Biochemical Engineering Journal 54 (2011) 141–150 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Ultrasound-assisted fermentation enhances bioethanol productivity Ahmad Ziad Sulaiman a,b , Azilah Ajit a,b , Rosli Mohd Yunus b , Yusuf Chisti a,∗ a School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand b Faculty of Chemical Engineering and Natural Resources, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Kuantan, Pahang, Malaysia article info Article history: Received 5 June 2010 Received in revised form 20 December 2010 Accepted 21 January 2011 Available online 24 February 2011 Keywords: Sonobioreactors Ultrasound Kluyveromyces marxianus -galactosidase Bioethanol Ethanol Fermentation abstract Production of ethanol from lactose by fermentation with the yeast Kluyveromyces marxianus (ATCC 46537) under various sonication regimens is reported. Batch fermentations were carried out at low-intensity sonication (11.8 W cm -2 sonication intensity at the sonotrode tip) using 10%, 20% and 40% duty cycles. (A duty cycle of 10%, for example, was equivalent to sonication for 1 s followed by a rest period (no sonication) of 10 s.) Fermentations were carried out in a 7.5 L (3 L working volume) stirred bioreactor. The sonotrode was mounted in an external chamber and the fermentation broth was continuously recirculated between the bioreactor and the sonication chamber. The flow rate through the sonication loop was 0.2 L min -1 . All duty cycles tested improved ethanol production relative to control (no sonication). A 20% duty cycle appeared to be optimal. With this cycle, a final ethanol concentration of 5.20 ± 0.68 g L -1 was obtained, or nearly 3.5-fold that of the control fermentation. Sonication at 10% and 20% cycles appeared to stimulate yeast growth compared to the control fermentation, but 40% duty cycle had a measureable adverse impact on cell growth. Sonication at 10% and 20% cycles enhanced both the extracellular and the intracellular levels of -galactosidase enzyme. Although at the highest duty cycle sonication reduced cell growth, cell viability remained at ≥70% during most of the fermentation. Sonication at a controlled temperature can be used to substantially enhance productivity of bioethanol fermentations. © 2011 Elsevier B.V. All rights reserved. 1. Introduction This study is concerned with the ultrasound-induced enhance- ment of the production of bioethanol from lactose using the yeast Kluyveromyces marxianus. Ultrasound, or sound of frequency ≥20 kHz, is generally asso- ciated with damage to cells and is widely used in laboratory protocols for breaking cell walls to release intracellular products [1]. Enzymes and other fragile macromolecules are known to be susceptible to damage by ultrasound [2]. Nevertheless, suitably applied ultrasound has the potential for enhancing the productivity of bioprocesses involving live cells and bioactive enzymes [3–10]. Effects of sonication for productivity enhancement have been previously reported for certain bacteria [3,5,6,11–16], filamentous fungi [7,8,17] and plant cells [18]. Bakers’ yeast (Saccharomyces cerevisiae) appears to have been the only yeast that has been assessed to some level in ultrasound irradiated fermentations [19–22]. Prior work on sonicated fermentations for producing bioethanol is pertinent to this study and is therefore reviewed here briefly. Nearly all such work focused on the yeast S. cerevisiae. Ultrasound intensity that is otherwise nonlethal to S. cerevisiae, appears to ∗ Corresponding author. Tel.: +64 6 350 5934; fax: +64 6 350 5604. E-mail address: y.chisti@massey.ac.nz (Y. Chisti). affect the integrity of the cell vacuole and rearrange the intracellu- lar contents [23]. The relatively low power diagnostic ultrasound of the frequency range 1–10 MHz is generally considered less dam- aging to cells than the power ultrasound (frequency range of 20–100 kHz); nevertheless, 2.2 MHz ultrasound applied continu- ously at an electrical power input of 14 W to a broth volume of 64 mL killed 25% of the S. cerevisiae cells exposed for 60 min [23]. Continuous sonication at 1 MHz and 10.5 W cm -2 has inhibited S. cerevisiae fermentation, but intermittent sonication at the same intensity was less damaging [19]. In production of wine, beer and sake from soluble sugars using immobilized cells of S. cerevisiae, extremely low intensity sonica- tion at 0.3 mW cm -2 and 43 kHz stimulated the fermentation to reduce the fermentation time to 50–64% [20]. Ultrasound (20 kHz) used at intensities of 0.2, 0.4 and 0.8 W cm -2 was claimed to accelerate the growth of S. cerevisiae in a medium that contained only dissolved nutrients [22], but the data did not clearly support this claim. Marginal improvements to S. cerevisiae growth were observed on controlled exposure to power ultrasound by Lanchun et al. [21]. Some bioethanol fermentations require pretreatment of the substrate. In pretreatment of starch, sonication in the absence of enzymes and microorganisms has been repeatedly shown to enhance the yield of fermentable sugars [24–26] and thereby increase the ethanol yield in a subsequent nonsonicated fermen- tation. This effect is of course a purely physical consequence 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.01.006