Hygienic Environment of Railway Stations – Investigation and Countermeasures for Reduction of Microbial Volatile Organic Compounds (MVOCs) Generated by Fungi in Railway Stations – T. Kyotani, T. Kawasaki, T. Ushiogi, Y. Izumi, K. Fujinami, H. Endo, H. Suzuki, and T. Hayakawa Railway Technical Research Institute, Kokubunji-shi, Tokyo, Japan

Abstract

It was made clear that many railway customers thought the major important factors which influence comfort of stations were “odors” and “clearness of air”. We also found that there was a high correlation between the evaluation of customers about odors and the amount of fungi floating in the air at several points of stations. So we consider that microbial volatile organic compounds (MVOCs) generated by fungi partially cause odors of stations. During cultivation of Aspergillus versicolor, Penicilluim sp., and Wallemia sp. MVOCs were detected by using SPME (solid phase microextraction) combined with GC-MS (gas chromatography-mass spectrometry). We tried to analyze odorous compounds in the air of underground platform of a railway station by using the same method. Many compounds could be detected, and some of them were detected during cultivation of fungi. The countermeasure we are now thinking about for reduction of odorous compounds and for inhibition of growth of fungi, is to introduce mirror duct or photocatalyst (TiO2, etc) to railway stations. We expect that more comfortable station space can be presented to customers with less labor by introducing them.

Introduction

The railway facilities, which are highly public, have many kinds of closed space (underground space, tunnels, etc.). So we think it is very helpful to understand, evaluate and improve hygienic environment in closed space of railway facilities, in giving railway customers safer and more comfortable railway services. Some studies on gaseous or hygienic environment of airplane and cars are reported [1, 2, 12, 13]. However, there are only a few cases of researches in which the railway customers’ consciousness and needs concerning the hygienic environment of railways are investigated or analyzed scientifically and technically [5, 14]. So we have thought that, in order to improve hygienic environments of railway facilities, it is necessary to understand customers’ sense of them and to extract factors which give influence on them. So we started the investigation about the customers’ sense of the hygienic environments in station buildings, and also about microbial and chemical environments [7, 8, 9]. As a result of the investigation of the customers’ sense by mail, it was found out that about 20% of the answerers pointed out “atmospheric environment” to the question, “What is the important factor do you think about the comfort of stations?”, and about 10% of them answered that it is “very uncomfortable”, to the question, “Do you have any sense about atmospheric environment in station buildings?” Furthermore, about 30% of them gave “fungous odors” as an example of bad odors [14]. So we carried out environmental investigation about microorganism which is considered as one of the factors to have influence on the quality of air of stations. As a result of investigation, we found out that more amounts of fungi floated in the air of underground platform than that in the air aboveground platform [6]. Also, we found high correlation between the subjective answers to the question, “Are you worried about odors of this place?”, and the amount of fungi floating in the air of the same place. On the other hand, we found low correlation between the same answers and the amount of bacteria in the air of the same place (data not shown). These findings suggest that microbial volatile organic compounds (MVOCs) generated by fungi may be a part of causes of odors of station yards.

In this paper, we will report, 1. the results of investigation of the kinds of fungi collected from the air of a railway station, 2. whether MVOCs were detected or not during experimental culture of these fungi, and 3. the results of experimental simple sampling and qualitative analysis of odorous compounds

in the air of a railway station We will also introduce briefly the countermeasures we are now thinking about for reduction of MVOCs and for inhibition of growth of fungi, namely the ones using mirror duct system in a station simulator or photocatalyst.

Research Methods

Railway station

Station A was chosen as a model station, because it has both concourses and platforms in underground and aboveground spaces respectively.

Collection and Identification of airborne fungi

An air sampler was used to collect airborne microorganisms [5, 6]. The air sampler was installed at a position about 150 cm above the floor as shown in Figure 1, and was set so as to take in 200 L of air. Airborne microorganisms in 200 L of air were captured on agar plates that were set in the air sampler. Dichloran-glycerol agar (DG18) media were used. The sample plates were cultivated at 25 °C for 5-14 days. After a pure culture of fungi was extracted from the original sample agar plates, the types of fungi were identified morphologically. Airborne microorganisms in 200 L air in the atmosphere immediately out side the station were also collected on the same day as a control.

Methods for analysis of MVOCs exhausted during cultivation of fungi

The fungi, collected from the air of the underground platform of Station A and purified, were cultured on about 50 ml of DG18 agar prepared in 500 ml Erlenmeyer flasks at 25 °C. During incubation, the volatile compounds in the headspace of the flasks were extracted with SPME (solid phase microextraction) fibres (75 μm Carboxen / PDMS (polydimethylsiloxane), Sigma-Aldrich, Figure 2) [3, 4, 10, 11, 15] for 1 hour, and were analyzed by using GC-MS (gas chromatography-mass spectrometry, Agilent Technologies, HP6890/5972, Figure 3). Before using, the fibres were conditioned in the GC injection port at 310 °C for about 1 hour. GC was equipped with a split / splitless injection port, a Merlin MicrosealTM High Pressure Septum, a 0.75 mm i.d. liner, and a HP-5MS (Agilent Technologies, 50 m × 0.2 mm i.d., 0.33 μm film thickness) as a GC column. Carrier gas was helium with a flow rate of 1.0 mL/min. The oven temperature was programmed as follows: held at 45 °C for 9 min, then raised to 140 °C at 6 °C /min, then further raised to 300 °C at 15 °C /min and held for 3 min. The temperature of injector was 310 °C; that of the transfer line to MS, 280 °C; that of MS ion source, 230 °C; and that of MS quadrupole, 150 °C. The injector was used in splitless mode, and desorption time was 1 min. The MS scan parameters were set to a range of 29-210 (m/z) and the acquisition mode was TIC (total ion chromatography).

Methods for analysis of airborne compounds in the air of the underground platform of Station A

At the end of the underground platform of Station A, SPME fibres (75 μm Carboxen / PDMS and 65 μm DVB (divinylbenzene) / PDMS) were installed at a height of about 150 cm above the floor, and made them extract airborne compounds for 3 hours (as shown in Figure 4). When we carried the fibres, we placed their needles in septa (Thermogreen LB-2 (diameter: 5 mm), Sigma-Aldrich) and put them in trays made of SUS to keep them from contamination. The conditions of analysis by GC-MS were described above.

Figure 1: Scene of collection of airborne microorganisms in the station.

Figure 2: SPME (Solid phase microextraction) fibre.

adsorbent

Figure 4: Scene of collection of airborne compounds with SPME fibres.

Figure 3: Image of analysis of MVOCs with SPME fibres.

Table 1: Detection of MVOCs exhausted during cultivation of fungi collected from air of the underground platform of Station A.

Figure 5: The example of GC-MS data in the headspace of flasks during cultivation of Aspergillus versicolor

Results and discussion

Fungi detected from the underground platform of Station A

Table 1 shows the fungi collected from the underground platform of Station A and whether MVOCs were detected during cultivation of these fungi. Among the detected fungi (5 species), 3 species, Aspergillus versicolor, Penicilluim sp., Wallemia sp., were detected to exhaust MVOCs. On the other hand, during cultivation of Cladosprium sp. and Aspergillus ochraceus, few amounts of MVOCs were detected. With respect to Wallemia sp., it had been found out that the rate of its existence in the air of station yards was very low [6]. We had also found that Aspergillus versicolor showed higher rate of detection in the air of the underground platform than in that of the aboveground platform [6]. So, at first, we carried out qualitative analysis of MVOCs exhausted during cultivation of Aspergillus versicolor.

Species of fungi Detection of MVOCs

(detected : ○、not detected : × )

Aspergillus versicolor ○

Penicilluim sp. ○

Wallemia sp. ○

Cladosporium sp. ×

Aspergillus ochraceus ×

Isobutanol

Camphene

2-Methyl-1-butanolIsoamylalcohol

1-Octen-3-ol

Camphor

Table 2: MVOCs identified from the data of Figure 5

Figure 6: The example of GC-MS data of the air in the underground platform of Station A.

MVOCs exhausted during cultivation of Aspergillus versicolor

Figure 5 shows the example of GC-MS data of gaseous compounds exhausted in the headspace of flasks during cultivation of Aspergillus versicolor collected from the air of the underground platform of Station A, and Table 2 shows MVOCs identified from the data. It was confirmed that many kinds of compounds, for example, alcohols and terpenes were exhausted to the headspace. Especially, some alcohols of which boiling points are relatively low (isobutanol which is regulated by Offensive Odor Control Law in Japan, isoamylalcohol (3-methyl-1-butanol), and 2-methyl-1-butanol) were found to have intensive peaks [9].

2-Methyl-3-buten-2-ol α-Pinene

Isobutanol Camphene

1-Butanol β-Pinene

Isoamylalcohol (3-Methyl-1-butanol) Limonene

2-Methyl-1-butanol Isoprene

1-Octen-3-ol Dichloromethane

3-Octanone 3-Methylfuran

Camphor (m-, or p-)Dichlorobenzene

Toluene

Ethylbenzene

XyleneStylene

Trimethylbenzene

2-Ethyl-1-hexanol

Cyclohexane

Table 3: Gaseous compounds identified from the data of Figure 6.

Analysis of the air in the underground platform of Station A

Figure 6 shows the example of GC-MS data of the air in the underground platform of Station A, and Table 3 shows compounds identified from the data. Many compounds were detected, and some of them were detected during cultivation of fungi (listed in Table 2). But the experiment in our laboratory was done with DG18, artificial broth, and the origins of generation of compounds detected from stations are considered to be not only fungi, but also building materials, paints, plants, and so on. Furthermore, some peaks were observed which correspond to some unknown compounds. Therefore, at present, we do not know the correlation between MVOCs exhausted by fungi and the compounds detected from the air of the underground platform of stations. We consider it is necessary to do experiments with other substrates for culture of fungi and to accumulate data of airborne compounds in the station to find correlation mentioned above.

Ethyl Acetate Stylene Methylpropylbenzene

Cyclohexane Cumene Diethylloluene

Trichloroethylene α-Pinene Camphor

Methylcyclohexane Ethyltoluene 2-Ethyl-1-hexanol

Methyl isobutyl ketone Trimethylbenzene Nonanal

Toluene Benzofuran Isomenthol

Tetrachloroethylene Dichlorobenzene (Alkanes)

Butyl acetate Indane (PAHs)

Ethylbenzene Indene (Sesquiterpenes)

Xylene Butyl Butylate

Station Simulator and Mirror Duct

We found that many kinds of compounds existed in the air of Station A. In future, we would like to investigate the followings: 1. Which compounds are the causes of unpleasant odors for railway customers? 2. To what extent the collected fungi contribute to production of these compounds? 3. What kind of countermeasure could we take? The countermeasure we are now thinking about for reduction of odorous compounds and for inhibition of growth of fungi, is to introduce mirror duct or photocatalyst (TiO2, etc) to railway stations. Mirror duct system (Material House Co. Ltd., Tokyo, Japan) is the one which takes sunlight into the duct of which inner surface is made of mirror, and by reflection, supplies the light into rooms, underground spaces, etc. without artificial energy (electricity, etc.). There are two types of mirror duct by the range of wavelength of the supplied light. One is called “normal type” for normal lighting which does not transmit UV light, and the other called “UV-highly-transmittable type” which can transmit UVA and UVB light. We expect that more comfortable station space can be presented to customers with less labor (ventilation, cleaning, etc.) by introducing “UV-highly-transmittable type” mirror duct to platforms, concourses, lavatories, etc. of railway stations. At present, there are not many facilities of which this system was installed because the cost for installing it is expensive and it is very difficult to install it in existing buildings. But it is expected to be used widely in future from the point of view of environmental

Figure 7: The overview of a station simulator.

protection of the earth. We have constructed a station simulator which reproduces an over-track station building in our institute (Figure 7), and have installed mirror ducts in it (Figure 8). We are going to examine the effect of them on reduction of odorous compounds and inhibition of growth of fungi (with and without photocatalyst) sampled from the air in stations in future. We are also under study whether the instrument for sampling gaseous compounds, which we are now using because of its small size and for its easiness to use, can be applied to searching the sources of odors in railway stations, by putting a lot of instruments to various positions of stations, and comparing the amounts of detected compounds. We are now doing preliminary examination in the station simulator described above. If this study generates practicable results, it will be expected that atmospheric environment of stations will be improved more effectively because the irradiation of light to the sources with mirror duct can be made more concentratively.

Conclusions

In future, we will investigate airborne fungi and gaseous compounds furthermore and make clear the correlation between them, in order to identify factors which produce a bad effect on the comfortable environment of stations. Then we will consider the countermeasures for reduction of the effect in order to improve comfort for passengers of railway stations.

Acknowledgements

We thank Dr. Noritoshi Ri, Hygiene & Microbiology Research Center, Tokyo, Japan, for cooperating with us in identifying and cultivating fungi. This research project is funded in part by the Japanese Ministry of Land, Infrastructure and Transport.

Figure 8: A mirror duct (UV-highly-transmittable type) in the station simulator shown in Figure 7.

References

[1] M. Dechow, et al, “Concentrations of selected contaminants in cabin air of Airbus aircrafts”, Chemosphere, 35, pp. 21-31, (1997)

[2] TS. Dumyahn, et al, “Comparison of the Environments of Transportation Vehicles: Results of two Surveys”, ASTM special tech. pub. pp. 3-25, (2000)

[3] K. Fiedler, et al, “Detection of microbial volatile organic compounds (MVOCs) produced by moulds on various materials”, Int. J. Hyg. Environ. Health, 204, pp. 111-121, (2001)

[4] M. Hippelein, “Analysing selected VVOCs in indoor air with solid phase microextraction (SPME): A case study”, Chemosphere, 65, pp. 271-277, (2006)

[5] T. Kawasaki, et al, “Fundamental examination of hygienic assessment of the railway station environment”, Proceedings at the 6th ICOH International Conference on Occupational Health Care Workers, Kitakyushu, Japan, p. 191, (2004)

[6] T. Kawasaki, et al, “Trial examination of a method to evaluate the environment in railway stations” presented at The 10th International Conference on Indoor Air Quality and Climate, Beijing, China, September 4-10, Paper pp. 859-863. (2005)

[7] T. Kawasaki, et al, “Hygienic assessment of the railway station environment,” The 79th annual meeting of Japan Society of Occupational Health, (2006, in Japanese)

[8] T. Kawasaki, et al, “Hygienic assessment of the railway station environment (2),” The 80th annual meeting of Japan Society of Occupational Health, (2007, in Japanese)

[9] T. Kyotani, et al, “Analysis of VOCs originated from fungus collected in a railway station by SPME-GCMS method,” The 80th annual meeting of Japan Society of Occupational Health, (2007, in Japanese)

[10] V. Larroque, et al, “Development of a solid phase microextraction (SPME) method for the sampling of VOC traces in indoor air”, J. Environ. Monit., 8, pp. 106-111, (2006)

[11] T. Nilsson, et al, “Application of head-space solid-phase microextraction for the analysis of volatile metabolites emitted by Penicillium species”, J. of Microbiol. Methods, 25, pp. 245-255, (1996)

Mirror duct

[12] LJ. Rose, et al, “Volatile organic compounds associated with microbial growth in automobile air conditioning systems”, Current Microbiol., 41, pp. 206-209, (2000)

[13] RB. Simmons, et al, “The occurrence and persistence of mixed biofilms in automobile air conditioning systems”, Current Microbiol., 39, pp. 141-145, (1999)

[14] H. Suzuki, et al, “”, RTRI Reports, 19-1, pp. 15-20, (2004, in Japanese) [15] L. Tuduri, et al, “Dynamic versus static sampling for the quantitative analysis of volatile

organic compounds in air with polydimethylsiloxane-Carboxen solid-phase microextraction fibers”, J. Chromatogr. A, 963, pp. 49-56, (2006)