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A pilot study of the effect of audible sound on the growth of Escherichia coli
Colloids and Surfaces B: Biointerfaces 78 (2010) 367–371
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
A pilot study of the effect of audible sound on the growth of Escherichia coli Gu Shaobin a,∗ , Ying Wu a , Kewei Li a , Shichang Li a , Shengyun Ma b , Qiannan Wang a , Rong Wang c a
College of Food and Bioengineering, Henan University of Science and Technology, No. 70, Tianjing Road, 471003 Luoyang, Henan, People’s Republic of China College of Life Sciences, Zhejiang University, 310029 Hangzhou, People’s Republic of China c College of Life Sciences, Wuhan University, 430072 Wuhan, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 25 December 2009 Received in revised form 22 February 2010 Accepted 25 February 2010 Available online 4 March 2010 Keywords: Audible sound Response Bacterial cell
a b s t r a c t Audible sound, one of the environmental factors, widely exists in natural world. However, the interaction between audible sound and biological materials is usually neglected in the field of biological research. Very little efforts have been put forth in studying the relation of organisms and audible sound. Here we investigated the response of Escherichia coli cells to the stimulation by audible sound under the normal condition and environmental stresses. The results showed that the audible sound treatment significantly increases the colony forming of E. coli under the normal growth condition. However, under osmotic stress induced by the sugar, audible sound stimulation may enhance the inhibitory effect of osmotic stress on E. coli growth. More interestingly, audible sound treatment seems to alleviate the inhibitory effect of salt stress on E. coli growth when the concentration of sodium chloride was increased to 30 g/l, although the action of sound waves of audible frequency is likely to evoke an inhibition of the growth of E. coli in the medium containing 20 g/l of sodium chloride. Some potential mechanisms may be involved in the responses of bacterial cells to audible sound stimulation. © 2010 Elsevier B.V. All rights reserved.
1. Introduction A sound wave is similar in nature to a slinky wave for a variety of reasons. Since a sound wave is a disturbance which is transported through a medium via the mechanism of particle interaction, a sound wave is characterized as a mechanical wave [1]. In the natural world almost all living organisms are “immersed” in a variety of sound waves, and interact with them [1,2]. In terms of the sound frequency, there are roughly three regimes: infrasound (10−4 –20 Hz), audible sound (20–2 × 104 Hz) and ultrasound (2 × 104 –1012 Hz). Ultrasound-induced biological effects and its biophysical mechanisms have been extensively investigated in recent decades [2–4]. It has already been successfully combined with biotechnology with the aim of enhancing the efficiency of bioprocesses [5]. Moreover, ultrasound is also used widely in medicine as both diagnostic and therapeutic tools [6]. Although infrasound induced bioeffects and its potential mechanism are still not very clear [7], some significant progresses have been made in the application field, e.g. infrasound diagnosis and therapeutic infrasound [8,9]. So far the study for audible sound is usually focused on how sound is produced, absorbed, reflected, as well as transmitted by objects. Little research concentrates on audible sound induced bio-
∗ Corresponding author. Tel.: +86 379 64283928; fax: +86 379 64282342. E-mail address: [email protected] (G. Shaobin). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.02.028
logical effects and its possible mechanism. Recently the biological effects induced by audible sound wave in plants were investigated [10–12]. To explain the potential mechanism, a hypothesis on plant meridian system was put forward [11]. However, the plant meridian system based on the human body meridian theory is complicated and imperfect. It is not proper to understand the mechanism of biological effects induced by audible sound waves. Furthermore, the complexity of multicellular organisms has also brought great difficulties to study of the audible sound waves biological effects mechanism. Here we attempted to use the single-cell organisms, Escherichia coli to investigate the biological effects of organism after audible sound waves stimulation and understand the potential mechanism. The research has positive significance for us to get more information about audible sound wave biological effects and reveal its underlying mechanism. 2. Materials and methods 2.1. Bacterial strain and culture conditions E. coli, laboratory-preserved strains, was first cultured in slant agar medium (0.5 g beef extract, 1 g peptone, 0.5 g NaCl, 2 g agar, 100 ml distilled water, pH 7.2) at 37 ◦ C for 18 h. Then cells expanded in a 250 ml Erlenmyer flask containing 100 ml of liquid medium (beef extract 0.5 g, peptone 1 g, NaCl 0.5 g, distilled water 100 ml, pH 7.2) with agitation of 200 rpm on a rotary shaking incubator at 37 ◦ C for 12 h.
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G. Shaobin et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 367–371
¯ c represents the average colonies number from five plates Here N in control group at 22 h after being placed in the sound load cham¯ i represents the average colonies number from five repeat ber. N plates. i represents different times after being put into the chamber. Each experiment had fivefold plate and was independently repeated at least three times. The data were presented as means of relative colony forming efficiency ± SD. 3. Results and discussion 3.1. The effects of audible sound waves stimulation on E. coli growth under normal conditions
Fig. 1. Schematic of sound wave load apparatus. Notes: a – wave generation unit; b – sound wave transfer wire; c – speaker; d – UV lamp (it is used for sterilization before the start of sound stimulation experiments); e – sample holder; f – outer wall of chamber (metal shell); g – inner wall of chamber (made with sound absorbing material); h – motor.
2.2. Sound wave load apparatus To investigate the bioeffects of biological material exposed to the acoustic environment, the sound wave load apparatus was designed (see Fig. 1). This equipment is composed of two parts. One is the sound wave generating unit. The other is sound wave load chamber. The former contains a waveform generator and the amplifying circuit. The signals produced by the waveform generator are amplified and then send to a speaker. The speaker along with UV lamp, rotating sample holder is installed in the sound load chamber whose inner walls are made with sound absorbing material. The rotating sample holder ensures that each sample can get the equal acoustic stimulation. The outer walls of chamber are wrapped by a metal shell in order to reduce influence of environmental noise on the experimental result. When the experiments are performed, the desired audible sound wave is generated by the apparatus and the frequency is precisely adjusted from 1 to 20 kHz. The sound intensity is set according to the experiment design.
People usually neglect the interaction between audible sound and living organisms. It is even more out of the question to study the relation of organisms and sound waves of audible frequency. However, audible sound, an important environmental factor, widely exists in the world. It inevitably affects the growth and metabolic behaviors of organism. Fig. 2 shows effects of audible sound waves stimulation on E. coli growth. The promotion of E. coli growth was obviously observed when bacteria cells were stimulated by audible sound wave. The relative colony forming efficiency reached 141.6%, 130.0% and 131.1% 22 h after stimulation by sound wave with the frequency of 1000, 5000 and 10,000 Hz, separately, which were significantly higher than that of the control (100%). With respect to the positive interaction between audible sound and biological cells, periodic oscillation of bacterial cell induced by audible sound forces may be the first factor to consider. As a mechanical wave, audible sound would produce a mechanical stress to E. coli cells, similar to an alternate pull and press forces. The alternative stress definitely causes the motion of the cell internal fluid and the deformation of cellular plasma membrane [13,14]. At the same time, the membrane fluidity would increase under the sound stimulation [15]. Moreover, sound stimulation makes significant changes on the membrane protein structure [16]. These processes are helpful to the membrane trafficking modulation [17], and the acceleration of metabolism activity [18]. In addition, we also note that the frequency of 1000 Hz of sound wave seems to be the best to promote the E. coli cells to grow.
2.3. Sound stimulation experiments After being cultured in liquid medium at 37 ◦ C for 12 h, the cells of E. coli were spread over to agar plates. Then the samples were put into the sound load chamber and stimulated by 90 dB sound wave with frequency of 1, 5 and 10 kHz separately. Duration of stimulation and interval time were 1 and 3 h, respectively. And the total treatment time (including stimulation and interval time) was 24 h. Samples without audible sound wave treatment served as a control group. The temperature within the sound wave load apparatus was maintained at 37 ◦ C. 2.4. Statistical analyses Samples were collected and colonies were scored after being put into the chamber for 10, 11, 12, 13, 14, 16, 18 and 22 h. The relative colony forming efficiency was calculated according to the following formula: relative colony forming efficiency =
¯i N × 100% ¯ Nc
Fig. 2. The influence of audible sound stimulation on E. coli growth under normal culture conditions. The medium consists of 0.5 g beef extract, 1 g peptone, 0.5 g NaCl, 2 g agar, 100 ml distilled water, and pH was controlled at 7.2. Each point is the average of four replications, and vertical bars represent one SD on either side of the average.
G. Shaobin et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 367–371
The results suggested that the action of audible sound may be non-linear, and show obvious frequency peculiarities. The investigations of Sun et al. and Sheng’s show that sound stimulation at a certain frequency and strength promoted the fluidity of membrane wall and membrane lipid in tobacco cells, which would benefit the absorb of nutriment and combination of DNA in S period, as well as synchronizing the cell cycle, which all benefit the growth of cells [19,20]. Zhao et al. found that the promotion effect on Dendranthema morifolium callus growth was most obvious in a frequency of 1000 Hz and strength of 100 dB sound stimulation [21]. It can obviously promote the soluble protein and sugar in cytoplast, which is a sign of high metabolism level and vigorous divide state in sound stimulation administrated D. morifolium callus. In spite of underlying mechanisms of frequency-dependent effects have still remained unknown, more and more investigations still showed that mechanical stress may play an important role in cellular metabolism, gene expression, cell growth and proliferation [18,22–25]. Consequently, concerning to cells, responding to audible sound forces may be the main reason to create biological effects, though it is not very clear that how the audible sound signal is taken over and then transferred to intracellular by the cells to bring about a further series of biological effects. 3.2. The influences of audible sound waves stimulation on E. coli growth under environmental stress The environmental stress response is an important physiological mechanism that protects cells and organisms from stressful changes in their environment. To ensure survival in the face of these afflictions, such as change in temperature, pH, osmolarity, radiation and the concentration of nutrients and toxins, organisms cannot but adapt to changes in their immediate vicinity by responding to the imposed stress. In order to have a thorough knowledge of audible sound waves biological effects, this part was focused on the study of the influences of audible sound waves stimulation on responses of E. coli to environmental stress. 3.2.1. The influence of audible sound stimulation on E. coli growth under glucose induced osmotic stress Under the osmotic stress induced by sugar, the influence of audible sound wave stimulation on the E. coli growth is shown in Fig. 3. The result implied that E. coli growth was severely depressed when glucose concentration remained at 90 g/l in medium. Moreover, audible sound stimulation may enhance the inhibitory effect of osmotic stress on E. coli growth. Compared with the sample without audible sound stimulation, the lower relative colony forming efficiency appeared in the treatment group. Under the osmotic stress induced by sugar, the actions of audible sound possible play a negative role in the stimulation of E. coli growth. Firstly, the opening of aquaporins in hypertonic conditions [26] may bring an inhibitory effect into the growth of E. coli. Secondly, the significant changes on the cellular plasma membrane structure and physical states induced by mechanical forces [14–16,27], was likely to be involved in the increase of water efflux in the abnormal concentration of sugar. Recent studies indicated that the change of plasma membrane protein structure is closely related to frequency of the sound wave [16]. With the increase of audible sound waves frequency, ␣-helix of membrane protein gradually increased, while decreasing -turn. The membrane protein conformation changes denote the increased interaction between membrane protein and lipids and the increased fluidity of membrane. There is no doubt that increases in fluidity correlate with increases in the water permeability of cellular plasma membrane [28]. The rise in efflux of water should be responsible for the enhancement of inhibitory effect of osmotic stress on E. coli growth. Consequently, it was not surprising that gradually reduced relative colony forming efficiency was eas-
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Fig. 3. The influence of audible sound stimulation on responses of E. coli to glucose induced osmotic stress. Notes: 1 – control; 2–9% glucose + 0 Hz; 3 – 9% glucose + 10,000 Hz; 4 – 9% glucose + 5000 Hz; 5 – 9% glucose + 1000 Hz. The culture was composed of 0.5% peptone, 0.5% dipotassium hydrogen phosphate, 9% glucose and 2% agar, and pH was controlled at 7.2. Data were means of relative colony forming efficiency ± SD at 22 h after being placed in the sound load chamber. Each experiment had fivefold plate and was independently repeated three times. Means of relative colony forming efficiency followed by the same letter are not significantly different (P > 0.05, LSD, SAS9.1.3, SAS Institute Inc., 2005).
ily observed with the increase of audible sound waves frequency in the above experiments. 3.2.2. The influence of audible sound waves stimulation on E. coli growth under salt stress The influence of audible sound wave stimulation on E. coli growth under salt stress is shown in Fig. 4. Even though in the absence of audible sound stimulation, the relative survival fraction of samples gradually decreased with the increase of sodium chloride concentration. Salinity stress can imbalance the osmotic potential in E. coli cells generating a water deficit and the influx of sodium may lead to metabolic toxicity. Under salt stress accompanied with audible sound stimuli, the increase in efflux of water by means of simple diffusion and water-selective channels (as mentioned) may be just one reason to lead to the enhancement of inhibitory effect of salt stress on E. coli growth. The activated mechanosensitive ion channel of large conductance (MscL) in response to physical stresses [29,30] was thought to provide an opportunity for an influx of sodium ion. A rise in influx of sodium ion through MscL and sodium channel proteins probably plays another role in elevation of inhibitory effect of salt stress on E. coli growth when sodium chloride concentration was 20 g/l in the medium. Therefore, with the increase of audible sound waves frequency, the inhibitory effect gradually increased (see Fig. 4A). However, compared with the sample without audible sound stimulation, it is interesting to note that the higher relative colony forming efficiency appeared in the treatment group when sodium chloride concentration reached 3% in the medium (see Fig. 4B). In terms of our experimental, the fact that with the gradually increasing of sodium chloride concentration, the total intracellular level of Na+ ion would keep going up under audible sound wave stimulation. We speculated that the gradual increase in sodium ion is likely to evoke a stimulation of the growth of E. coli by means of some unknown mechanisms, such as mazEF system, which takes charge of programmed cell death in bacteria under stressful conditions [31], may be inactivated or suppressed, some special stress-related enzymes are possibly induced [32], and/or the Ms channel may be partly closed, and so on. Matsuhashi et
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passing audible sound waves from air to liquid medium, in our previous study, we also failed to observe biological effects of audible sound waves on bacterial cells grown in liquid culture under the above-mentioned experimental conditions. But, the great interest has been aroused by the influence of audible sound stimuli on the growth of E. coli in plates. In order to better understand the biological effects of audible sound waves, here we investigated the response behavior of bacterial cells to audible sound under both normal conditions and environmental stresses. The results showed that the audible sound treatment significantly promote the growth of E. coli under the normal growth condition. However, under the osmotic stress induced by sugar, audible sound stimulation markedly inhibited the E. coli growth. More interestingly, action of audible sound wave probably swayed the results of responses of E. coli to salinity stress. On one hand, the audible sound stimulation may evoke an inhibition of the growth of E. coli when sodium chloride concentration was 20 g/l in the medium. On the other hand, with increasing NaCl concentration up to 30 g/l, the action of audible sound waves may play a positive role in the alleviation of inhibitory effect of salt stress on E. coli growth. Some potential mechanisms may be involved in the responses of bacterial cells to audible sound stimulation: Firstly, audible sound stimulation may induce the motion of the cell internal fluid and make significant changes on the cellular plasma membrane structure and physical states. These processes are helpful to the modulation of membrane traffics, and the acceleration of metabolism activity. Secondly, under the osmotic stress, the actions of audible sound possible play a positive role in the increase of water efflux. The rise in efflux of water should be responsible for the enhancement of inhibitory effect of osmotic stress on E. coli growth. Thirdly, under salt stress accompanied with audible sound stimuli, the increase in efflux of water may be just one reason to lead to the enhancement of inhibitory effect of salt stress on E. coli growth. A rise in influx of sodium ion through MscL and sodium channel proteins probably plays another role in the subsequent biological effects. In addition, other unknown complicated mechanisms should not be ignored. They may also be involved in the interaction between bacterial cells and audible sound. Our future research will be directed toward more in-depth understanding of audible sound-biophysics mechanisms in single-cell organism’s research system. Fig. 4. The influence of audible sound stimulation on responses of E. coli to salt stress. The medium consists of 0.5% beef extract, 1% peptone and 2% agar besides NaCl, and pH was controlled at 7.2. (A) Experimental result when sodium chloride was maintained by 2%. (B) Experimental result when sodium chloride was maintained by 3%. Each point is the average of four replications, and vertical bars represent one SD on either side of the average.
al. also reported similar findings that single sine sound waves with appropriate frequency produced by a speaker could dramatically promote colony formation of Bacillus carboniphilus under non-permissive stress conditions of high potassium chloride concentration and high temperature [33,34]. Moreover, as well as our experimental phenomena, their results imply that the sound waves with low frequency can significantly accelerate cell proliferation, while the sound waves with relatively high frequency slow it down. 4. Conclusions Audible sound wave, one of the environmental factors, widely exists in the world. It undoubtedly interacts with all living beings and affects the growth and metabolic behaviors of organism. Very little efforts have been put forth in studying the relation of organisms and audible sound, although many phenomena supporting the claim that living organisms acknowledge and respond to music have been observed. Owing to the most energy is reflected when
Acknowledgements This investigation was supported by the Startup Foundation for Advanced Talents of Henan University of Science and Technology (06-021) and Natural Science Foundation of Henan Educational Committee (2008A180007). We gratefully acknowledge Dr Burong Hu for helpful discussions and critical reading of the manuscript. References [1] A.F. D˘anet¸, Environmental Pollution Monitoring: Pollution, Analysis, Legislation, Quality Assurance and Managing, S.C. Pro Act Birotic S.R.L., Bucharest, 2005, pp.56–64. [2] A. Davies, Acoustic trauma: bioeffects of sound. http://schizophonia.com/ installation/trauma/trauma thesis/index.htm, 2009. [3] T.G. Leighton, What is ultrasound? Prog. Biophys. Mol. Biol. 93 (2007) 3–83. [4] W.D. O’Brien Jr., William, Ultrasound–biophysics mechanisms, Prog. Biophys. Mol. Biol. 93 (2007) 212–255. [5] E.V. Rokhina, P. Lens, J. Virkutyte, Low-frequency ultrasound in biotechnology: state of the art, Trends Biotechnol. 27 (2009) 298–306. [6] T.A. Whittingham, Medical diagnostic applications and sources, Prog. Biophys. Mol. Biol. 93 (2007) 84–110. [7] G. Leventhall, What is infrasound? Prog. Biophys. Mol. Biol. 93 (2007) 130–137. [8] A.J. Anastassiades, A.D. Petounis, Infrasonic analysis of carotid vibration as a diagnostic method in carotid insufficiency syndrome, Phys. Med. Biol. 21 (1976) 128–133. [9] P. Raven, Therapeutic Infrasound: A Collection of 3 Research Papers, Copyright © 2002–2009 Lulu, Inc., 2007, pp. 107–212.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Short communication
A pilot study of the effect of audible sound on the growth of Escherichia coli Gu Shaobin a,∗ , Ying Wu a , Kewei Li a , Shichang Li a , Shengyun Ma b , Qiannan Wang a , Rong Wang c a
College of Food and Bioengineering, Henan University of Science and Technology, No. 70, Tianjing Road, 471003 Luoyang, Henan, People’s Republic of China College of Life Sciences, Zhejiang University, 310029 Hangzhou, People’s Republic of China c College of Life Sciences, Wuhan University, 430072 Wuhan, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 25 December 2009 Received in revised form 22 February 2010 Accepted 25 February 2010 Available online 4 March 2010 Keywords: Audible sound Response Bacterial cell
a b s t r a c t Audible sound, one of the environmental factors, widely exists in natural world. However, the interaction between audible sound and biological materials is usually neglected in the field of biological research. Very little efforts have been put forth in studying the relation of organisms and audible sound. Here we investigated the response of Escherichia coli cells to the stimulation by audible sound under the normal condition and environmental stresses. The results showed that the audible sound treatment significantly increases the colony forming of E. coli under the normal growth condition. However, under osmotic stress induced by the sugar, audible sound stimulation may enhance the inhibitory effect of osmotic stress on E. coli growth. More interestingly, audible sound treatment seems to alleviate the inhibitory effect of salt stress on E. coli growth when the concentration of sodium chloride was increased to 30 g/l, although the action of sound waves of audible frequency is likely to evoke an inhibition of the growth of E. coli in the medium containing 20 g/l of sodium chloride. Some potential mechanisms may be involved in the responses of bacterial cells to audible sound stimulation. © 2010 Elsevier B.V. All rights reserved.
1. Introduction A sound wave is similar in nature to a slinky wave for a variety of reasons. Since a sound wave is a disturbance which is transported through a medium via the mechanism of particle interaction, a sound wave is characterized as a mechanical wave [1]. In the natural world almost all living organisms are “immersed” in a variety of sound waves, and interact with them [1,2]. In terms of the sound frequency, there are roughly three regimes: infrasound (10−4 –20 Hz), audible sound (20–2 × 104 Hz) and ultrasound (2 × 104 –1012 Hz). Ultrasound-induced biological effects and its biophysical mechanisms have been extensively investigated in recent decades [2–4]. It has already been successfully combined with biotechnology with the aim of enhancing the efficiency of bioprocesses [5]. Moreover, ultrasound is also used widely in medicine as both diagnostic and therapeutic tools [6]. Although infrasound induced bioeffects and its potential mechanism are still not very clear [7], some significant progresses have been made in the application field, e.g. infrasound diagnosis and therapeutic infrasound [8,9]. So far the study for audible sound is usually focused on how sound is produced, absorbed, reflected, as well as transmitted by objects. Little research concentrates on audible sound induced bio-
∗ Corresponding author. Tel.: +86 379 64283928; fax: +86 379 64282342. E-mail address: [email protected] (G. Shaobin). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.02.028
logical effects and its possible mechanism. Recently the biological effects induced by audible sound wave in plants were investigated [10–12]. To explain the potential mechanism, a hypothesis on plant meridian system was put forward [11]. However, the plant meridian system based on the human body meridian theory is complicated and imperfect. It is not proper to understand the mechanism of biological effects induced by audible sound waves. Furthermore, the complexity of multicellular organisms has also brought great difficulties to study of the audible sound waves biological effects mechanism. Here we attempted to use the single-cell organisms, Escherichia coli to investigate the biological effects of organism after audible sound waves stimulation and understand the potential mechanism. The research has positive significance for us to get more information about audible sound wave biological effects and reveal its underlying mechanism. 2. Materials and methods 2.1. Bacterial strain and culture conditions E. coli, laboratory-preserved strains, was first cultured in slant agar medium (0.5 g beef extract, 1 g peptone, 0.5 g NaCl, 2 g agar, 100 ml distilled water, pH 7.2) at 37 ◦ C for 18 h. Then cells expanded in a 250 ml Erlenmyer flask containing 100 ml of liquid medium (beef extract 0.5 g, peptone 1 g, NaCl 0.5 g, distilled water 100 ml, pH 7.2) with agitation of 200 rpm on a rotary shaking incubator at 37 ◦ C for 12 h.
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G. Shaobin et al. / Colloids and Surfaces B: Biointerfaces 78 (2010) 367–371
¯ c represents the average colonies number from five plates Here N in control group at 22 h after being placed in the sound load cham¯ i represents the average colonies number from five repeat ber. N plates. i represents different times after being put into the chamber. Each experiment had fivefold plate and was independently repeated at least three times. The data were presented as means of relative colony forming efficiency ± SD. 3. Results and discussion 3.1. The effects of audible sound waves stimulation on E. coli growth under normal conditions
Fig. 1. Schematic of sound wave load apparatus. Notes: a – wave generation unit; b – sound wave transfer wire; c – speaker; d – UV lamp (it is used for sterilization before the start of sound stimulation experiments); e – sample holder; f – outer wall of chamber (metal shell); g – inner wall of chamber (made with sound absorbing material); h – motor.
2.2. Sound wave load apparatus To investigate the bioeffects of biological material exposed to the acoustic environment, the sound wave load apparatus was designed (see Fig. 1). This equipment is composed of two parts. One is the sound wave generating unit. The other is sound wave load chamber. The former contains a waveform generator and the amplifying circuit. The signals produced by the waveform generator are amplified and then send to a speaker. The speaker along with UV lamp, rotating sample holder is installed in the sound load chamber whose inner walls are made with sound absorbing material. The rotating sample holder ensures that each sample can get the equal acoustic stimulation. The outer walls of chamber are wrapped by a metal shell in order to reduce influence of environmental noise on the experimental result. When the experiments are performed, the desired audible sound wave is generated by the apparatus and the frequency is precisely adjusted from 1 to 20 kHz. The sound intensity is set according to the experiment design.
People usually neglect the interaction between audible sound and living organisms. It is even more out of the question to study the relation of organisms and sound waves of audible frequency. However, audible sound, an important environmental factor, widely exists in the world. It inevitably affects the growth and metabolic behaviors of organism. Fig. 2 shows effects of audible sound waves stimulation on E. coli growth. The promotion of E. coli growth was obviously observed when bacteria cells were stimulated by audible sound wave. The relative colony forming efficiency reached 141.6%, 130.0% and 131.1% 22 h after stimulation by sound wave with the frequency of 1000, 5000 and 10,000 Hz, separately, which were significantly higher than that of the control (100%). With respect to the positive interaction between audible sound and biological cells, periodic oscillation of bacterial cell induced by audible sound forces may be the first factor to consider. As a mechanical wave, audible sound would produce a mechanical stress to E. coli cells, similar to an alternate pull and press forces. The alternative stress definitely causes the motion of the cell internal fluid and the deformation of cellular plasma membrane [13,14]. At the same time, the membrane fluidity would increase under the sound stimulation [15]. Moreover, sound stimulation makes significant changes on the membrane protein structure [16]. These processes are helpful to the membrane trafficking modulation [17], and the acceleration of metabolism activity [18]. In addition, we also note that the frequency of 1000 Hz of sound wave seems to be the best to promote the E. coli cells to grow.
2.3. Sound stimulation experiments After being cultured in liquid medium at 37 ◦ C for 12 h, the cells of E. coli were spread over to agar plates. Then the samples were put into the sound load chamber and stimulated by 90 dB sound wave with frequency of 1, 5 and 10 kHz separately. Duration of stimulation and interval time were 1 and 3 h, respectively. And the total treatment time (including stimulation and interval time) was 24 h. Samples without audible sound wave treatment served as a control group. The temperature within the sound wave load apparatus was maintained at 37 ◦ C. 2.4. Statistical analyses Samples were collected and colonies were scored after being put into the chamber for 10, 11, 12, 13, 14, 16, 18 and 22 h. The relative colony forming efficiency was calculated according to the following formula: relative colony forming efficiency =
¯i N × 100% ¯ Nc
Fig. 2. The influence of audible sound stimulation on E. coli growth under normal culture conditions. The medium consists of 0.5 g beef extract, 1 g peptone, 0.5 g NaCl, 2 g agar, 100 ml distilled water, and pH was controlled at 7.2. Each point is the average of four replications, and vertical bars represent one SD on either side of the average.
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The results suggested that the action of audible sound may be non-linear, and show obvious frequency peculiarities. The investigations of Sun et al. and Sheng’s show that sound stimulation at a certain frequency and strength promoted the fluidity of membrane wall and membrane lipid in tobacco cells, which would benefit the absorb of nutriment and combination of DNA in S period, as well as synchronizing the cell cycle, which all benefit the growth of cells [19,20]. Zhao et al. found that the promotion effect on Dendranthema morifolium callus growth was most obvious in a frequency of 1000 Hz and strength of 100 dB sound stimulation [21]. It can obviously promote the soluble protein and sugar in cytoplast, which is a sign of high metabolism level and vigorous divide state in sound stimulation administrated D. morifolium callus. In spite of underlying mechanisms of frequency-dependent effects have still remained unknown, more and more investigations still showed that mechanical stress may play an important role in cellular metabolism, gene expression, cell growth and proliferation [18,22–25]. Consequently, concerning to cells, responding to audible sound forces may be the main reason to create biological effects, though it is not very clear that how the audible sound signal is taken over and then transferred to intracellular by the cells to bring about a further series of biological effects. 3.2. The influences of audible sound waves stimulation on E. coli growth under environmental stress The environmental stress response is an important physiological mechanism that protects cells and organisms from stressful changes in their environment. To ensure survival in the face of these afflictions, such as change in temperature, pH, osmolarity, radiation and the concentration of nutrients and toxins, organisms cannot but adapt to changes in their immediate vicinity by responding to the imposed stress. In order to have a thorough knowledge of audible sound waves biological effects, this part was focused on the study of the influences of audible sound waves stimulation on responses of E. coli to environmental stress. 3.2.1. The influence of audible sound stimulation on E. coli growth under glucose induced osmotic stress Under the osmotic stress induced by sugar, the influence of audible sound wave stimulation on the E. coli growth is shown in Fig. 3. The result implied that E. coli growth was severely depressed when glucose concentration remained at 90 g/l in medium. Moreover, audible sound stimulation may enhance the inhibitory effect of osmotic stress on E. coli growth. Compared with the sample without audible sound stimulation, the lower relative colony forming efficiency appeared in the treatment group. Under the osmotic stress induced by sugar, the actions of audible sound possible play a negative role in the stimulation of E. coli growth. Firstly, the opening of aquaporins in hypertonic conditions [26] may bring an inhibitory effect into the growth of E. coli. Secondly, the significant changes on the cellular plasma membrane structure and physical states induced by mechanical forces [14–16,27], was likely to be involved in the increase of water efflux in the abnormal concentration of sugar. Recent studies indicated that the change of plasma membrane protein structure is closely related to frequency of the sound wave [16]. With the increase of audible sound waves frequency, ␣-helix of membrane protein gradually increased, while decreasing -turn. The membrane protein conformation changes denote the increased interaction between membrane protein and lipids and the increased fluidity of membrane. There is no doubt that increases in fluidity correlate with increases in the water permeability of cellular plasma membrane [28]. The rise in efflux of water should be responsible for the enhancement of inhibitory effect of osmotic stress on E. coli growth. Consequently, it was not surprising that gradually reduced relative colony forming efficiency was eas-
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Fig. 3. The influence of audible sound stimulation on responses of E. coli to glucose induced osmotic stress. Notes: 1 – control; 2–9% glucose + 0 Hz; 3 – 9% glucose + 10,000 Hz; 4 – 9% glucose + 5000 Hz; 5 – 9% glucose + 1000 Hz. The culture was composed of 0.5% peptone, 0.5% dipotassium hydrogen phosphate, 9% glucose and 2% agar, and pH was controlled at 7.2. Data were means of relative colony forming efficiency ± SD at 22 h after being placed in the sound load chamber. Each experiment had fivefold plate and was independently repeated three times. Means of relative colony forming efficiency followed by the same letter are not significantly different (P > 0.05, LSD, SAS9.1.3, SAS Institute Inc., 2005).
ily observed with the increase of audible sound waves frequency in the above experiments. 3.2.2. The influence of audible sound waves stimulation on E. coli growth under salt stress The influence of audible sound wave stimulation on E. coli growth under salt stress is shown in Fig. 4. Even though in the absence of audible sound stimulation, the relative survival fraction of samples gradually decreased with the increase of sodium chloride concentration. Salinity stress can imbalance the osmotic potential in E. coli cells generating a water deficit and the influx of sodium may lead to metabolic toxicity. Under salt stress accompanied with audible sound stimuli, the increase in efflux of water by means of simple diffusion and water-selective channels (as mentioned) may be just one reason to lead to the enhancement of inhibitory effect of salt stress on E. coli growth. The activated mechanosensitive ion channel of large conductance (MscL) in response to physical stresses [29,30] was thought to provide an opportunity for an influx of sodium ion. A rise in influx of sodium ion through MscL and sodium channel proteins probably plays another role in elevation of inhibitory effect of salt stress on E. coli growth when sodium chloride concentration was 20 g/l in the medium. Therefore, with the increase of audible sound waves frequency, the inhibitory effect gradually increased (see Fig. 4A). However, compared with the sample without audible sound stimulation, it is interesting to note that the higher relative colony forming efficiency appeared in the treatment group when sodium chloride concentration reached 3% in the medium (see Fig. 4B). In terms of our experimental, the fact that with the gradually increasing of sodium chloride concentration, the total intracellular level of Na+ ion would keep going up under audible sound wave stimulation. We speculated that the gradual increase in sodium ion is likely to evoke a stimulation of the growth of E. coli by means of some unknown mechanisms, such as mazEF system, which takes charge of programmed cell death in bacteria under stressful conditions [31], may be inactivated or suppressed, some special stress-related enzymes are possibly induced [32], and/or the Ms channel may be partly closed, and so on. Matsuhashi et
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passing audible sound waves from air to liquid medium, in our previous study, we also failed to observe biological effects of audible sound waves on bacterial cells grown in liquid culture under the above-mentioned experimental conditions. But, the great interest has been aroused by the influence of audible sound stimuli on the growth of E. coli in plates. In order to better understand the biological effects of audible sound waves, here we investigated the response behavior of bacterial cells to audible sound under both normal conditions and environmental stresses. The results showed that the audible sound treatment significantly promote the growth of E. coli under the normal growth condition. However, under the osmotic stress induced by sugar, audible sound stimulation markedly inhibited the E. coli growth. More interestingly, action of audible sound wave probably swayed the results of responses of E. coli to salinity stress. On one hand, the audible sound stimulation may evoke an inhibition of the growth of E. coli when sodium chloride concentration was 20 g/l in the medium. On the other hand, with increasing NaCl concentration up to 30 g/l, the action of audible sound waves may play a positive role in the alleviation of inhibitory effect of salt stress on E. coli growth. Some potential mechanisms may be involved in the responses of bacterial cells to audible sound stimulation: Firstly, audible sound stimulation may induce the motion of the cell internal fluid and make significant changes on the cellular plasma membrane structure and physical states. These processes are helpful to the modulation of membrane traffics, and the acceleration of metabolism activity. Secondly, under the osmotic stress, the actions of audible sound possible play a positive role in the increase of water efflux. The rise in efflux of water should be responsible for the enhancement of inhibitory effect of osmotic stress on E. coli growth. Thirdly, under salt stress accompanied with audible sound stimuli, the increase in efflux of water may be just one reason to lead to the enhancement of inhibitory effect of salt stress on E. coli growth. A rise in influx of sodium ion through MscL and sodium channel proteins probably plays another role in the subsequent biological effects. In addition, other unknown complicated mechanisms should not be ignored. They may also be involved in the interaction between bacterial cells and audible sound. Our future research will be directed toward more in-depth understanding of audible sound-biophysics mechanisms in single-cell organism’s research system. Fig. 4. The influence of audible sound stimulation on responses of E. coli to salt stress. The medium consists of 0.5% beef extract, 1% peptone and 2% agar besides NaCl, and pH was controlled at 7.2. (A) Experimental result when sodium chloride was maintained by 2%. (B) Experimental result when sodium chloride was maintained by 3%. Each point is the average of four replications, and vertical bars represent one SD on either side of the average.
al. also reported similar findings that single sine sound waves with appropriate frequency produced by a speaker could dramatically promote colony formation of Bacillus carboniphilus under non-permissive stress conditions of high potassium chloride concentration and high temperature [33,34]. Moreover, as well as our experimental phenomena, their results imply that the sound waves with low frequency can significantly accelerate cell proliferation, while the sound waves with relatively high frequency slow it down. 4. Conclusions Audible sound wave, one of the environmental factors, widely exists in the world. It undoubtedly interacts with all living beings and affects the growth and metabolic behaviors of organism. Very little efforts have been put forth in studying the relation of organisms and audible sound, although many phenomena supporting the claim that living organisms acknowledge and respond to music have been observed. Owing to the most energy is reflected when
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