fuel consists of the following steps: disruption of the protective lignin barrier by pretreatment, hydrolysis of cellulose and hemicellulose into fermentable sugars using biocatalysts, fermentation of sugars to biofuels or biochemicals, and distillation or other separation technologies for purification of the product [4, 5]. Cellulases (a complex cocktail of at least three different enzymes: exoglucanases, endoglucanases, and β-glucosidases) play a significant role in the process of catalyzing the hydrolysis of cellulose into fermentable sugars [6]. However, literature has shown that enzymatic hydrolysis has several problems, which can be divided into either substrate- or enzyme-related challenges [7, 8]. Substrate-related difficulties are essentially a link to the lignocellulosic material structure, which must be subjected to a harsh pretreatment in order to improve the accessibility of cellulose within the biomass sheath to enzyme attack [9]. For enzyme challenges, the low specific activity, high propensity for loss of activity, and difficulty recovering activity lead to elevated costs and are commonly cited as a major barrier that hinders the economic commercialization of lignocellulosic biorefining [10, 11]. It has been estimated that the cost of cellulases accounts for approximately 20 % of the total cost of a biomass-to-biorenewable product process [12, 13]. In the past decade, many studies were conducted with the objective to reduce the amounts of enzyme used during the hydrolysis step [14–16]. These studies showed several methods to decrease the amounts of enzymes used, including optimizing the hydrolysis conditions (pH, temperature, etc.); increasing the specific activity of cellulases through enzyme engineering; and enhancing the complex enzyme cocktail composition through the addition of minority enzymes, optimizing enzyme ratios, and addition of other non-enzyme excipients [17]. Several studies have shown that the sugar yield from enzymatic hydrolysis is affected by factors such as the initial substrate concentration [18, 19]; the amount of lignin within the substrate [20, 21]; the presence of inhibitors such as furfural, hydroxymethyl furfural, acetic acid, and polyphenols produced during the pretreatment step [22–24]; and the concentration of hydrolyzed products such as glucose and cellobiose [25, 26]. Thus, lower concentrations of these compounds are beneficial for increased enzymatic conversion efficiencies. Another effective way to reduce the amount of fresh enzyme required is to recover and reuse the cellulases. A substantial amount of work has been carried out on the recovery and recycle of the enzyme cocktail. From these studies, it has been shown that the cellulase activity can be recovered from either the non-cellulosic solid residue remaining at the end of hydrolysis [20, 27, 28], the residual solid cellulosic substrate [29–32], or the liquid supernatant [27, 33–35] at the termination of the hydrolysis reactions. However, the yield of enzymatic activity is often very low (below 25 % of the originally added amount). Several strategies have been attempted to increase the yields of enzyme recovery following cellulose hydrolysis [21, 27, 36–40]. These methods usually use ultra-filtration, readsorption of free enzymes onto fresh substrate, and/or surfactant techniques to recover the cellulases from the supernatant along with enzymes bound to the residual substrate. Ultra-filtration appears to be a viable option for enzyme reclamation, with up to 66–75 % recovery [36–38]; however, the method is limited by its relatively slow processing time, the high cost, and the inevitable fouling of the filtration membranes, requiring frequent replacement [41, 68, 69]. Readsorption of free cellulases that remain in the liquid and/or solid phases after hydrolysis onto fresh substrate can recover 80 % of the residual liquid-phase enzymes [35, 39, 40]. Unfortunately, the increase in residual lignin content when added to fresh substrate and the accompanying viscosity elevation of the slurry in successive rounds of recycling affect the hydrolysis yields due to the high lignin content and ineffective mass transfer [42]. The addition of surfactants to desorb the enzymes associated with the solids can also increase the

Appl Biochem Biotechnol

hydrolysis yields up to 86 % [39]. However, such surfactants can lead to excessive foam formation during yeast fermentation and often require an expensive recovery step [43]. Recovery and recycling of cellulase by adsorption/desorption using ion exchange and other adsorption mechanisms are an attractive alternative method for its dual application of separating enzymes and inhibitory compounds. An ion exchange chromatography column has been extensively used for separation and purification of cellulase complexes by NaCl gradient elution [44–46]; on the other side, ion exchange resins have also been known to remove organic acids, aldehydes, and lignin-derived inhibitors [47–49]. Similarly, polyethylene imine (PEI), either alone or in combination with other separation technologies, has allowed for high-efficiency removal of organic acids, aldehydes, and phenolics [22, 50, 51]. The objective of the present study is to evaluate the effects of continuous or fed-batch enzyme addition at lower dosages along with the continuous removal of end product inhibitors (especially glucose and cellobiose) on the hydrolysis rate and yields. At the same time, it is also a primary goal to investigate the application of ion exchange resin to remove enzymatic and fermentation inhibitors within the continuous process along with the feasibility of recovering cellulolytic enzymes involved in the hydrolysis.

Materials and Methods Raw Material and Dilute Acid Pretreatment Ponderosa pine wood (∼35 % moisture) was kindly provided by Baker Timber (Rockerville, SD) in sawdust form. The sawdust was sieved to obtain a dust size of

Continuous enzymatic hydrolysis of lignocellulosic biomass with simultaneous detoxification and enzyme recovery.

Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical ...
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