Accepted Manuscript

Title: Ultrasound-assisted fermentation enhances bioethanol

productivity

Authors: Ahmad Ziad Sulaiman, Azilah Ajit, Rosli Mohd

Yunus, Yusuf Chisti

PII: S1369-703X(11)00020-9
DOI: doi:10.1016/j.bej.2011.01.006
Reference: BEJ 5269

To appear in: Biochemical Engineering Journal

Received date: 5-6-2010

Revised date: 20-12-2010
Accepted date: 21-1-2011

Please cite this article as: A.Z. Sulaiman, A. Ajit, R.M. Yunus, Y. Chisti, Ultrasound-
assisted fermentation enhances bioethanol productivity, Biochemical Engineering
Journal (2010), doi:10.1016/j.bej.2011.01.006

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 1

Ultrasound-assisted fermentation enhances bioethanol productivity

Ahmad Ziad Sulaiman,1,2 Azilah Ajit1,2, Rosli Mohd Yunus2, Yusuf Chisti1,*

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1
School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New

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Zealand

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2
Faculty of Chemical Engineering and Natural Resources, Universiti Malaysia Pahang,

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Lebuhraya Tun Razak, 26300 Kuantan, Pahang, Malaysia

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Correspondence to: Yusuf Chisti, School of Engineering, Massey University, Private Bag 11

222, Palmerston North, New Zealand

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Keywords: Sonobioreactors; ultrasound; Kluyveromyces marxianus; β-galactosidase;

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bioethanol; ethanol; fermentation

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 Macro Kinetic model screening for water gas shift reaction carried out with CFD

 The results are validated against experimental literature data

 Langmuir Hinshelwood model have been found to better predict the CO conversion

Page 1 of 40
 2

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)

t
using 10%, 20% and 40% duty cycles. (A duty cycle of 10%, for example, was equivalent to

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sonication for 1 s followed by a rest period (no sonication) of 10 s.) Fermentations were

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carried out in a 7.5 liter (3 L working volume) stirred bioreactor. The sonotrode was mounted

in an external chamber and the fermentation broth was continuously recirculated between the

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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).

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A 20% duty cycle appeared to be optimal. With this cycle, a final ethanol concentration of
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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
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fermentation, but 40% duty cycle had a measureable adverse impact on cell growth.
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Sonication at 10% and 20% cycles enhanced both the extracellular and the intracellular levels

of β-galactosidase enzyme. Although at the highest duty cycle sonication reduced cell growth,
p

cell viability remained at 70% during most of the fermentation. Sonication at a controlled
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temperature can be used to substantially enhance productivity of bioethanol fermentations.

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Page 2 of 40
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1. Introduction

This study is concerned with the ultrasound-induced enhancement of the production of

bioethanol from lactose using the yeast Kluyveromyces marxianus.

Ultrasound, or sound of frequency 20 kHz, is generally associated with damage to

t
cells and is widely used in laboratory protocols for breaking cell walls to release intracellular

ip
products [1]. Enzymes and other fragile macromolecules are known to be susceptible to

cr
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].

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Effects of sonication for productivity enhancement have been previously reported for

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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
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to some level in ultrasound irradiated fermentations [19–22].

Prior work on sonicated fermentations for producing bioethanol is pertinent to this

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study and is therefore reviewed here briefly. Nearly all such work focused on the yeast S.
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cerevisiae. Ultrasound intensity that is otherwise nonlethal to S. cerevisiae, appears to affect

the integrity of the cell vacuole and rearrange the intracellular contents [23]. The relatively
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low power diagnostic ultrasound of the frequency range 1–10 MHz is generally considered
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less damaging to cells than the power ultrasound (frequency range of 20–100 kHz);

nevertheless, 2.2 MHz ultrasound applied continuously at an electrical power input of 14 W to

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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 sonication 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

Page 3 of 40
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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].

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Some bioethanol fermentations require pretreatment of the substrate. In pretreatment

ip
of starch, sonication in the absence of enzymes and microorganisms has been repeatedly

cr
shown to enhance the yield of fermentable sugars [24–26] and thereby increase the ethanol

yield in a subsequent nonsonicated fermentation. This effect is of course a purely physical

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consequence of the sonication-induced rupture of the starch granules and does not involve any

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biological activity. Similar phenomena have been observed in bacterial fermentations for

producing ethanol. For example, a 20% enhancement in ethanol yield was reported by
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intermittent sonication of a paper pulp slurry being enzymatically hydrolyzed and fermented

in a combined saccharification-fermentation process that used the bacterium Klebsiella

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oxytoca [14]. Productivity enhancements have been claimed by sonication in some other S.
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cerevisiae fermentations [27]. Power ultrasound has been claimed to enhance the permeability

of S. cerevisiae cell to proteases [28] and Ca2+ [29].

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The present study used the well known yeast Kluyveromyces marxianus as a model
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system to investigate the sonication regimens that may be used to enhance cell growth and

ethanol production from lactose, a completely soluble substrate. K. marxianus has been
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formerly referred to as Kluyveromyces fragilis [30–32]. K. marxianus has been widely used to

produce ethanol from lactose-containing media [31–40], but in conventional nonsonicated

fermentations.

2. Materials and Methods

2.1 Microorganism, maintenance and preparation

Page 4 of 40
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Kluyveromyces marxianus ATCC 46537 was obtained from the American Type Culture

Collection, USA (www.atcc.org). The yeast was supplied as a freeze-dried powder in a glass

vial. The cell were rehydrated in sterile YM broth, incubated at 30 °C for 24-h and then

inoculated on agar slants. After a further incubation period (30 C, 24 h), the slants were

t
stored at 4 C. The maintenance agar medium was made using deionized water and had the

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following composition [31] (g L1): lactose 50; yeast extract 2; (NH4)2SO4 6.25;

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MgSO47H2O 2; KH2PO4 4; and agar 15. The medium was sterilized by autoclaving (121 C,

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15-min). The slants were kept at 4 °C and subcultured every 2-months. This stock culture was

used for inoculum preparation throughout this study.

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Agar plates were prepared from slants in the usual way. Seed cultures were prepared

by inoculating a single colony from an agar plate into 80 mL of a sterile medium contained in
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a 250 mL shake flask. The medium was as described above, but without the agar, and had

been sterilized as mentioned above. The culture was incubated (30 C) in an orbital shaking
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incubator (180 rpm) for 24 h. This culture (50 mL) was used to inoculate 150 mL of the
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earlier specified sterile medium contained in a 1000 mL shake flask. The flask was incubated

as specified above. After the specified incubation period, the inoculum had a
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spectrophotometric absorbance of 0.7 at 620 nm (Ultraspec 2000, model 80-2106-00

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spectrophotometer; Pharmacia Biotech Inc., Piscataway, NJ, USA) and contained 4107
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cells mL1. All subsequent fermentations were inoculated using the above inoculum at a level

of 5% by volume.

2.2 Bioreactor fermentations and ultrasound equipment

A 7.5-L stirred bioreactor (BIOFLO 110 New Brunswick Scientific, East Brunswick, NJ,

USA, www.nbsc.com) was used (Figure 1). The working volume was 3-L. The internal

diameter of the jacketed glass bioreactor vessel was 0.18 m. The vessel was fully baffled with

Page 5 of 40
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4 vertical baffles spaced equidistance around the periphery. The baffle width was 19 mm. A

central shaft supported two 6-bladed Rushton disc turbine agitators. The agitators were

identical with a diameter of 59.6 mm and were spaced 0.15 m apart on the shaft. The lower

agitator was located 59.6 mm above the bottom of the vessel. A single hole sparger was used

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for aeration. The sparger hole diameter was 4.3 mm and it was located directly below the

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lower agitator, about 30 mm above the base of the vessel.

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All fermentations were run as aseptic aerobic batch cultures. The air inlet and exhaust

ports on the bioreactor were installed with sterile hydrophobic membrane filters (0.2 m;

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either Sartorious, Gottingen, Germany, or Millipore, Bedford, MA, USA). The assembled

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bioreactor filled with the earlier specified liquid medium was autoclaved (121 C, 20 min)

with the pH and the dissolved oxygen electrodes installed. The pH electrode (Ingold gel-filled
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electrode, model no. 465-35-SC-P-K9/270/9848; Mettler-Toledo, www.mt.com) had been

calibrated using pH 7.0 and pH 4.0 buffers prior to autoclaving.

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The concentration of dissolved oxygen (DO) in the broth was measured online using a
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polarographic electrode (model In Pro 6800 sensor 12/25 mm; Mettler-Toledo,

www.mt.com). The DO electrode had been calibrated at 30 C in the sterilized culture

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medium. For the calibration, the liquid medium was first bubbled with nitrogen until the
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dissolved oxygen reading failed to decline further. The DO readout was then adjusted to read

0%. Nitrogen flow was then replaced with a preset air flow of 2.67 vvm, with the impeller
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rotating at 500 rpm. Once the measured concentration of dissolved oxygen had stabilized, it

was adjusted to an air saturation value of 100%.

A 20 kHz, 600 W maximum power, Misonix Sonicator 3000 (Misonix, Inc.,

Farmingdale, NY, USA, www.misonix.com) ultrasound generator was used in combination

with a standard tapped sonic horn (Misonix, Inc., part no. 200 with 12.7 mm tip diameter, 127

mm length), or sonotrode, installed in an external 800B Misonix Flocell with a 3.175 mm

Page 6 of 40
 7

diameter inlet orifice (Figure 2). The horn had a replaceable flat tip made of titanium alloy

(Misonix, Inc., part no. 406). The flow cell, with the sonic horn in place, was autoclaved (121

C, 20 min), cooled to room temperature, and connected to the bioreactor aseptically using

sterile silicone tubing as shown in Figure 1. The broth from the bioreactor was recirculated

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continuously through the sonic chamber using a peristaltic pump (Masterflex model no. 7554-

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60; Cole Parmer Instrument Co., Chicago, IL, USA). The recirculation flow rate was fixed at

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0.2 L min1. The recirculation commenced after the fermenter had been inoculated and briefly

mixed. All fermentations were carried out with recirculation of the broth through the sonic

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chamber, but ultrasound was not applied to the control fermentation.

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The sterile bioreactor was inoculated with 150 mL (5% by vol) of the earlier specified

inoculum. The final volume of the broth in the fermenter after inoculation was 3,150 mL. The
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fermentation temperature was controlled at 30.00.2 °C. The agitation speed and aeration rate

were maintained at 500 rpm and 2.67 vvm, respectively. The pH and the dissolved oxygen
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concentration were monitored, but not controlled. Sterile (121 C, 15 min) antifoam emulsion
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(catalog no. A 6426-100G, 10 g/100 mL of water; Sigma-Aldrich, St. Louis, MO, USA) was

added to the fermenter in response to a foam sensor to automatically suppress severe foaming.
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Each batch fermentation was run for 24 hours. Samples were taken periodically. The optical
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density and the cell viability were measured immediately after sampling, as specified later in
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this paper. For the other measurements, the samples were centrifuged at 2000 g for 10-min

(model 0008931 centrifuge; Eppendorf AG, Germany, www.eppendorf.com) immediately

after collection and the supernatant was stored at 4 °C for further analysis. The storage period

did not exceed 3 days.

2.3 Sonobioreactor fermentations

Page 7 of 40
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For ultrasound-assisted fermentations, the ultrasound power level could be varied by adjusting

the amplitude setting of the sonotrode and the cumulative average ultrasound dose could be

varied by adjusting the duty cycle. The amplitude was set at position 2 to correspond to a

power input P of 15 W, or a sonication intensity I of 11.8 W cm2. The sonication intensity

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was calculated using the following equation:

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P
I (1)

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A

where A (cm2) was the area of the sonotrode tip. The A value was 1.27 cm2.

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The cumulative sonic energy imparted to the fluid depended on the duty cycle of

sonication. The duty cycle determined the proportion of the time that the sonication was “on”.

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A duty cycle of 10% was equivalent to sonication for 1 s followed by a rest period (no

sonication) of 10 s. A sonication duty cycle of 100% meant uninterrupted sonication. The

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time units of seconds were used in setting the duty cycle. Duty cycles of 10%, 20% (1 s

sonication, 5 s rest period) and 40% (2 s sonication, 5 s rest period) were used.
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2.4 Analyses
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2.4.1 Biomass concentration

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Biomass concentration was determined by measuring the optical density of the fermentation

broth at 620 nm (A620) with a spectrophotometer (Ultraspec 2000, model 80-2106-00;

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Pharmacia Biotech Inc., Piscataway, NJ, USA) against a blank of sterile medium. A 1 mL

sample of the broth was diluted with 24 mL of the sterile medium prior to measurement. This

way the spectrophotometric absorbance was always 0.7. A calibration curve was used to

convert the optical density data to the dry biomass concentration. The equation of the

calibration curve was the following:

A620
Dry biomass concentration (g/L)  (2)
6.95  10 2

Page 8 of 40
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2.4.2 Lactose concentration

Lactose concentration was estimated using a modified dinitrosalicylic acid (DNS) method

based on Miller [41]. Thus, a 1% (w/v) solution of DNS reagent was prepared by dissolving

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10 g DNS and 2 g of phenol in 1000 mL of a solution of sodium hydroxide (10 g L1) and

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sodium sulfite (0.5 g L1). The broth supernatant sample containing lactose was appropriately

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diluted with deionized water. The diluted sample (3 mL) was mixed with 3 mL of DNS

reagent and heated for 15 min on a boiling water bath. One milliliter of Rochelle salt solution

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(potassium-sodium tartrate, 400 g L1) was added and the resulting mixture was cooled to

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ambient temperature in a cold water bath. The absorbance of the cooled solution was

measured at 575 nm (Ultraspec 2000, model 80-2106-00 spectrophotometer; Pharmacia

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Biotech Inc., Piscataway, NJ, USA) against a blank that had been prepared using deionized

water instead of the sample. The absorbance was converted to lactose concentration using a
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standard curve. The standard curve had been prepared using lactose solutions of known
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concentrations. The equation of the standard curve was the following:

A575
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Lactose concentration ( g/mL)  (3)

5.2  10 3
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where A575 was the spectrophotometric absorbance at 575 nm. The above equation applied to

an absorbance range of 0 to 0.7.

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2.4.3 Ethanol concentration

Ethanol concentration in the broth supernatant was determined using gas chromatography

(model GC 6000 Vega Series 2; Carlo Erba Instruments, Milan, Italy) fitted with a flame

ionization detector and chromato-integrator (model D-2500; Hitachi, Tokyo, Japan). The

carrier gas was nitrogen at a flow rate of 40 mL min1. The column temperature was 200 C.

Page 9 of 40
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Standard ethanol solutions were prepared in the concentration range of 2 to 8 g L1 by diluting

absolute ethanol with deionized water. The sample volume injected was 2 μL. The sample had

been prefiltered through a 0.45 m membrane filter. The ethanol concentration of the culture

supernatant sample was calculated by measuring the relative area under the ethanol peak and

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comparing it with the standard curve prepared using the standard solutions.

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2.4.4 Cell viability

Cell viability was determined using the methylene blue staining method [42]. A 10 μL aliquot

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of serially diluted freshly sampled yeast broth was mixed with of 10 μL of a methylene blue

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solution and incubated for 5 min [42]. The cell suspension was then counted on a

hemacytometer at 400 magnification. The viability was calculated as the ratio of the
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unstained cell count and the total count. In prior unpublished work, this method had been

rigorously validated for K. marxianus using the highly reliable but cumbersome colony
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forming unit counts on Petri dishes.

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2.4.5 Activity of -galactosidase

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Activity of the extracellular β-galactosidase was measured in the cell-free culture supernatant
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as specified in the Sigma enzymatic assay for β-galactosidase [43]. The activity was
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determined using the synthetic substrate o-nitrophenyl-β-D-galactopyranoside, ONPG

(catalog no. N1127-25G; Sigma-Aldrich, St. Louis, MO, USA). One unit of β-galactosidase

activity was defined as the amount of the enzyme that liberated 1.0 μmol of o-nitrophenol

from 5 mM ONPG per minute at pH 3.5 and 25 °C.

The intracellular β-galactosidase activity was measured according to the method

described by Wang and Sakakibara [13]. A 35 mL sample of the broth was centrifuged

(3300 g, 10-min) to recover the cells. The cells were washed (235 mL) with 0.1 M

Page 10 of 40
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phosphate buffer, pH 6.5. The washed cells were resuspended in 35 mL of deionized water

using a vortex mixer. The suspension was cooled in an ice-water bath at 4 °C and sonicated at

550 W, 20 kHz, for 30 s (Misonix Sonicator 3000, Misonix, Inc., Farmingdale, NY, USA).

The sonicated suspension was centrifuged (12000 g, 30-min; Hitachi CR-22GII refrigerated

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centrifuge, Hitachi Koki Co., Ltd., Tokyo, Japan) at 4 °C. The supernatant was collected and

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analyzed in accordance with the procedure given above for the determination of the

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extracellular β-galactosidase activity.

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3. Results and Discussion

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3.1 Baseline determination (Nonsonicated batch fermentation)

The results of duplicate nonsonicated batch fermentations are shown in Figure 3 as baseline
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data for comparison with the sonicated fermentations. The fermentation was essentially

complete by 24 h (Figure 3). The biomass growth, the ethanol production and lactose
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consumption profiles are consistent with expectations for an aerated fermentation. The error
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bars in Figure 3 demonstrate a good reproducibility of the fermentations. The baseline

fermentation kinetic parameters determined from Figure 3 are compared later (Table 1) with
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those of the sonicated fermentations.

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3.2 Effects of ultrasound

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Sonication at 11.8 W cm2 and the specified duty cycle commenced 9.5 h after inoculation of

a batch fermentation. The profiles of biomass growth, lactose consumption and the dissolved

oxygen concentration are shown in Figure 4 in comparison to controls. All the profiles were

comparable prior to the beginning of sonication. Sonication at duty cycles of 10% and 20%

substantially improved the biomass growth rate and final concentration relative to control, but

increasing the duty cycle to 40% adversely affected the growth rate and the final biomass

Page 11 of 40
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concentration (Figure 4a). The reduced biomass growth and final concentration at the highest

duty cycle were clearly reflected in a slower rate of lactose consumption and a higher

concentration of the residual lactose for this fermentation (Figure 4b). Lactose consumption of

the fermentations conducted at duty cycles of 10 and 20% was comparable to that of the

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control (Figure 4b).

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The adverse effect of sonication at 40% duty cycle was reflected also in the dissolved

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oxygen concentration profiles (Figure 4c). Thus, at the 40% duty cycle, because of a reduced

rate of consumption of lactose, the decline in the dissolved oxygen concentration during

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exponential growth was less than for the other fermentations and the oxygen concentration

recovered earlier (Figure 4c) suggesting an earlier end to exponential growth even though

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plenty of lactose remained in the broth. Clearly, even at a relatively high intensity of 11.8 W

cm2, ultrasound stimulated growth of K. marxianus on a soluble substrate so long as the duty
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cycle was appropriately selected. Each sonication event had to be followed by a recovery
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period of no sonication to prevent adverse impact on the yeast. No other work has been
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reported on sonication of K. marxianus, but continuous sonication of S. cerevisiae with

diagnostic ultrasound (1 MHz) at a lower intensity (10.5 W cm2) than used by us, has proved
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to be inhibitory [19] while intermittent sonication was less damaging.

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The effects of pulsed sonication on ethanol production are shown in Figure 5 in

comparison to the control fermentation. All duty cycles tested improved ethanol production
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relative to control, but the duty cycles of 10% and 20% were clearly the most effective. With

the best duty cycle of 20%, the final ethanol concentration of 5.200.68 g L1 was nearly 3.5-

fold that of the control fermentation. For this sonication regimen, the ethanol yield on lactose

was 0.109 g g1 compared to a yield of 0.034 g g1 for the control culture. The ethanol

productivity of the culture sonicated at a duty cycle of 20% was 3.5-fold greater than for the

control.

Page 12 of 40
 13

Ultrasonication is known to improve interfacial mass transfer. Mass transfer

enhancements have been attained at power intensities as low as 2.2 W cm2 [3]. Therefore, a

plausible improved gas-liquid mass transfer of oxygen as a consequence of sonication [44]

may potentially explain the observed increase in the concentration of the biomass (Figure 4a)

t
relative to control; however, it does not explain the increased concentration of ethanol (Figure

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5) that is normally produced optimally under conditions of a low dissolved oxygen

cr
concentration [32]. In the present study, the dissolved oxygen concentration did not drop to

much less than 20% of air saturation as shown in Figure 4c.

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Improved production of ethanol (Figure 5) must therefore have a different explanation.

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One of the products of the fermentation is carbon dioxide. Elevated concentrations of

dissolved carbon dioxide are known to inhibit S. cerevisiae [45, 46] and have a similar effect
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on K. marxianus [32]. Improved gas-liquid mass transfer may have contributed to improved

removal of the highly soluble carbon dioxide from the broth to enhance the ethanol
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productivity relative to control. Rapid desorption of carbon dioxide from a fermentation broth
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commonly produces foaming, as it does in a glass of beer. The fermentation broth was indeed

observed to foam within minutes of commencing sonication as shown Figure 6. At the recycle
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rate used, nearly 63% of the broth in the bioreactor had passed through the sonication
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chamber at least once by 10-min when the picture (Figure 6b) was taken. Foaming may also

be attributed to release of intracellular proteins, but up to a sonication duty cycle of 20%

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biomass growth was in fact better than in the control culture (Figure 4a), suggesting little or

no cell lysis. No distinct pH changes attributable to a possible change in the concentration of

dissolved carbon dioxide could be observed. The pH values for the different sonciation

regimens were generally within 0.2 pH units of the measured value (Figure 7).

The kinetic parameters for the various fermentations are compared in Table 1. The

equations used in calculating the parameters [47] were as follows:

Page 13 of 40
 14

Specific growth rate, 

1 X
 ln 2 (4)
t2  t1  X 1

t
where X1 is the biomass concentration at time t1 (= 8 h) and X2 is the biomass concentration at

ip
time t2 (= 14 h) during exponential growth.

cr
Average specific lactose consumption rate, qs

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S
qs   (5)
Xt

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where S is the substrate consumed by time t (= 22 h) and X is the increase in biomass

concentration by time t.
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Maximum biomass yield on substrate, Yx/s
d

X
Yx / s   (6)
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S

where Yx/s is calculated at the instance of the maximum biomass concentration Xmax.
p
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Maximum biomass productivity, Px

X max  X 0
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Px  (7)
t

where Px is calculated at the instance t of the maximum biomass concentration in the

fermentation. In Eq. (7), X0 is the biomass concentration at the begining of the fermentation.

Final ethanol yield on substrate, Yp/s

P
Yp / s   (8)
S

Page 14 of 40
 15

where P is the change in ethanol concentration during the fermentation.

Final ethanol productivity, PE

E f  E0
PE  (9)
tf

t
ip
where E0 is the initial concentration of ethanol, Ef is the final concentration of ethanol and tf is

cr
the duration of the fermentation.

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Average specific ethanol production rate, qp

E

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qp  (10)
X maxt

where qp is calculated at the instance t of the maximum biomass concentration. In Eq. (10) E
M
is the increase in ethanol concentration by time t during the fermentation.
d

Under the best sonication regimen of a 20% duty cycle, the sonicated fermentation
te

was substantially superior to the control culture (Table 1). For example, compared to control,
p

the biomass yield on lactose was 33% greater for the sonicated culture; the maximum biomass
ce

concentration was 42% greater; the maximum biomass productivity was 57% greater; the

final ethanol yield on lactose was 3-fold greater; the final ethanol concentration was 3.5-fold
Ac

greater; and the final ethanol productivity was 3.5-fold greater (Table 1).

Cell viability profiles for the fermentations are shown in Figure 8. Prior to the

beginning of sonication at 9.5 h, the cell viability in all fermentations exceeded >90%, but in

all cases, the viability continuously declined as the fermentations progressed. For the control

culture, this decline could be explained by a progressive accumulation of ethanol, a well

known inhibitor of yeasts [48, 49] including K. marxianus [32]. The beginning of the viability

decline (Figure 8) coincided with the instance of the rapid increase in ethanol concentration

Page 15 of 40
 16

around 9.5 h (Figure 3). The viability decline of the sonicated cultures was also due to

accumulation of ethanol (Figure 5), but sonication appears to have been an additional

contributing factor. Thus, at any instance after the sonication began, the viability was

progressively reduced with the increasing value of the duty cycle of sonication (Figure 8).

t
Although sonication enhanced the viability decline, by the end of the fermentation >65% of

ip
the yeast cells were still viable in the culture that was sonicated at a duty cycle of 40% (Figure

cr
8). Ethanol is known to affect the structure of cell membranes [49] and this likely explained

the increased susceptibility of cells to ultrasound once the ethanol concentration had

us
increased.

Under certain conditions, ultrasound is known to affect the morphology of cells

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without causing a loss in viability [23, 16, 17]. Therefore, the cell morphology was examined
M
photographically at 22 h of various fermentations (Figure 9). By this time the yeast broth had

passed through the sonication chamber 50 times. Compared to nonsonicated culture (Figure
d

9a), no morphological changes were discerned in cells sonicated at 10 and 20% duty cycles
te

(Figure 9b, 9c). However, the culture that had been sonicated at the 40% duty cycle contained

many ghost cells (i.e. cells that had lost most or all of their contents) and cells with clearly
p

broken envelopes (Figure 9d). This concurred with the lower biomass concentration (Figure
ce

4a) and cell viability (Figure 8) in this fermentation, as discussed earlier.

Transport of lactose into cells of K. marxianus is mediated by a lactose permease [32].

Ac

Once internalized, the lactose is hydrolyzed by -galactosidase and the resulting glucose and

galactose are metabolized by separate biochemical pathways [32]. As most of the lactose is

hydrolyzed intracellularly, most of the -galactosidase activity resides within the cells. The

observed sonication-dependent changes in growth metabolism and ethanol production may be

potentially linked to possible effects of sonication on the enzyme -galactosidase.

Page 16 of 40
 17

Considering this, the activity of the intercellular and extracellular -galactosidase was

measured in the various fermentations (Figure 10).

Until the beginning of sonication at 9.5 h, the profiles for all fermentations were

identical for both the extracellular and the intracellular enzyme activity (Figure 10).

t
Irrespective of the fermentation, the extracellular enzyme activity was relatively small

ip
compared to the intracellular activity at any given instance (Figure 10), as expected. The

cr
extracellular -galactosidase was a consequence of either cell leakage or an ongoing lysis of a

small fraction of the growing cell population. Sonication at 10 and 20% duty cycles appears to

us
have stimulated the production of the enzyme inside the cells relative to control (Figure 10b),

an
whereas sonication at the 40% duty cycle appears to have suppressed enzyme synthesis. In

fact these apparent effects are entirely explained by the differences in the biomass
M
concentrations of the various fermentations (Figure 4a) and not by any direct effect of

sonication on the production or release of the enzyme. This is confirmed in Figure 11 where
d

the measured extracellular and intracellular activities of -galactosidase are plotted per unit of
te

dry cell mass present at any given instance during fermentation. From 9.5 h onwards, all the

sonicated cultures had nearly the same biomass specific enzyme activity as did the control
p

culture. Therefore, sonication had no effect at all on production or release of -galactosidase.

ce

During exponential growth, i.e. prior to 9.5 h, the biomass always had a much higher enzyme

activity than later in the fermentation. This was likely because production of -galactosidase
Ac

was up regulated during rapid growth that demands a rapid hydrolysis of lactose to feed the

resulting sugars into the energy consuming metabolic pathways.

For the experimental system used, the bioreactor could always be considered to be

well mixed. This could be readily demonstrated by comparing the mixing time in the

bioreactor with the residence time of the recycle flow in the reactor. Thus, the residence time

tR of the recycle stream was calculated as follows:

Page 17 of 40
 18

VL
tR  (11)
QL

where VL is the working volume (3 L) in the bioreactor and QL is the previously specified

recycle flow rate. The residence time was always 15 min. The mixing time in the bioreactor

was calculated using the following equation [50]:

t
ip
 ln1   
t  2.17 0.5
(12)
D T 

cr
1.06 N    
T  H

where t is the time required to attain a fractional homogeneity of  (e.g. a -value of 0.99 is

us
equivalent to 99% of the fully mixed state), N is the rotational speed of the impeller, D is the

an
diameter of the impeller, T is the diameter of the mixing vessel and H is the depth of fluid in

the tank. For the earlier specified bioreactor geometry and H = T, the mixing time for
M
attaining a 99% homogeneity was found to be 0.096 min. Thus, the residence time in the

bioreactor was nearly 150-fold greater than the time required for mixing.
d
te

4. Concluding remarks

Intermittent sonication with power ultrasound (20 kHz) at duty cycles of 20% stimulated
p

biomass production, lactose metabolism and ethanol production in K. marxianus at a

ce

relatively high sonication intensity of 11.8 W cm2. Increasing the duty cycle to 40% had a
Ac

clear adverse impact on the yeast. Under the best conditions, sonication enhanced the final

ethanol concentration by nearly 3.5-fold relative to control. This corresponded to a 3.5-fold

enhancement in ethanol productivity, but required 952 W of additional power input per cubic

meter of broth through sonication. This additional requirement for energy was certainly within

acceptable operational norms for bioreactors and, for high value products, could be easily

compensated by the increased productivity. In view of the potential benefits of sonication and

Page 18 of 40
 19

its cost effectiveness in some processes, a wider investigation of its applications in

biotechnology based processing is warranted.

5. Acknowledgements

t
This research was made possible with a scholarship from the Ministry of Higher Education,

ip
Malaysia, and support from Universiti Malaysia Pahang (UMP).

cr
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Figure captions

Figure 1. Ultrasound assisted batch fermentation system.

Figure 2. The ultrasonic flow cell. Dimensions in mm.

Figure 3. A typical control fermentation profile.

t
Figure 4. Effects of sonication on: (a) biomass concentration; (b) lactose concentration; and

ip
(c) dissolved oxygen concentration. Except for the nonsonicated control, the sonication

cr
intensity was 11.8 W cm2.

Figure 5. Ethanol concentration profiles. The sonication intensity was 11.8 W cm2 except for

us
the nonsonicated control culture.

an
Figure 6. Foaming behavior of the fermentation: (a) just before sonication commenced 9.5 h

after inoculation; (b) the same fermentation 10 min after sonication commenced at a power
M
intensity of 11.8 W cm2 and a duty cycle of 20%.

Figure 7. The pH profiles. The sonication intensity was 11.8 W cm2 except for the
d

nonsonicated control culture.

te

Figure 8. Cell viability profiles. The sonication intensity was 11.8 W cm2 except for the

nonsonicated control culture.

p

Figure 9. Yeast cell morphology (1000 magnification) at 22 h of fermentation: (a) control (no
ce

sonication); (b) sonication at 10% duty cycle; (c) sonication at 20% duty cycle; (d) sonication at
Ac

40% duty cycle. The sonication intensity was always 11.8 W cm2.

Figure 10. -Galactosidase activity profiles during fermentation: a) extracellular enzyme

activity; b) intracellular enzyme activity. The sonication intensity was 11.8 W cm2 except for

the nonsonicated control culture.

Figure 11. Biomass specific -galactosidase activity profiles during fermentation: a)

extracellular enzyme activity; b) intracellular enzyme activity. The sonication intensity was

12.5 W cm2 except for the nonsonicated control culture. For clarity, lines are plotted only

Page 25 of 40
 26

through the data for the control culture (solid lines) and the culture sonicated at the 40% duty

cycle (dashed lines).

t
ip
cr
us
an
M
d
p te
ce
Ac

Page 26 of 40
 Ac
ce
pte
d
M
an
us
cr
ip
t
27

Page 27 of 40
 t
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28

cr
us
Table 1. Comparison of fermentation kinetics

Kinetic parameter Sonication regimens (duty cycle)a

an
Control 10% 20% 40%

(no sonication)

M
Maximum specific growth rate, μ (h1) 0.2030.011 0.2060.027 0.2170.007 0.1790.017

Average specific lactose uptake rate, qs (g g1 h1) 0.2060.003 0.1510.010 0.1720.006 0.2080.009

Maximum biomass yield on lactose, Yx/s (g g1)

Maximum biomass concentration, Xmax (g L1) ed 0.2200.003

9.7120.076
0.3000.020

13.7550.850
0.2920.010

13.8130.443
0.2180.010

8.3880.315
pt
Maximum biomass productivity, Px (g L1 h1) 0.4410.003 0.6250.039 0.6930.022 0.3810.014
ce

Final ethanol yield on substrate, Yp/s (g g1) 0.0340.001 0.0960.009 0.1090.014 0.0520.002

Final ethanol concentration (g L1) 1.4790.036 4.4210.042 5.1990.677 2.0030.086

Ac

Final ethanol productivity, PE (g L1 h1) 0.0620.002 0.1840.017 0.2170.028 0.0830.004

Average biomass specific ethanol production rate, qp (g g1 h1) (6.350.16)103 (13.391.47)103 (15.682.10)103 (9.950.57)1

03

Page 28 of 40
 t
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29

cr
us
a
Except for the control culture, the sonication power intensity was always 11.8 W cm2

an
M
ed
pt
ce
Ac

Page 29 of 40
 t
ip
30

cr
us
Exhaust Motor

Filter Air flow

an
Nitrogen flow

Bioreactor

M
control unit

Ultrasound Nitrogen
Sampling 2 cylinder
chamber
bottle

ed 1
pt
3
Pumps
ce

Pump

Bioreactor
Ac

1. Dissolved oxygen probe connection

2. pH probe connection

3. Temperature probe connection

Fig. 1

Page 30 of 40
 31

Ø 35

t
ip
cr
Ø 34 Outlet

us
96.5
50
Ø 12.7

an
M
10
d
p te
ce

Inlet
Ac

Fig. 2

Page 31 of 40
 32

50 12 2.0

Biomass concentration (g L )
Lactose concentration (g L ) 10

Ethanol concentration (g L )
1
1

1
40
1.5
8

t
30

ip
6 1.0

cr
20
Lactose 4
Biomass
0.5
Ethanol

us
10
2

0 0 0.0

an
0 5 10 15 20 25

Fermentation time (h)

M
Fig. 3
d
p te
ce
Ac

Page 32 of 40
 33

14 a) Ultrasonication

Biomass concentration (g/L)

12

10

t
6

ip
4

cr
2

0
0 5 10 15 20 25 30

us
Fermentation time (h)

50 b)

an
Lactose concentration (g/L)

Control (no sonication)

10% duty cycle
40
20% duty cycle
40% duty cycle
M
30

20
d

10
te

0
0 5 10 15 20 25 30

Fermentation time (h)

p

120
c)
ce
Dissolved oxygen level (%)

100

80
Ac

60

40

20

0
0 5 10 15 20 25 30

Fermentation time (h)

Fig. 4

Page 33 of 40
 34

Ultrasonication Duty cycle

6
Ethanol concentration (g L )
1

20%

t
5

ip
10%
4

cr
3

us
2 40%
Control

an
1

0
0 5 10 15 20 25 30
M
Fermentation time (h)
d
te

Fig. 5
p
ce
Ac

Page 34 of 40
 35

t
ip
cr
us
a) b)

an
M
d

Fig. 6
pte
ce
Ac

Page 35 of 40
 36

5.5
Ultrasonication
5.0

t
Control (no sonication)

ip
10% duty cycle
4.5 20% duty cycle
40% duty cycle

cr
4.0
pH

us
3.5

3.0

2.5
an
M
2.0
0 5 10 15 20 25

Fermentation time (h)

d
p te
ce

Fig. 7
Ac

Page 36 of 40
 37

100
Ultrasonication

90

t
ip
Cell viability (%)

80

cr
70

us
60
Control (no sonication)

an
10% duty cycle
50 20% duty cycle
40% duty cycle
M
40
0 5 10 15 20 25

Fermentation time (h)

d
te

Fig. 8
p
ce
Ac

Page 37 of 40
 t
ip
38

cr
a) b)

us
an
M
c)
ed d)
pt
ce
Ac

Fig. 9

Page 38 of 40
 39

12
Duty cycle

Extracellular enzyme activity (U mL )

a)
1
10 20%
10%

t
8

ip
6

cr
Control

us
2 Ultrasonication
40%

0
0 5 10 15
an
Fermentation time (h)
20 25 30
M
35
Ultrasonication
Intracellular enzyme activity (U mL )

b)
d
1

30
te

25
p

20
ce

15
Ac

10 Control (no sonication)

10% duty cycle
5 20% duty cycle
40% duty cycle

0
0 5 10 15 20 25 30

Fermentation time (h)

Fig. 10

Page 39 of 40
 40

25000
Extracellular enzyme activity (U g )
1
a) Ultrasonication

20000

t
ip
15000 Control (no sonication)
10% duty cycle
20% duty cycle

cr
40% duty cycle
10000

us
5000

0
0 5 10
an
15 20 25 30
M
Fermentation time (h)

50000
b)
d
Intracellular enzyme activity (U g )

Ultrasonication
1

te

40000
p

30000
ce

20000
Ac

10000

0
0 5 10 15 20 25 30

Fermentation time (h)

Fig. 11

Page 40 of 40