Metabolomics (2012) 8:670678 DOI 10.

1007/s11306-011-0360-x

ORIGINAL ARTICLE

Sonic vibration affects the metabolism of yeast cells growing in liquid culture: a metabolomic study
Raphael Bastos Mereschi Aggio Victor Obolonkin as Silas Granato Villas-Bo

Received: 18 July 2011 / Accepted: 19 August 2011 / Published online: 4 September 2011 Springer Science+Business Media, LLC 2011

Abstract It is undeniable that music and sounds can affect our emotions and mood, but so far the study of physical stimuli provoked by sound on living organisms has been mostly focused on brain and sensorimotor structures rather than cellular metabolism. Using metabolomics, we compared the physiology of yeast cells growing in dened liquid medium exposed to music, high and low frequency sonic vibration and silence. All sonic stimuli tested not only increased the growth rate of the yeast cells by 12% but they also reduced biomass production by 14%. The intra- and extracellular metabolite proles differed signicantly depending on the sonic stimulus applied showing that different metabolic pathways are affected differently by different sound frequency. Therefore, our results clearly demonstrate that sound does affect microbial cell metabolism when growing in liquid culture, opening an entirely new perspective for scientic investigation interfacing acoustics, biophysics and biochemistry. Keywords Metabolomics Metabolite proling Bioacoustics Yeast physiology Cell metabolism

1 Introduction Although humanity has puzzled over the effect of sound on animal behaviour and physiology for centuries we have
Raphael Bastos Mereschi Aggio and Victor Obolonkin contributed equally to this work. as (&) R. B. M. Aggio V. Obolonkin S. G. Villas-Bo Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, 3A Symonds Street, Private Bag 92019, Auckland 1142, New Zealand e-mail: s.villas-boas@auckland.ac.nz

limited knowledge of the role of sound on single cells. Audible sounds, consist of oscillating pressure waves transmitted through solids, liquids, or gases, that if sufciently strong stimulate mechanosensory cells (Pickett et al. 2000). Sound can govern emotions and mood via structures involved in cognitive, sensorimotor, and emotional processing, and music has even been proposed as an alternative form of therapy to treat various illnesses (Koelsch et al. 2010). To date little is known about the effects of sonic waves on the cellular metabolism. Nonetheless, some scientic studies have reported that plants respond to different music (Xiujuan et al. 2003) and high frequency sound waves can signicantly increase protective antioxidative enzyme expression in plants (Xiujuan et al. 2003; Li et al. 2008). Sonic waves at specic frequencies have also been found to stimulate intestinal contractions and duodenal passage of barium in humans (Polous and Kurko 1991) and Syroeshkin et al. (1998) demonstrated that sonic waves induce contractive conformational changes of the mitochondrial transmembrane ATPase, affecting ATP production. Despite this tantalising evidence, audible sounds intrinsic impact on cellular metabolism has received limited attention from the scientic community. Although biological interactions of sonic and ultrasonic vibration appears to have been explored since 1900 (Naimark et al. 1951), since the 1920s the work has almost exclusively focused on ultrasound (\20 kHz; Wood and Loomis 1927; Harvey and Loomis 1928; Harvey et al. 1928). Using a single cell organism as a model, we have exposed bakers yeast, Saccharomyces cerevisiae to sound, to test whether sonic waves of different frequencies affect basal cell metabolism and growth. We choose a unicellular microorganism to exclude the complexity of multicellular

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organisms, and to eliminate the inuence of nervous and auditory systems of animals. Moreover the genome, biochemistry and metabolome of S. cerevisiae are extremely well characterised (Sherman 1997), and as such provide us a powerful model to test the role of sound on cell metabolism.

2 Materials and methods 2.1 Yeast strain Saccharomyces cerevisiae strain VIN13 was maintained on YPD (yeast extract, peptone, dextrose) agar plates at 30C and used to prepare all pre-inoculums. 2.2 Shake ask cultures The yeast strain was cultivated aerobically in triplicate, using shake-asks containing glucose (20 g l-1) as single carbon source, (NH4)2SO4 (5.0 g l-1), MgSO47H2O (0.5 g l-1), KH2PO4 (3.0 g l-1), vitamins and trace elements according to Verduyn et al. (1992). The culture asks were inoculated with an overnight grown pre-inoculum prepared in the same growth medium. Cultivations were performed using the same rotatory shaker and incubated in the same incubation room at 28 1C and 190 rev./min, in 500 ml shake asks containing 100 ml of medium and cotton plugs. Each sound treatment was carried out at different days but using broth medium prepared in a single bulk to ensure identical composition and inoculated with identical initial optical density using a preinoculum grown overnight in the same medium. 2.3 Sound stimuli The experimental set up as shown in Fig. 1 consisted of two middle-high frequency speakers and low frequency subwoofer connected to a computer. They were placed on at panel shaker surrounding the 500 ml shake-asks. The distance between the speakers and shake-asks did not exceed 20 cm. Due to the small within-group variation (samples from the different asks clustered close together and presented small standard deviations in the different measurements, n = 15), we assumed the sound pressure to be roughly equal for all technical replicates. The yeast cultures were exposed to four different conditions: (HF) high frequency, 10 kHz, recording 89 dBA, sampled at 705 Kbps; (LF) low frequency, 100 Hz, recording 92 dBA, sampled at 705 Kbps; broad-band music, sampled at 320 Kbps and 8092 dBA; and silencecontrol conditions including only the mechanical noise of the incubator room at approx 60 dBA. The sound stimuli were played continuously for the duration of the experiment.

Fig. 1 Scheme of experimental set up. The experimental set up consisted of two middle-high frequency speakers and low frequency subwoofer connected to a computer. They were placed on at panel shaker surrounding the 500 ml shake-asks. The distance between the speakers and shake-asks did not exceed 20 cm

2.4 Fermentation parameters Optical density (OD) of culture broth was measured at 600 nm using a Hitachi (model U-1100) spectrophotometer each hour until the stationary growth phase was reached. Presence of contamination was monitored using light microscopy and growth rate (l) was calculated as l 2:303logOD logOD0 t t0

Table 1 Classication table obtained from leave-one-out cross validation of 15 samples containing intracellular metabolites Original source Predicted source HF HF LF M S 15 0 0 0 LF 0 15 0 0 M 0 0 15 0 S 0 0 0 15

HF high frequency sonic wave, LF low frequency sonic wave, M music, S silence

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Fig. 2 Growth curves and growth rates of Saccharomyces cerevisiae at different experimental conditions. Cultivations occurred aerobically in shake asks at 28C under constant agitation at 190 rev. min-1. Flasks in triplicate were subject to different sound

stimuli: Low frequency sonic waves (100 Hz at 92 dBA), high frequency sonic waves (10 kHz at 89 dBA), broad-band music (sampled at 320 kbps at 8092 dBA), and silence (background noise at *60 dBA). Each data point represents mean values of three asks

where l is the growth rate, OD is the optical density measured at the end of the exponential growth, OD0 is the optical density measured in the beginning of the exponential growth, t is the time at the end of the exponential growth, and t0 is the time at the beginning of the exponential growth. 2.5 Sampling for metabolome analysis Five 5 ml cellular samples were rapid harvested from each ask when the cells had reached an OD600 = 2.32.4 (early to middle exponential growth phase). The cell metabolism was quenched using cold-glycerol saline solution (-23C) followed by cold centrifugation at -20C. Internal standard, 20 ll of 2,3,3,3-d4 alanine (10 mM), was added to the cell pellets and the intracellular metabolites were extracted using freeze/thaw cycles and cold methanol water (1:1v/v) solution. Detailed protocol can be found in our previously published work (Smart et al. 2010).

2.6 Metabolite analyses The cell extracts containing intracellular metabolites were freeze-dried using a 12L Labconco Freeze Dryer (Labconco Corporation). The freeze-dried solids were resuspended in 200 ll of sodium hydroxide solution (1 M) and derivatized according to our standard laboratorial procedure (Smart et al. 2010). The derivatized samples were analyzed using a GC MS system (GC7890 coupled to a MSD5975, Agilent technologies), with a quadrupole mass selective detector (EI) operated at 70 eV. The column used for all analyzes was a ZB-1701 (Phenomenex), 30 m 9 250 m (internal diameter) 9 0.15 (lm thickness), with 5 m guard column. The MS was operated in scan mode (start after 6 min; mass range 38650 a.m.u. at 1.47 scans/s (Smart et al. 2010). 2.7 Compound identication We have used the Automated Mass Spectral Deconvolution and Identication System (AMDIS) to identify compounds

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Table 2 All intracellular metabolites identied in samples of Saccharomyces cerevisiae growing aerobically on glucose at different growth conditions (sound stimuli) Mean of relative intensity (n = 15) standard deviationa Silence Aspartate Oleate Proline Citrate Isoleucine Leucine 14-methylpentadecanoate Glutamine Succinate Phenol derivative Glutamate Stearate Caproicate Tyrosine 2-methoxysuccinate Asparagine NADPH/NADP? NADH/NAD? Caprylate Myristate 2-oxoglutarate Malate Caprinate 2-aminobutyrate Benzoate Fumarate Nicotinate Histidine Indol derivative Alanine Serine 4-aminobutyrate Adipate Cis-aconitate Levulinate Norvaline Phenylpropanoate Lysine Glycine Norleucine Threonine Valine Hydroxybenzoate Methionine 10,13-dimethyltetradecanoate Phenylalanine 0.041 0.008 0.063 0.017 0.231 0.034 0.000 0.000 0.090 0.020 0.103 0.018 0.043 0.014 0.098 0.023 0.009 0.002 0.138 0.055 0.171 0.029 0.039 0.012 0.015 0.002 0.017 0.003 0.071 0.017 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.017 0.001 0.000 0.000 0.000 0.000 0.000 0.000 0.033 0.004 0.004 0.0004 0.005 0.0007 0.000 0.000 0.000 0.000 0.033 0.011 0.177 0.032 0.000 0.000 0.019 0.001 0.008 0.0008 0.000 0.000 0.003 0.001 0.000 0.000 0.005 0.0005 0.224 0.043 0.027 0.004 0.043 0.002 0.046 0.007 0.021 0.003 0.009 0.001 0.000 0.000 0.045 0.012 0.014 0.003 Music 0.018 0.001 0.060 0.017 0.084 0.011 0.000 0.000 0.040 0.010 0.051 0.005 0.035 0.008 0.040 0.005 0.004 0.001 0.089 0.021 0.069 0.016 0.021 0.006 0.009 0.005 0.013 0.004 0.033 0.009 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.015 0.002 0.002 0.001 0.000 0.000 0.000 0.000 0.003 0.001 0.031 0.014 0.126 0.022 0.000 0.000 0.015 0.008 0.000 0.000 0.000 0.000 0.000 0.000 0.009 0.0005 0.000 0.000 0.176 0.052 0.014 0.002 0.037 0.003 0.020 0.004 0.071 0.072 0.004 0.0002 0.000 0.000 0.038 0.009 0.000 0.000 Low frequency 0.068 0.013 0.032 0.007 0.049 0.008 0.068 0.011 0.047 0.007 0.042 0.006 0.032 0.005 0.035 0.021 0.039 0.007 0.035 0.005 0.479 0.073 0.023 0.004 0.045 0.004 0.012 0.002 0.011 0.001 0.015 0.003 0.022 0.009 0.020 0.009 0.023 0.002 0.006 0.001 0.008 0.001 0.004 0.001 0.008 0.001 0.002 0.0001 0.004 0.004 0.004 0.001 0.005 0.001 0.007 0.002 0.005 0.004 0.359 0.031 0.003 0.001 0.003 0.001 0.005 0.0007 0.002 0.001 0.003 0.001 0.000 0.000 0.001 0.000 0.204 0.042 0.009 0.001 0.000 0.000 0.018 0.003 0.216 0.020 0.000 0.000 0.003 0.0003 0.028 0.007 0.348 0.032 High frequency 0.103 0.031 0.053 0.039 0.058 0.023 0.076 0.020 0.058 0.009 0.049 0.009 0.040 0.018 0.027 0.010 0.029 0.006 0.043 0.013 0.558 0.202 0.029 0.010 0.029 0.004 0.015 0.006 0.012 0.006 0.017 0.009 0.017 0.008 0.020 0.010 0.014 0.002 0.007 0.003 0.008 0.001 0.006 0.002 0.009 0.001 0.006 0.0008 0.003 0.001 0.004 0.002 0.004 0.001 0.004 0.002 0.005 0.001 0.319 0.035 0.004 0.001 0.002 0.001 0.003 0.0003 0.002 0.001 0.003 0.001 0.002 0.0001 0.001 0.001 0.174 0.071 0.013 0.006 0.064 0.005 0.020 0.005 0.196 0.021 0.000 0.000 0.005 0.001 0.038 0.022 0.523 0.057

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674 Table 2 continued Mean of relative intensity (n = 15) standard deviationa Silence 4-aminobenzoate Pyruvate Lactate Malonate Carbamate Glutarate Ornithine Pyroglutamate
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Music 0.000 0.000 0.000 0.000 0.017 0.010 0.000 0.000 0.016 0.008 0.000 0.000 0.304 0.052 0.000 0.000

Low frequency 0.004 0.000 0.004 0.0005 0.155 0.014 0.004 0.004 0.000 0.000 0.002 0.0003 0.196 0.029 0.253 0.072

High frequency 0.000 0.000 0.003 0.0004 0.149 0.030 0.001 0.000 0.000 0.000 0.000 0.000 0.106 0.037 0.162 0.089

0.002 0.0002 0.000 0.000 0.037 0.019 0.000 0.000 0.089 0.040 0.000 0.000 0.570 0.069 0.000 0.000

Mean of GCMS peak normalized by internal standard

present in each sample. AMDIS is a software freely distributed by the National Institute of Standards and Technology and has been largely applied to metabolomics. Although AMDIS algorithm is considered powerful in deconvoluting and identifying chromatographic peaks, it produces some inaccuracies in relation to compound quantication. Therefore, we used an in-house R (www. r-project.org) script which recalculates the correct intensity of each compound previously identied by AMDIS. A detailed description of this process and our in-house R script can be found in Aggio et al. (2011). 2.8 Data normalization As described above, many technical steps precede the generation of metabolomics data (e.g. sampling, quenching, metabolite extraction and freeze-drying). Each one of these steps is a potential source of variability, which can possibly interfere in the identication of trends and correlations present in our data-set. Therefore, we used the 2,3,3,3-d4 alanine as internal standard to minimise this variability. This way, we divided the intensity of each metabolite by the intensity of the internal standard detected in each sample. More details about data normalization procedure can be found in Smart et al. (2010). 2.9 Metabolomic data analysis The difference in metabolite proles of the different samples was assessed by three different statistical methods. Firstly we short-listed the relative level of each identied metabolite in all the growth conditions. Conditions for which the level of a metabolite was below the detection limit of the method it was assigned absent. The qualitative results are summarised in the Venn diagram (Fig. 3). Secondly, the metabolites that were detected in all samples from all conditions were selected for Fisher discriminant analysis (FDA). The projections of samples to three dimensions were computed by

Fig. 3 Fisher discriminant analysis (FDA) for sample visualisation. GCMS metabolite data successfully distinguishes the different growth conditions. Projections of the log transformed intracellular metabolite data from 60 samples into 3D space shows four distinct clusters of the four data classes. For each sample the projection values were calculated as the linear combination of metabolite values determined by FDA. Only metabolites that were detected in all samples of all four growth conditions were used for the analysis

dicrcoord function from fpc package (http://www.homepa ges.ucl.ac.uk/*ucakche/). The visual clustering was achieved by plotting the rst three FDA projections. lda function from the MASS package was used to classify samples into four experimental categories. The results were validated using leave-one-out cross validation technique, using a single observation as the testing data for the FDA and the remaining data as the training set. The procedure was repeated until all observations were used as the testing samples. Results are showed in Table 1. We achieved 100% correct classication for all of the experimental conditions, which validates the separation of samples in the four data classes (growth conditions). Finally each metabolite

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expression level was tested for the difference between growth conditions with univariate analysis of variance (ANOVA). The data was log-transformed to t the normal distribution criteria. Post-hoc turkey multiple comparison test was used to estimate signicantly different pairs of conditions. Analysis was performed using R 2.9.0 software ( http://www.r-project.com).

3 Results and discussion A strain of S. cerevisiae commonly used in wine fermentation was cultured in a chemically dened broth with glucose as the only carbon source. All culture asks were inoculated with freshly prepared (exponentially growing) pre-inoculum at identical initial optical density and were incubated at 28C under constant agitation using the same shaker-incubator. Three different sound stimuli were applied constantly to the culture asks in triplicate throughout the incubation period (Fig. 1): (LF) low frequency sonic waves (100 Hz at 92 dBA), (HF) high frequency sonic waves (10 kHz at 89 dBA), and (M) broad-band music (sampled at 320 kbps at 8092 dBA); which were compared to culture asks growing in the silence (background noise at *60 dBA). The yeast cells growing under sonic stimuli presented longer lag

phase (?1 h) and shorter exponential growth (-2 h) when compared to cells growing in the silence (Fig. 2). In addition, the growth rate of cells grown under sound stimuli was found to be signicantly different than the growth rate of yeast cells growing in the silence (P value \ 0.005Students t-test) (Fig. 2). Yeasts growing under music presented the highest growth rate of 0.362 h-1, which was 12.4% faster (P \ 0.005) than cells growing in the silence (0.322 h-1). The growth rate of cells growing under low frequency (0.350 h-1) and high frequency sonic waves (0.359 h-1) grew also signicantly faster than in the silence (P \ 0.005). Interestingly, the increased growth rates of sound exposed yeast resulted in up to 14% reduction in biomass production (Fig. 2). The maximum optical densities (600 nm) reached by cultures incubated under sound stimuli were between 3.44 and 3.72 while the cultures kept in the silence reached 4.03 (P \ 0.05). We reasoned that sonically modulated growth and biomass accumulation must have impacted cellular metabolism and metabolites. Therefore we explored the metabolome of cultures from each sound treatment. Cells from all treatments were harvested at middle exponential growth phase (OD600nm 2.32.4) and promptly quenched at -23C to stop cell metabolism (Smart et al. 2010). Cells were washed and intracellular metabolites extracted at low temperature prior

Fig. 4 Venn visualization of the different identied metabolites comparing experimental conditions. Experimental conditions: S silence, M music, LF low frequency, and HF high frequency

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to be chemical derivatized and analysed by gas chromatography-mass spectrometry (Smart et al. 2010). Approximately 50 different metabolites, which play important metabolic roles in the central carbon metabolism, lipid and amino acid biosynthesis, were identied among hundreds of detected compounds (Table 2). To distinguish samples among classes, we projected the identied metabolite level data from each sample to a lower dimensional space. Two often-used data projection methods are PCA (principal component analysis) and FDA (Fisher discriminant analysis). PCA maximizes variation in the reduced dimensions, whereas FDA maximizes separa as et al. 2005). For this tion between classes (Villas-Bo reason, we applied FDA to visualize samples in an attempt to distinguish them among classes, which revealed very clear separation as shown in Fig. 3. Our metabolomics data successfully demonstrated that each data class (sound stimuli and silence) present distinct metabolite proles, with samples from same data class clustering very close to each other. For instance, we achieved 100% correct classication for all of the experimental conditions using the leave-one-out cross validation technique (Table 1)

55 different intracellular metabolites were consistently identied from over 120 metabolites searched for in our MS library (Table 2). Of these 32 metabolites were detected at signicant different levels in each growth condition and 9 were unique to specic conditions (Fig. 5). On comparison of the most signicantly changed metabolite levels among growth conditions, some metabolites detected in all four conditions showed a linear decrease in levels when comparing silence, music, low frequency and high frequency sonic waves (e.g. ornithine, glutamine, 2-methoxysuccinate). On the other hand, several compounds were detected only in samples growing under xed sonic waves such as NADP?, NAD?, myristate, 2-oxoglutarate, malate, fumarate, and others (Fig. 5), whereas fewer metabolites were only detected in music and silence (Figs. 4, 5). Although non-detection for a given condition illustrates that some metabolites were below our detection limits, differential metabolite level data still permits interpretation and provides proof-of-concept that sound impacts on cellular metabolism of yeast. Using our recently developed approach for predicting metabolic pathway activity based

Fig. 5 Some of the metabolites detected at signicant different levels. Stripcharts of the metabolites shows statistically signicant difference between experimental conditions tested using univariate ANOVA. Arrows represent 95% turkey condence intervals for

multiple comparisons between growth conditions. Only metabolites showing greatest difference were selected for the plot. Experimental conditions: S silence, M music, LF low frequency, and HF high frequency

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Fig. 6 Comparative metabolic pathway activities of Saccharomyces cerevisiae under different growth conditions based on intracellular metabolomic data. The activity scores (AS) for each pathway were calculated using our Pathway Activity Proling (PAPi) algorithm (Aggio et al. 2010). PAPi calculates for each metabolic pathway listed in KEEG database an activity score based on the number of

metabolites identied from each pathway and their relative abundances. As a result, the activity score represents the likelihood that a metabolic pathway is active inside the cell and, consequently, allows the comparison of metabolic pathway activities using metabolite prole data

on metabolite prole data (Aggio et al. 2010), we have explored metabolic traits evident in our data (summarised in Fig. 6). Based on the hypothesis generating algorithm (PAPi), which explores metabolite prole data and the KEGG database to predict and compare metabolic pathway activities among different experimental conditions (Aggio et al. 2010), it is apparent that different sound stimuli have distinct effects on cell metabolism (Fig. 6). For instance, low frequency sound induces an apparent increase in activity of numerous metabolic pathways, with exception of glycolysis. All sound stimuli inhibited the activity of pantothenate and CoA biosynthetic pathways, whilst the activity of ve other pathways under sonic treatment (e.g. pentose phosphate pathway and pentose-glucoronate interconversions) increased. Interestingly, only music stimulus negatively impacted lysine biosynthesis and cysteine and methionine metabolism. High frequency sound depressed apparent activities of several pathways generally related to aromatic amino acid biosynthesis and metabolism. Ultrasonic vibrations have been long recognized as a powerful energy source able to affect cell structures and

growth (Jomdecha and Prateepasen 2006). There is some evidence that bacteria can enhance the proliferation of stressed neighbouring cells by emitting ultrasound over distances of 30 cm (Matsuhashi et al. 1998). Xiujuan et al. (2003) observed that the H?-ATPase activity was perturbed by low frequency sound and this redistributed cytoplasmic calcium in plant cells (Xiujuan et al. 2003). The protective antioxidant peroxidase isoenzymes also increased on sonic treatment (Xiujuan et al. 2003). These data suggest that specic sound intensities and frequencies altered membrane ion permeabilities and may alter free radical formation. Sound frequencies of 170 and 340 Hz also drove ATP formation in bovine F1/F0 ATPase function in submitochondrial particles (Syroeshkin et al. 1998). Potentially sound may alter ATP rotation, or membrane proton gradients given that other transmembrane enzymes and transporters might also be susceptible to sonic vibrations. Future studies should certainly involve the measurement of redox potential of the cells and their nucleotide levels during sound stimuli under continuous growth cultures, where the growth rate can be kept constant. This will certainly assist in the understanding of the mechanism behind the effect of sound on yeast cell metabolism.

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R. B. M. Aggio et al. process. In 12th Asia-Pacic Conference on NDT, 5th10th November 2006, Auckland, New Zealand. Koelsch, S., Offermanns, K., & Franzke, P. (2010). Music in the treatment of affective disorders: An exploratory investigation of a new method for music-therapeutic research. Music Perception, 27, 307316. Li, B., Wei, J., Tang, K., Liang, Y., Shu, K., & Wang, B. (2008). Effect of sound wave stress on antioxidant enzyme activities and lipid peroxidation of Dendrobium candidum. Colloids and Surfaces B: Biointerfaces, 63, 269275. Matsuhashi, M., Pankrushina, A. N., Takeuchi, S., Ohshima, H., Miyoi, H., Endoh, K., et al. (1998). Production of sound waves by bacterial cells and the response of bacterial cells to sound. The Journal of General and Applied Microbiology, 44, 4955. Naimark, G. M., Klair, J., & Mosher, W. A. (1951). A bibliography on sonic and ultrasonic vibration: Biological, biochemical and biophysical applications. Journal of The Franklin Institute, 251, 279299. Pickett, J. P., et al. (2000). The American Heritage Dictionary of the English Language (4th ed.). Boston: Houghton Mifin. Polous, Y. U. M., & Kurko, V. S. (1991). Sound-wave stimulation of duodenal motility in chronic duodenal ileus. Klinicheskaya Meditsina, 69, 4244. Sherman, F. (1997). Yeast genetics. In R. A. Meyers (Ed.), The encyclopaedia of molecular biology and molecular medicine (Vol. 6, pp. 302325). Weinheim: VCH Publisher. as, S. G. Smart, K. F., Aggio, R. B. M., Van Houtte, J. R., & Villas-Bo (2010). Analytical platform for metabolome analysis microbial cells using methyl chloroformate derivatization followed by gas chromatographymass spectrometry. Nature Protocols, 5, 17091729. Syroeshkin, A. V., Bakeeva, L. E., & Cherepanov, D. A. (1998). Contraction transitions of F1F0 ATPase during catalytic turnover. Biochimica et Biophysica Acta, 1409, 5971. Verduyn, C., Postma, E., Scheffers, W. A., & van Dijken, J. P. (1992). Effect of benzoic acid on metabolic uxes in yeasts: A continuous-culture study on regulation of respiration and alcoholic fermentation. Yeast, 8, 501517. kesson, M., Stephanopoulos, G., & as, S. G., Moxley, J. F., A Villas-Bo Nielsen, J. (2005). High-throughput metabolic state analysis: The missing link in integrated functional genomics of yeasts. Biochemical Journal, 388, 669677. Wood, R. W., & Loomis, A. L. (1927). The physical and biological effects of high frequency sound waves of great intensity. The London, Edinburgh, and Dublin Philosophical Magazine, 4, 417436. Xiujuan, W., Bochu, W., Yi, J., Defang, L., Chuanren, D., Xiaocheng, Y., et al. (2003). Effects of sound stimulation on protective enzyme activities and peroxidise isoenzymes of chrysanthemum. Colloids and Surfaces B: Biointerfaces, 27, 5963.

4 Conclusions By using a metabolomic approach, we demonstrate that audible frequency sound inuences yeast cell metabolism. We not only showed that the intracellular metabolite prole of yeasts cells harvested at same growth phase (early exponential growth) but growing at different sound stimuli were signicantly different as demonstrated by FDA analysis (Fig. 3), we also showed that some identied metabolites were only detected in specic group of samples (sound treatments, n = 15) (Fig. 4), while those metabolites detected in all samples also presented signicant different levels (Fig. 5). Moreover, our data indicate that different sound frequencies induce different metabolic and physiological responses in yeast cells. This opens new perspectives for scientic investigation interfacing acoustics, biophysics and biochemistry, and perhaps provides powerful tools for manipulating cell metabolism and growth control (i.e.; in bioreactors) and proliferation (i.e.; cancer treatment).
Acknowledgments We are very grateful to Gregory Cook, Matthew Goddard, Richard Gardner, and Vladimir Obolonkin for valuable comments and to Anthony Hickey for discussion and critical reading of this manuscript. We also thank Farhana Pinu and Sang Kim for technical assistance with media and sample preparation.

References
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