Noise is ubiquitous in present-day civilization and it reached an alarming level [1, 2], especially in cities, industrial settings, and traffic, but also in other areas of human activity. Commonly used machines like tractors and locomotives produce 95db (load) and 84db (idle) at a 3m distance, respectively (personal measurements, PCE-MSL1 (PCE Instruments)) with many more examples as chain saw, lawn mover, leaf vacuum/blower, etc. All of these machines are used in soil proximity having an opportunity to influence soil organisms. The influence of sound is well documented on vertebrates [3] including humans in which the effects of rhythm were also documented [4]. On the other side effect of sound on microbes was rarely studied. In a recent mini-review Robinson et al [5] presented only eight papers. Besides a couple of papers investigating sound effects on gut microbiomes of rats [6] and birds [7], we are aware of only one paper that investigated sound effects on bacteria isolated from soil [8]. To the best of our knowledge, we are the first that measured the effects of noise on soil microbial communities.
In soil, microbes often form aglomerates of bacterial cells/biofilms which offer protection and survival in changing environmental conditions while at the same time influencing soil properties by decreasing soil porosity [9] or by increasing cohesion between soil particles [10]. So by influencing soil biofilm formation sound could change soil properties degrading or improving soil quality.
We hypothesized that microbes from agricultural soils would be less affected by noise compared to forest soils of the same soil type. Also, we investigated the effects of different sound types like music and noise, and rhythm on the biofilm-forming ability of soil microbes.
The sound apparatus consisted of extruded polystyrene foam boxes (34x31x25cm, wall thickness 3cm) with the speaker on top of it (Magnat MCOS80 CP460). Sound sources were two laptops and a mobile phone while we were using three amplifiers: Fender Rumble 15, Ibanez GTA10, and Panasonic SA-HE7. The distance between the sample and the speaker was 20cm and the noise level between replicates was checked by a sound level meter PCE-MSL1. To prevent noise leaking, the box was placed inside a larger expanded polystyrene foam box (60x50x30cm, 10cm wall thickness). To minimize the temperature difference between treatment and control boxes (maximum difference was 0.5°C) control speaker was connected to a 1.5V direct current source instead to an amplifier. Each treatment was repeated three times with three technical replicates per treatment.
The sound patterns tested were different regarding their complexity. For a test of dose-response (duration of exposure), we have chosen a sine wave at 4000Hz (90dB SPL; for 2, 4, 6, and 21h). In the preliminary test, this frequency had the largest effect on biofilm-forming ability (compared to 200Hz and 1000Hz). We also tested the classical music of Mozart and Beethoven (90-115dB SPL; for 24h), the noise of road and construction works (90-100dB SPL; for 8h), and regular (120 beats per minute, 4/4) and irregular rhythm (120 beats per minute, 7/8) played on drums (90dB SPL; 6h). A list of all used music, sound patterns of construction, road work noise, and regular and irregular rhythm is in the online supplementary material.
Soil samples of two soil types (luvisol and gley) were collected in deciduous forest and surrounding fields. Dry soil was rewetted seven days before the experiment. Soil samples were weighed before the experiment (305 ±5mg) in sterile plastic centrifuge tubes (2ml) and exposed to noise with an open cap. The preliminary experiment showed no significant evaporation occurred during a time course of experiments (6 to 24h).
The biofilm-forming ability (BFA) of the soil was determined by the method of Golby et al [11] with few modifications. The flat-bottom polystyrene 48-well plates were grinded [12] with autoclaved gravel (0.8-1.2mm) on a rotary shaker at 400rpm for 30 minutes, rinsed with 96% ethanol, and dried before use. The growth medium consisted of 2ml BG110 medium, 10µl MgSO4 1M, 50µl glucose 22.2mM, 10µl nutrient broth, 50µl 100mM arginine, and 2.88ml of sterile distilled water. After noise exposure, soil samples were mixed with 1.5ml of sterile distilled water and vortexed and further processed as described elsewhere [13].
Statistical analysis was conducted in Microsoft Excel and R-Studio. The results were analyzed by conducting a Mann-Whitney U-test and Kruskal-Wallis test followed by corrected pairwise comparisons.
In the dose-response experiment forest soil responded quicker than the field soil (Fig. 1). Music of Mozart decreased the BFA of gley soil type (Fig. 2a). Noise of construction and road works decreased BFA in all samples but significantly only from the field (Fig. 2b). Regular rhythm increased BFA in all samples but in one sample increase was not statistically significant (Fig. 2c).
To the growing list of soil pollutants (e.g. fertilizers, pesticides, nanomaterials, microplastics, or microglass) we should add noise as one of the pollutants with the potential to significantly impact soil microbial communities. The effect of sound on bacteria was scarcely investigated even in pure culture conditions and the effect of sound on the “in situ” microbial communities was reserved for investigations of gut microbiomes of different groups of vertebrates [6, 7]. We present the first evidence that functional properties of the different soil microbial communities developed in different soil types and conditions of agricultural and forest soils were impacted by audible sound on sound levels and environmentally relevant time intervals. Dose-response experiments support our hypothesis that agricultural soil is less affected by noise compared to forest soil of the same soil type. This was only partially corroborated when more complex sound patterns were tested. Noise from construction and road works destimulated BFA only in samples from agricultural soils, while the classical music of Mozart destimulated BFA only in gley samples. But the most striking was the effect of the regular rhythm which increased BFA regardless of the soil type and use up to 83% compared to the control. A stimulatory effect on biofilm formation was noted in pure cultures at 800Hz [14] while Gu et al [15] found stimulation of bacterial cell size and biomass at 8000Hz. Gu et al [16] tried to explain the effects of audible sound on bacterial cells by modulation of membrane traffic which could be related to biofilm formation as bacterial mechanosensitive channels are related to biofilm formation [17]. In which way regularity of sound stimulation enhances biofilm formation is yet to be investigated.
An important feature of all conducted experiments is that sound initiated change in BFA which lasted after exposure to noise ended which means that sound represented a signal that induced changes in the soil microbial community that were not related to physical effects of vibration, e.g. vibration enhancement of gas exchange [18]. This raises questions like how long change in BFA lasts after noise exposure ended, or what are the effects of other sound properties like sound pressure level, frequency, duration of exposure, or rhythm properties like beats per minute? These are all questions that should be addressed in the future to better understand the entirety of soil microbial ecology simply because nature does not have a remedy for noise pollution in the sense it has for chemical pollutants which would be degraded with time while the environment is self-purified.