Covalent immobilization of enzymes and yeast: Towards a continuous simultaneous saccharification and fermentation process for cellulosic ethanolby Clara T.H. Tran, Neil Nosworthy, Marcela M.M. Bilek, David R. McKenzie

Biomass and Bioenergy


Analysis of biomass cellulose in simultaneous saccharification and fermentation processes

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Article history:

Accepted 9 July 2015

Available online xxx

The immobilization of enzymes and yeast cells is a key factor for establishing a continuous process of tion process and a simultaneous saccharification and fermentation process. This paper investigates the matic catalysis, there are three common designs of processes: efficient in carrying out all of these processes are rare in Nature and in which the hydrolysis step occurs before fermentation. The separation of these two steps allows the optimum operation of each step to be achieved [20]. However, the separation of these two steps also causes the accumulation of hydrolysis products in the

Takagi et al. [19] proposed a SSF process in 1977 and since then, it nol production. In s and fermentation

The simultaneous e hydrolysis effiysis products that y enzymes. Ghosh y 13e30% by using ocess that allows glucose to accumulate. Ohgren et al. [14] also reported a 13% higher

However, some disadvantages of the SSF process have been reported in the literature. Ooshima et al. [16] found that when the ethanol concentration exceeded 0.2 M, it disturbed the adsorption of exoglucanase on cellulose and depressed the saccharification rate. In addition, the presence of cellulase enzyme mixtures in the same vessel significantly influences yeast growth [17]. Since the hydrolysis and fermentation steps have so far been carried out in* Corresponding author.

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Biomass and w.e

Biomass and Bioenergy 81 (2015) 234e241E-mail address: (C.T.H. Tran).must be created by genetic engineering [11]. SHF is a simple process yield of ethanol from the SSF process compared to SHF process.consolidated bioprocessing (CBP), separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). CBP is a process in which enzyme production, cellulose hydrolysis and fermentation are all accomplished by microorganisms [15]. Apart from having fermentation capability, the microorganisms selected for CBP must be capable of expressing enzymes for hydrolysing cellulose (i.e. cellulases) as well as enzymes for converting glucose into ethanol. Microorganisms that are has been widely used for studying cellulosic etha the SSF process of Takagi et al., enzyme hydrolysi occur at the same time and in the same vessel. saccharification and fermentation enhances th ciency by reducing the accumulation of hydrol would otherwise impede the activity of the ke et al. [7] reported an increase in hydrolysis rate b

SSF compared to a normal saccharification prIn ethanol production from cellulosic biomass based on enzy- To overcome the disadvantages of both of these processes,Keywords:

Cellulosic ethanol

Covalent immobilization

Plasma treatment

Continuous saccharification and fermentation 1. Introduction 0961-9534/? 2015 Elsevier Ltd. All rights reserved.soluble substrates such as cellobiose and carboxymethyl cellulose. The immobilization was achieved by incubating enzymes and yeast cells on polystyrene surfaces which had been treated by nitrogen ion implantation. The saccharification by immobilized enzymes and the fermentation by immobilized yeast cells were conducted in two separate vessels connected by a pump. During the experiments, glucose concentrations were always maintained at low levels which potentially reduce product inhibition effects on the enzymes. Covalent immobilization of enzymes and yeast cells on the plasma treated polymer reduces loss by shear flow induced detachment. The potential for continuous flow production of ethanol and the influence of daughter yeast cells in the circulating flow on the immobilized enzyme activity are discussed. ? 2015 Elsevier Ltd. All rights reserved. hydrolysis vessel which inhibit enzyme activity, consequently reducing the reaction rate over time [1].Received in revised form 19 May 2015 use of cellulase enzyme and yeast cell immobilization under a flow regime of ethanol production fromReceived 18 August 2014 cellulosic ethanol production, which can combine the benefits of a separated hydrolysis and fermenta-Research paper

Covalent immobilization of enzymes and simultaneous saccharification and ferme ethanol

Clara T.H. Tran*, Neil Nosworthy, Marcela M.M. Bile

Applied and Plasma Physics (A28), University of Sydney, Sydney, NSW 2006, Australia a r t i c l e i n f o a b s t r a c t journal homepage: http : / /wweast: Towards a continuous tation process for cellulosic

David R. McKenzie

Bioenergy lsevier .com/locate/biombioe the same vessel, the process is often operated in the temperature range between 30 and 40 C due to the restricted thermostability of yeast. Removing this constraint by separating the yeast from the enzyme processes in separate vessels would enable the use thermophilic enzymes to achieve higher activities by allowing operation at higher temperatures. Here we report on the operation of such a process in which the yeast and enzyme reaction steps are carried out in separated zones, while allowing a continuous flow process to be operated.

The proposed separated continuous simultaneous saccharification and fermentation (continuous SSF) process is shown in Fig.1. In this process, cellulosic biomass is continuously pre-treated and pumped to the hydrolysis vessel where a mixture of cellulases converts it into fermentable sugars. The fermentation substrates are pumped continuously to the fermentation vessel where the sugars are fermented to produce ethanol. Ethanol is continuously separated from the bulk by an ethanol selective membrane or continuous distillation. The remaining liquids, consisting of water and ethanol with incompletely reacted substrates, are recycled to the hydrolysis tank, and combinedwith newly pre-treated biomass, for further hydrolysis. The use of a circulating flow of the liquid medium allows the separation of saccharification and fermentation into two vessels while the concentrations of sugars and ethanol can be maintained stable over time, hence reducing the end product inhibition and enabling the optimum temperature to be used for each step. The activities of the enzymes used in hydrolysis and the