Journal of Occupational Health

Advance Publication Journal of Occupational Health

Accepted for Publication: Mar 24, 2012 J-STAGE Advance Published Date: Apr 26, 2012 Bacterial hazards of sludge brought ashore by the tsunami after the Great East

Japan Earthquake of 2011

Koji Wada1*, Kazumasa Fukuda2, Toru Yoshikawa3, Toshio Hirose4, Takako Ikeno2,

Toshiyuki Umata5, Toshiya Irokawa6, Hatsumi Taniguchi 2, Yoshiharu Aizawa7

1. Department of Public Health, Kitasato University School of Medicine,

Sagamihara, Japan

2. Department of Microbiology, University of Occupational and Environmental

Health, Kitakyushu, Japan

3. International Cooperation Center, Institute for Science of Labour, Kawasaki, Japan 4. Sendai Nishiki‐cho Clinic and Occupational Health Center, Sendai, Japan

5. Radioisotope Research Center, University of Occupational and Environmental

Health, Kitakyushu, Japan

6. Center for Environmental Conservation and Research Safety, Tohoku University

and Department of Occupational Health, Tohoku University, Graduate School of

Medicine, Miyagi, Japan

7. Department of Preventive Medicine, Kitasato University School of Medicine,

Sagamihara, Japan

*Corresponding author: Koji Wada, MD, MSc, PhD, Department of Public Health,

Kitasato University School of Medicine, 1‐15‐1 Kitasato, Minami‐ku, Sagamihara,

Kanagawa 252‐0374, Japan

Tel: +81‐42‐778‐9352; Fax: +81‐42‐778‐9257; E‐mail: kwada‐[email protected]

Article type: Original article

Running title: Bacterial hazards of sludge

Number of tables: 5

Number of figures: 1

Abstract

Objectives: The aim of this study was to identify bacteria in sludge brought by the

2011 tsunami in Japan to determine the necessary precautions for workers who

handle the sludge.

Methods: Two sludge samples and one water sample were collected from each of

two sites in Miyagi Prefecture in June 2011. We also obtained control samples from a paddy field and a dry beach in Fukuoka, Japan. The samples were subjected to physicochemical analyses, conventional cultivation methods, and molecular methods for bacterial flora analysis. The bacterial floras were analyzed using a clone library method employing fragments of the 16S ribosomal RNA gene (rDNA)

amplified with universal primers.

Results: We detected 51−61 genera in sludge samples and 14 and 17 genera in water samples collected in the tsunami‐affected areas. In sludge samples collected in the tsunami‐affected areas, more genera belonged to Proteobacteria than to Bacteroidetes, but in water samples collected in these areas, more genera belonged to Bacteroidetes

than to Proteobacteria. Non‐O1, non‐O139 V. cholerae (non‐agglutinable vibrio) was

found at approximately 104 cells/mL near the coast of the tsunami affected area.

Sulfate‐reducing bacteria were detected in sludge collected from the paddy field,

and a relatively high concentration of sulfate ions was found in the water sample

(258 mg/L).

Conclusions: Sludge brought by the tsunami contained some pathogens; therefore, frequent hand washing is recommended for workers who have direct contact with the sludge to minimize their risk of infection. Under the anaerobic conditions of paddy fields, hydrogen sulfide could be produced by sulfate‐reducing bacteria

metabolizing sulfate ions.

Keywords: sludge, tsunami, NAG vibrio, hydrogen sulfide, bacterial flora

Introduction

At 14:46 on March 11, 2011, an earthquake occurred off the coast of eastern

Japan. This earthquake, which has since been named the Great East Japan

Earthquake, caused a massive tsunami that resulted in approximately 20,000 deaths

1) and left 23 million tons of debris. The tsunami impacted buildings at elevations of up to 40.5 m in Miyako, Iwate Prefecture2). Many workers, volunteers, and residents

of tsunami‐affected areas have become involved in cleanup efforts.

Sludge brought by the tsunami could contain pathogens with the potential to

harm workers 3). Fukuda et al. analyzed sediment samples collected from Doukai

Bay in Kitakyushu using a culture‐independent clone library method and reported

that the genera Vibrio, Staphylococcus, and Mycobacterium were present4). Using the

same molecular method, Taniguchi et al. identified sulfate‐reducing bacteria that

could produce hydrogen sulfide, which is very harmful 5). To control bacterial hazards associated with sludge brought by the tsunami, it is necessary to identify pathogens and other health risks associated with the sludge. However, to our knowledge, no studies of bacteria in sludge brought by the tsunami have been conducted to date. Therefore, this study was conducted to identify bacteria in sludge brought by the tsunami to determine what precautions workers removing the sludge should take.

Methods

Collection of samples

We collected three samples at each of two sites in Miyagi Prefecture on June 19,

2011: 1) a paddy field impacted by the tsunami located 300 m from the coast in

Shichigahama (A‐1 (wet sludge), A‐2 (dry sludge), and A‐3W (water)) and 2) a waste

dump in Tagajo, where debris and sludge were stored for subsequent transport to a

final disposal site (B‐1 (dry sludge), B‐2 (wet sludge), and B‐3W (water)). Figure 1 shows a map of the locations from which samples were taken.

We also obtained control samples in the city of Fukutsu, Fukuoka Prefecture,

Japan, which was not affected by the tsunami, on June 24, 2011. Two samples (C‐1

and C‐2W) were taken at a paddy field and two samples (C‐3 and C‐4W) were obtained at a dry beach along the coast of the Genkai Sea.

All samples were collected from the surface at each site and immediately stored in sterile tubes at 4°C until analysis.

Physicochemical tests

The pH, electrical conductivity, total organic carbon, total nitrogen, anions

(Cl−, SO42−), and leachability of the samples were determined. Briefly, leachates were

obtained from 5 g aliquots of dried soils that had been added to 45 mL of water and

then vertically shaken at 170 rpm for 6 h. The leachate was then passed through a

filter with a mean pore diameter of 1 μm. All samples were subsequently analyzed

using an Iwaki pH meter (model UB‐10), a Digital Instruments conductivity meter

(model CD‐4307), a Shimadzu total organic carbon and total nitrogen analyzer

(model TNPC‐4110), a Dionex ion chromatograph (model DX‐AQ), and a Seiko

inductively coupled plasma analyzer (model SPS 1500R). Analytical grade reagents

such as metal and anion standards were purchased from Wako Pure Chemical

Industries (Osaka, Japan).

Epifluorescence staining and cell lysis efficiency

Cell concentrations (cells/mL (water) or g (soil)) were determined by

epifluorescence staining using ethidium bromide5‐7). Briefly, 100 μL samples were

added to 900 μL of ethidium bromide solution (100 μg/mL) and left to stand for 10

min at room temperature, after which the mixture (1.0 mL) was passed through a 0.2

μm pore filter (Millipore, Bedford, MA, USA). Objects shaped like bacteria on the

filter were counted with the aid of an Olympus BX50 microscope (Olympus Optical,

Tokyo, Japan), and the number of bacteria per milliliter of solution or gram of soil

was calculated.

After DNA extraction (described in the “DNA extraction” section), the

remaining bacteria were counted in the same manner. The cell lysis efficiency was calculated as the ratio of the number of bacteria remaining after the DNA extraction treatment to the total number before treatment (100 − [postextraction number/pre‐extraction number] × 100). DNA extracted from samples that showed

more than 80% cell lysis was used for analysis of bacteria.

Cultivation methods

Aerobic bacteria were counted after incubation on standard agar medium

(yeast extract, 0.5 g/L; peptone, 1.0 g/L; glucose, 0.2 g/L; agar, 15 g/L) at 30°C for 6 days 4, 5). Samples were also cultivated on bromothymol blue (BTB) lactose agar

medium, thiosulfate−citrate−bile salts−sucrose (TCBS) agar medium, SSB agar

medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and

Wadowsky−Yee−Okuda agar medium supplemented with α‐ketoglutarate (WYOα)

(Eiken Chemical Co., Ltd., Tokyo, Japan) for at least 48 h at 37°C under aerobic conditions. Colonies on TCBS agar medium were Gram stained, after which the

Gram‐negative samples were identified using an API 20E system according to the manufacturer’s instructions (bioMérieux, Marcy lʹEtoile, France). Colonies on WYOα

agar medium were subcultured on sheep blood agar to identify Legionella spp.

Serotyping of V. cholerae and PCR of cholera toxin gene

One yellow colony on TCBS agar medium was subjected to serotyping using

a V. cholerae AD Seiken kit (Denka Seiken Co., Ltd.) and V. cholerae O139 antiserum

(Denka Seiken Co., Ltd.). The colony was also analyzed for the presence of the V. cholerae toxin gene by PCR using the VCT‐1&2 primers (Takara Bio Inc.).

DNA extraction

To detect pathogenic bacteria that are difficult to culture, such as

mycobacteria, Legionella spp., anaerobes, viable but nonculturable bacteria,

sulfur‐reducing bacteria, and sulfur‐oxidizing bacteria, the bacterial flora of each

sample was analyzed by the clone library method with PCR using universal primers

specific for the 16S ribosomal RNA gene. The soil sample (0.3 g) was vigorously agitated in 3.0 mL of distilled water. A 900 μL aliquot of sample solution was mixed

with 100 μL of 30% SDS solution and approximately 0.3 g of glass beads. The mixtures were then shaken for 5 min at 4500 rpm using a Micro Smash MS‐100 apparatus (Tomy Seiko Co., Ltd., Tokyo, Japan). Next, the samples were centrifuged at 20,000 g for 5 min at room temperature, after which the supernatants were

collected. This DNA extraction procedure was conducted three times, and the mixture of three supernatants was subsequently extracted with an equal volume of phenol−chloroform−isoamyl alcohol (25:24:1 vol/vol). The DNA in the aqueous phases was then concentrated to 30 μL of TE buffer using a Montage PCR centrifugal filter device (Millipore, Bedford, MA, USA).

Clone library construction and nucleotide sequencing analysis

Using the DNA extracts obtained as described above, a fragment of the 16S

rDNA (550 bp) was amplified with the primers E341F

(5’‐CCTACGGGAGGCAGCAG‐3’) and E907R (5’‐CCGTCAATTCMTTTRAGTTT‐3’).

PCR amplification was then conducted using a GeneAmp PCR system 9700 thermal

cycler (Applied Biosystems, Foster City, CA, USA). The cycling conditions were

96 °C for 5 min, followed by 30 cycles of 96°C for 30s, 53°C for 30s, and 72°C for 1 min, with a final elongation step at 72°C for 7 min. The PCR products were then

cloned into Escherichia coli TOP10 cells using a TOPO TA Cloning Kit (Invitrogen,

Carlsbad, CA, USA). A total of 96 white colonies from each sample were randomly

selected, and the inserted PCR product was amplified with M13 forward and reverse

primers. Sequencing reactions were subsequently conducted using a BigDye

Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) with the M13 forward

primer. Nucleotide sequences were determined with a 3130xl Genetic Analyzer

(Applied Biosystems).

Highly accurate sequences were trimmed from the primer and vector regions.

Only sequences showing good quality and whose primer sequences were

successfully trimmed were used for homology analysis. The remaining sequences

were compared with an in‐house database containing only 16S rRNA gene sequences of type strains (5,878 species) obtained from the Ribosomal Database

Project II (http://rdp.cme.msu.edu/) and the DNA Data Bank of Japan

(http://www.ddbj.nig.ac.jp/) using the BLAST algorithm.

Ethics

Ethics committee approval was not required for this study.

Results

Physicochemical tests

Some environmental physicochemical factors influence bacterial multiplication; therefore, we examined five such factors (Table 1). We found that samples A‐2 and A‐3W were slightly alkaline. Additionally, the electrical conductivity of A‐3W was high (1346 mS/m). Total organic carbon was also high in

A‐3W (47.3 mg/L). The chloride (Cl−) concentrations were very high in all samples, especially A‐3W (8503 mg/L) and B‐3W (3396 mg/L), which suggested condensation

of seawater. The sulfate (SO42−) concentrations were high in A‐3W (258 mg/L) and B‐1

(329 mg/L).

Bacterial enumeration

The cell lysis efficiencies varied from 76.9% to 99.8%. Bacteria were enumerated

by three methods (Table 2). Bacterial cell numbers were estimated by epifluorescence

microscopy, which revealed concentrations of 108–109 cells/g in soil and 106–107

cells/mL in water (including control samples). Aerobic culture on BTB lactose agar

medium at 37°C was used to identify lactose‐utilizing enteric bacteria. Samples A‐1,

A‐2, B‐1, B‐2, and C‐1 showed approximately 105 colony forming units (CFU) per milliliter (water) or gram (soil), but lactose‐utilizing bacteria were found only in B‐2, where they accounted for 10% of the colonies. Bacteria were also enumerated by aerobic culture on environmental standard agar medium at 30°C. The results revealed concentrations of 106–108 CFU/g in soil and about 106 CFU/mL in water,

even for the control samples.

Cultivation test for pathogenic bacteria

Cultivation and Gram staining were conducted to detect Escherichia, Salmonella,

Shigella, Vibrio, and Legionella spp. Samples were cultured on SSB agar for detection

of Escherichia, Salmonella, and Shigella spp. Aliquots from samples A‐3W, B‐2, and

B‐3W produced 104 CFU/mL on SSB agar; however, none of these colonies were

suspected pathogens. Aliquots from A‐3W produced approximately 104 CFU/mL of

suspected V. cholerae on TCBS agar. To discriminate between V. cholerae and other

Vibrio species, API 20E tests for Vibrio spp. were conducted. One colony had a 99.5%

probability of being V. cholerae and one colony had a 76.8% probability of being V.

fluvialis. The suspected V. cholerae colony was then analyzed to determine whether it

was serotype O1 and O139. In addition, the colonies were analyzed by PCR to

determine whether the cholera toxin gene was present. The analyzed colonies were

non‐O1, non‐O139, and cholera toxin gene negative V. cholerae (non‐agglutinable

vibrios). Analysis of the samples for the presence of Legionella spp. revealed no

positive samples.

Bacterial flora analyses

Nucleotide sequences of 86 to 95 clones from each sample (total 918 clones)

were determined (Table 3). Of these clones, 5%−57% and 77%−100% showed more than 97% and 80% homology to strains present in an in‐house database, respectively.

To cover the widest possible range of clones, 80% homology was selected; therefore,

taxa above the rank of genus were used for further analyses. The numbers of taxa

indicated that the bacterial communities of soil samples were more diverse than

those of water samples (Table 3). The numbers of genera detected were as follows: 61

(A‐1), 53 (A‐2), 57 (B‐1), 51 (B‐2), 48 (C‐1), and 44 (C‐3) in soils, and 17 (A‐3W), 14

(B‐3W), 49 (C‐2W), and 45 (C‐4W) in water samples. At the phylum level, 10 (A‐1), 9

(A‐2), 6 (B‐1), 4 (B‐2), 9 (C‐1), and 8 (C‐3) taxa were detected in soils, and 4 (A‐3W), 3

(B‐3W), 7 (C‐2W), and 5 (C‐4W) taxa were detected in water samples. Most of the genera belonged to Proteobacteria and Bacteroidetes (Table 4). In sludge samples taken in the tsunami‐affected areas, more genera belonged to Proteobacteria (37%−51%) than

to Bacteroidetes (14%−27%), but in water samples taken in these areas, more genera

belonged to Bacteroidetes (38%, 45%) than to Proteobacteria (22%, 32%). In control

water samples, more genera belonged to Proteobacteria (37%, 43%) than to

Bacteroidetes (16%, 21%).

At the family level, some sulfate‐reducing bacteria and sulfur‐oxidizing bacteria were detected (Table 5). In sample A‐1 (wet sludge), 10 clones (11%) of sulfate‐reducing bacteria and one clone (2%) of sulfur‐oxidizing bacteria were

detected among 95 tested clones. One clone of Mycobacterium elephantis

(nontuberculous mycobacteria) (97% homology with AJ010747) was also detected in

A‐1. In sample B‐1 (dry sludge), seven clones (8%) of sulfate‐reducing bacteria, 11 clones (12%) of sulfur‐oxidizing bacteria, and one clone of Legionella impletisoli (94%

homology) were detected among 92 tested clones. In sample B‐2, two clones (2%) of

Clostridiaceae that were possible sulfate‐reducing bacteria, two clones (2%) of

Vibrionaceae, and one clone of Massilia timonae (97% homology with U54470) were

identified among 88 tested clones. In sample B‐3W, only sulfur‐oxidizing bacteria

were detected. Two pathogens were identified in control samples: C‐1 contained one clone of Mycobacterium moriokaense (98% homology with AJ429044) and C‐4W contained one clone of Vibrio natriegens (100% homology with X74714). Samples C‐3 and C‐4W, taken from the beach, contained balanced numbers of sulfur‐oxidizing and sulfur‐reducing bacteria (13 and 14 clones for C‐3 and 6 and 9 clones for C‐4W).

Discussion

We analyzed sludge brought by the tsunami that occurred after the Great

East Japan Earthquake in 2011 to identify bacterial hazards. To detect bacteria causing enteric or respiratory infection, we used both conventional culture and molecular methods. No significant bacterial proliferation was observed in the samples; however, non‐O1, non‐O139, and cholera toxin gene negative V. cholerae

(non‐agglutinable vibrio) was identified by the culture method, and Massilia timonae

was found using the molecular method. Additionally, some samples were found to

contain sulfate‐reducing bacteria and sulfate at concentrations sufficient to generate hydrogen sulfide, which is very toxic to humans. No pathogens that require government notification were identified.

We collected samples from a coastal area in which sludge was stagnant and

from a waste dump that contained sludge and debris. Water samples collected from both places contained high levels of chloride ions, which could have been brought from the sea (Table 1). Total organic carbon, which is a nonspecific indicator of water

quality, was high in water samples, possibly supporting bacterial growth or metabolic activity8).

Non‐agglutinable vibrios, which cause sporadic cases or outbreaks of

diarrhea9), have often been found in seawater and were still active in stagnant

seawater in the paddy field 3 months after the tsunami (Table 5). We also identified

Vibrionaceae in samples B‐2 and C‐4W. In areas in which the samples for the present

study were collected, frequent hand washing and avoiding close contact with the

sludge are recommended to prevent infection. There is also potential for the bacteria

to proliferate over the summer, when the weather is hot and humid.

Mycobacterium elephantis and Massilia timonae, which have been reported as

human pathogens10‐13), were found in sludge samples collected from tsunami‐affected

areas (Table 5). The most probable reservoir of these bacteria is the outer environment, as we also identified Mycobacterium moriokaense in sample C‐114). The

site from which sample A was collected was completely immersed after the tsunami

and we also identified various bacteria usually found in the sea, such as

Marinobacterium (A‐1), Neptunomonas (A‐1), Marinicola seohaensis (B‐2), and

Marinobacter (B‐2). However, we could not determine whether Mycobacterium

elephantis (A‐1) and Massilia timonae (B‐2) were transferred from the sea or originated

from the sample site. These bacteria cause infection, especially among

immunocompromised hosts. Therefore, avoiding exposure to the site and frequent

hand washing are recommended, especially for immunocompromised individuals.

In addition, we identified Legionella impletisoli (B‐1) even though the homology was

94%, which has been found in industrial wastes15).

Sulfate‐reducing bacteria live in oxygen‐deficient environments such as

seawater, deep wells, and plumbing systems. They reduce sulfate to form hydrogen

sulfide, a colorless gas with an offensive odor. Sulfate‐oxidizing bacteria pose no

identified health risk and convert hydrogen sulfide gas to sulfuric acid 16). In control

samples taken from the beach and the sea (C‐3 and C‐4W), similar numbers of clones

were identified for sulfate‐reducing and sulfur‐oxidizing bacteria. However, in a

control sample taken from the sludge of a paddy field (C‐1), there were more

sulfate‐reducing bacteria. This was also observed for the sample taken from the

sludge of a paddy field in the tsunami‐affected area (A‐1). Since the sulfate ion levels in sample A‐3W were high (Table 1), and this sample was collected near sample A‐1, hydrogen sulfide could be produced if these samples became mixed under anaerobic

conditions. Even though the sulfate levels in sample B‐1 (taken from a waste dump)

were higher, hydrogen sulfide would not be produced because the amount of sulfate‐reducing bacteria was equal to the amount of sulfate‐oxidizing bacteria.

It should be noted that our study had a few limitations. Specifically, a small number of samples were selected for analysis. Further sample analysis is required to clarify the health risks associated with sludge. In addition, the storage method that we used did not allow us to identify cold sensitive bacteria such as Neisseria

gonorrhoeae and Neisseria meningitides. At the end, we used a clone library that was limited to bacteria; therefore, no conclusions regarding the presence of viruses, fungi, and protozoa can be drawn.

In conclusion, sludge brought by the tsunami following the 2011 earthquake in Japan contained some pathogens. Accordingly, frequent hand washing is

recommended for workers that have direct contact with the sludge to minimize the

risk of infection. Additionally, hydrogen sulfide could be produced by

sulfate‐reducing bacteria under anaerobic conditions such as those present in paddy

fields. Overall, the results of this study indicate that the sludge should be cleaned up

as soon as possible.

References

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8) Gergel SE, Turner MG, Kratz TK. Dissolved organic carbon as an indicator of the scale of watershed influence on lakes and rivers. Ecological Applications 1999; 9: 1377‐90. 9) Igbinosa EO, Okoh AI. Vibrio fluvialis: an unusual enteric pathogen of increasing public health concern. Int J Environ Res Public Health 2010; 7: 3628‐43. 10) Potters D, Seghers M, Muyldermans G, Pierard D, Naessens A, Lauwers S. Recovery of Mycobacterium elephantis from sputum of a patient in Belgium. J Clin Microbiol 2003; 41: 1344. 11) Tortoli E, Rindi L, Bartoloni A, Garzelli C, Mantella A, Mazzarelli G, et al. Mycobacterium elephantis: not an exceptional finding in clinical specimens. Eur J Clin Microbiol Infect Dis 2003; 22: 427‐30. 12) Lindquist D, Murrill D, Burran WP, Winans G, Janda JM, Probert W. Characteristics of Massilia timonae and Massilia timonae‐like isolates from human patients, with an emended description of the species. J Clin Microbiol 2003; 41: 192‐6. 13) Van Craenenbroeck AH, Camps K, Zachee P, Wu KL. Massilia timonae infection presenting as generalized lymphadenopathy in a man returning to Belgium from Nigeria. J Clin Microbiol 2011; 49: 2763‐5. 14) Adekambi T, Raoult D, Drancourt M. Mycobacterium barrassiae sp. nov., a Mycobacterium moriokaense group species associated with chronic pneumonia. J Clin Microbiol 2006; 44: 3493‐8. 15) Kuroki H, Miyamoto H, Fukuda K, Iihara H, Kawamura Y, Ogawa M, et al. Legionella impletisoli sp. nov. and Legionella yabuuchiae sp. nov., isolated from soils contaminated with industrial wastes in Japan. Syst Appl Microbiol 2007; 30: 273‐9. 16) Kantachote D, Charernjiratrakul W, Noparatnaraporn N, Oda K. Selection of sulfur oxidizing bacterium for sulfide removal in sulfate rich wastewater to enhance biogas production. Electronic Journal of Biotechnology 2008; 11: 107‐18.

Conflict of interest

The authors have declared that no competing interests exist.

Funding

This study was funded by a research fund of the Fit‐test research committee. The

funder had no role in study design, data collection and analysis, decision to publish,

or preparation of the manuscript.

Authors’ contributions KW, HT, KF, TH, and TY conceived and designed the experiments. HT, KF, TI, and TU performed the experiments. KW, KF, and HT analyzed data and drafted the manuscript. All the authors read and approved the final manuscript.

Figure legends

Figure 1. Map of the locations where samples were taken.

A and B on the inset map signify the locations where samples A and B were taken.

Figure 1.

Table 1. Results of physicochemical analyses of samples.

A‐1 A‐2 A‐3W B‐1 B‐2 B‐3W C‐1 C‐2W C‐3 C‐4W (wet (dry （water (dry (wet (water (wet (water) (wet (water) sludge) sludge) ） sludge) sludge) ) sludge) sludge)

Potential hydrogen (PH) － 7.5 8.6 8.3 7.5 7.2 7.8 6.8 7.2 6.9 7.5 Electric conductivity (EC) mS/m 221 37 1346 198 225 196 6 ND 100 ND Total organic carbon (TOC) mg/ℓ 13.5 24.6 47.3 9.7 5.5 24.9 5.4 20.9 8.2 3.7 Total nitrogen (TN) mg/ℓ 1.6 2.2 3.7 1.5 0.9 10.4 1.6 2.9 1.8 0.6 Chloride ion (Cl‐) mg/l 555 28 8503 337 246 3396 7 278 771 1091 2‐ Sulfate ion (SO4 ) mg/l 71 24 258 329 117 0 7 133 148 ND

ND: not detected Table 2. Cell lysis efficiency and cell concentration determined using three methods.

A‐1 (wet A‐2 A‐3W B‐1 B‐2 B‐3W C‐1 C‐2W C‐3 C‐4W sludge) (dry （water） (dry (wet (water) (wet (water) (wet (water) sludge) sludge) sludge) sludge) sludge) Cell lysis efficiency (%) 79.0 (±2.3) 96.3 (±1.4) 98.6 (±2.3) 92.7 (±0.9) 99.1 (±0.5) 99.8 (±0.1) 76.9 (±0.8) 85.0 (±3.0) 85.9 (±1.1) 92.7 (±2.5)

Epifluorescence staining 6.2(±0.4)× 1.0(±0.1)× 9.9(±0.7)× 7.8(±0.1)× 2.9(±0.6)× 5.3(±0.6)× 1.3(±0.2)× 7.9(±0.3)× 6.8(±0.8)× 7.6(±2.5)× method (cells/mL (water) 108 109 106 108 108 106 109 106 108 106 or g (soil))

Aerobic culture on BTB medium at 37℃ (colony 8x104 1x105 3x104 1x105 6x105 3x104 7x105 2x104 1x104 1x102 forming units/mL (water) or g (soil))

Aerobic culture on standard medium for environmental bacteria at 1.0(±0.2)× 6.3(±1.0)× 2.0(±0.1)× 5.9(±4.0)× 9.2(±0.3)× 1.0(±0.2)× 1.0(±0.03) 1.4(±0.4)× 1.8(±0.03) 3.1(±0.2)× 30℃ (colony forming 107 107 106 107 107 106 ×108 106 ×106 106 units/mL (water) or g (soil)) Mean±standard deviation Table 3. Number of clones with homology to 16S rDNA sequences of type strains and number of taxa identified within each sample.

A‐1 A‐2 A‐3W B‐1 B‐2 B‐3W C‐1 C‐2W C‐3 C‐4W (wet (dry （water） (dry (wet (water) (wet (water) (wet (water) sludge) sludge) sludge) sludge) sludge) sludge) Number of tested clones 95 90 86 92 88 94 94 94 93 92 Number of clones with > 97% homology *1 (% of the 17 (18%) 27 (30%) 4 (5%) 30 (32%) 50 (57%) 36 (38%) 23 (25%) 21 (22%) 6 (6%) 14 (15%) total tested clones)

Number of clones with > 80% homology *2 (% of the 73 (77%) 73 (81%) 70 (81%) 77 (84%) 82 (93%) 94 (100%) 82 (87%) 75 (80%) 72 (77%) 72 (78%) total tested clones) Number of phyla 109 4 6 439 785 Number of classes 16 15 6 11 10 7 15 11 14 9 Number of orders 30 27 10 25 24 10 23 23 24 17 Number of families 43 37 13 40 33 12 40 33 32 25 Number of genera 61 53 17 57 51 14 48 49 44 45

*1: Number of clones with nucleotide sequences that showed > 97% homology to those present in the database. *2: Number of clones with nucleotide sequences that showed > 80% homology to those present in the database. Table 4. Percentage of genera in each sample belonging to each of 13 phyla. A‐1 A‐2 A‐3W B‐1 B‐2 B‐3W C‐1 C‐2W C‐3 C‐4W (wet (dry （water） (dry (wet (water) (wet (water) (wet (water) sludge) sludge) sludge) sludge) sludge) sludge) Acidobacteria 21 914171144 Actinobacteria 59 8 96 Bacteroidetes 18 20 38 14 27 45 26 16 12 21 Chloroflexi 122 Cyanobacteria 211182 Deferribacteres 11 Firmicutes 13 2 3 3 8 6 2 2 2 Gemmatimonadetes 2 Lentisphaerae 0 0 0 000001 0 Nitrospira 22 1 1 Proteobacteria 38 37 22 51 44 32 35 37 43 43 Spirochaetes 21 Thermomicrobia 11 1

Unclassified* 11 15 14 14 5 12 19 21 20

* ʹUnclassified bacteriaʹ indicates the clones whose nucleotide sequence homologies with standard strains of the database were less than 80% Table 5. Identified sulfur‐reducing bacteria, sulfur‐oxidizing bacteria, and pathogens at the family level. Samples Identified microbes No of clones A‐1 (wet sludge) Mycobacteriaceae (M. elephantis) (P) 1 Peptococcaceae (Desulfotomaculum) (SRB) 1 Desulfobacteraceae (SRB) 5 Desulfobulbaceae (SRB) 2 Syntrophobacteraceae(Desulfovirga) (SRB) 1 Clostridiaceae (Clostridium) (SRB) 1 Ectothiorhodospiraceae (SOB) 1

A‐2 (dry sludge) Hydrogenophilaceae (SOB) 1 Chromatiaceae (SOB) 1

A‐3W （water） Chromatiaceae (SOB) 1

B‐1 (dry sludge) Desulfobacteraceae (SRB) 5 Desulfobulbaceae (SRB) 2 Hydrogenophilaceae (SOB) 3 Chromatiaceae (SOB) 5 Ectothiorhodospiraceae (SOB) 2 Piscirickettsiaceae (SOB) 1

B‐2 (wet sludge) Clostridiaceae (Clostridium) (SRB) 2 Oxalobacteraceae (Massilia‐timonae) (P) 1 Vibrionaceae (V. ichthyoenteri, V. natriegens) （ P) 2

B‐3W (water) Chromatiaceae (SOB) 6 Ectothiorhodospiraceae (SOB) 6 Thiotrichaceae (SOB) 4

C‐1 (wet sludge) Mycobacteriaceae (M. moriokaense) (P) 1 Clostridium‐aldrichii (SRB) 1 Clostridium‐alkalicellum (SRB) 1 Clostridium‐cellobioparum (SRB) 1 Clostridium‐chartatabidum (SRB) 2 Desulfovibrio‐inopinatus (SRB) 1 Ectothiorhodospiraceae (SOB) 1

C‐2W （water） Hydrogenophilaceae (SOB) 1 Chromatiaceae (SOB) 1 Ectothiorhodospiraceae (SOB) 1 C‐3 (wet sludge) Desulfobacterium‐catecholicum (SRB) 2 Desulfobacterium‐indolicum (SRB) 1 Desulfosarcina‐cetonica (SRB) 3 Desulfobulbus‐propionicus (SRB) 3 Desulfobulbus‐rhabdoformis (SRB) 1 Desulforhopalus‐singaporensis (SRB) 1 Desulfotalea‐psychrophila (SRB) 2 Chromatiaceae (SOB) 6 Ectothiorhodospiraceae (SOB) 6 Thiotrichaceae (SOB) 4

C‐4W (water) Desulfobacter‐postgatei (SRB) 1 Desulfobacterium‐catecholicum (SRB) 1 Desulfococcus‐biacutus (SRB) 1 Desulfosarcina‐cetonica (SRB) 1 Desulfobulbus‐propionicus (SRB) 2 Chromatiaceae (SOB) 3 Ectothiorhodospiraceae (SOB) 6 Vibrionaceae (V. natriegens) （ P) 1

SRB: Sulfur Reducing Bacteria, SOB: Sulfur Oxidizing Bacteria, P: Pathogen (P): M. elephantis and Massilia timonae showed 97% homology to the database type strain.