From April 2016 to June 2017, we collected water and sediment samples from various water environments, namely two locations around Tokyo University of Marine Science and Technology, in Tokyo, and 12 locations in Yokohama, Kanagawa prefecture, Japan. All the samples were collected in sterile tubes, and the pH value of each sample was measured. As for the sediment sample, a small amount of distilled water was added and the sample was vortexed, then the pH value was measured. The samples were carried and stored at low temperatures in our laboratory at Tokyo University of Marine Science and Technology.

For cultivation, we used an R2A medium and a YPD medium. The R2A medium consisted of 0.1% yeast extract (Becton Dickinson, Franklin Lakes, NJ, USA), 0.1% proteose peptone (Becton Dickinson), 0.1% casamino acid (Nihon Pharmaceutical, Tokyo), 0.1% D-(+)-glucose, 0.1% soluble starch (Kokusan Chemicals, Tokyo), 0.06% sodium pyruvate, 0.03% K₂HPO₄, and 0.005% MgSO₄∙7H₂O. The YPD medium consisted of 1.0% yeast extract (Becton Dickinson), 2.0% proteose peptone (Becton Dickinson), and 2.0% D-(+)-glucose. We prepared neutral media or acidic media adjusted to pH 3.0 with sulfuric acid, and the solid media were constructed by adding 1.2% gellan gum (Kanto Chemical, Tokyo) to both R2A and YPD liquid media. The neutral media contained 0.01% chloramphenicol to prevent bacteria growth.

For the isolation of yeasts, we first used both R2A (1) medium (neutral R2A solid medium) and YPD (1) medium (neutral YPD solid medium). The water samples were filtered with a 0.45-µm FTFE membrane-filter (Advantec, Tokyo), and microorganisms were trapped on the filter. They were dispersed into the portion of the filtrate; thus, we obtained about a 100-fold-concentrated population of the microorganisms in the water samples. The sediment samples were diluted to 10-fold with physiological saline (0.8%w/v NaCl). A 200 µl volume of each preparation was spread on the R2A and YPD media and incubated at 25˚C. After several days, we picked up growing yeast-like colonies: i.e., we attached the colonies to the tip of a needle and transferred them to a new medium.

To isolate acid-tolerant yeasts, the obtained colonies were inoculated into acidic R2A (2) medium (R2A solid medium adjusted to pH 3.0 with sulfuric acid) or acidic YPD (2) medium (YPD solid medium adjusted to pH 3.0 with sulfuric acid) and then incubated at 25˚C.

Colonies formed on acidic solid medium were removed and inoculated into R2A (3) medium (R2A plate medium adjusted to pH 3.0 with sulfuric acid which contained 0.02% bromocresol purple, which is yellow in acidic conditions and purple in neutral and basic conditions). After several days’ incubation at 25˚C, we separated the yeasts that were capable of neutralizing acid (hereafter referred to as acid-neutralizing yeasts) by observing the change of the medium color from yellow to purple.

The yeasts inoculated on YPD (1) medium formed colonies that were over 2 cm in diameter (giant colonies) after several days’ incubation at 25˚C. First, we distinguished yeast species among the isolates by observing the appearance of giant colonies with the naked eye and observing the cell morphologies under a microscope. Second, with the yeasts thus obtained, we carried out the gene identification. The 26S rRNA genes of the yeasts were amplified by polymerase chain reaction (PCR) using the forward primer NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3'), and the reverse primer NL-4 (5'-GGTCCGTGTTTCAAGACGG-3'), and Premix Ex Taq (Takara Bio, Shiga, Japan). The PCR reaction program was performed in a TaKaRa PCR Thermal Cycler Dice TP600 with an initial denaturation at 95˚C for 5 min, followed by 25 cycles of denaturation at 94˚C for 30 s, annealing at 56˚C for 30 s, extension at 72˚C for 1 min and final extension at 72˚C for 4 min. The D1/D2 domain sequences of the 26S rRNA genes in the yeasts were deposited in DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and GenBank.

To investigate the neutralization ability of the yeasts, we carried out a neutralization test using the isolated acid-neutralizing yeasts. The yeasts were precultured at 25˚C for 48 h in 10 mL of R2A liquid medium at pH 4.0 with shaking. The yeast pellet obtained by the centrifugation at 3000 rpm was washed three times with saline adjusted to pH 4.0 with sulfuric acid. The washed pellet was added to 10 mL of 0.5% (w/v) casamino acid solution adjusted to pH 4.0 with sulfuric acid, and incubated at 25˚C for 72 h with shaking. We prepared four same 10 ml samples. One of them was picked up and centrifuged at 3000 rpm, and the pH value of the supernatant was then measured every 24 h.

A total of 177 yeast-like strains were obtained from all water and sediment samples. Forty-six strains changed the color of the R2A (2) medium containing a pH indicator from yellow to purple (an example of the change of color is shown in Figure 1). As a result of the microscopic observation, 12 bacteria-like strains were excluded, and 34 yeast-like strains were finally isolated as acid-neutralizing yeasts. Figure 2 shows micrograph examples of acid-neutralizing yeasts. With these 34 strains, we performed giant colony observation to distinguish their species. After that, when species that greatly resembled other species were excluded, 26 strains were identified, and the results are shown in Table 1 and Table 2. As a result, four strains (r-w2, r-w3, r-w4, and fi-m10) showed sequences identical

^aThe total number of colonies isolated from samples. ^bObtained strains that were acid-neutralizing yeasts.

to Aureobasidium pullulans. The strains h-m7, so-w34, and n-w33 were found to be Candida parapsilosis, C. cylindracea, and C. oleophila, respectively, and two strains (h-m8 and om-w46) were found to be Candida sp. Two strains (ko-w20, om-w42) were Cryptococcus sp. and two strains (m-m25 and n-w28) were Cryptococcus flavescens. Two strains (si-w12 and n-w29) were Rhodotorula sp. Strain mi-w17 was Hannaella pagnoccae, and strain sm-w38 was H. coprosmae. Strain sm-w37 was found to be Pseudozyma antarctica, and sm-w39 was P. tsukubaensis. Strains mi-w16, si-w13, om-41, om-w44, sm-w40, ks-w31, and mr-w1 were Filobasidium magnum, Leucosporidium golubevii, Auriculibuller sp., Papiliotrema flavescens, Microbotryozyma collariae, Bullera alba, and Meyerozyma guilliermondii, respectively. The phylogenetic tree of these species was constructed on the 26s rRNA sequences by the maximum likelihood algorithm in MEGA version 6.06, as shown in Figure 3.

Among the 26 identified strains, 10 strains (h-m7, ko-w20, ks-w31, m-m25, mi-w16, mi-w17, n-w29, r-w2, r-w3, and si-w13) changed the color of R2A (2) medium faster than the others. With these 10 strains, we performed neutralizing tests using casamino acid solution adjusted to pH 4.0 with sulfuric acid, and the results are shown in Figure 4. All strains elevated the pH from 4.0 to approximately 7.5 after 24 h, and from 24 h to 72 h the pH increased gradually up to approximately 8.0.