Acid-tolerant yeasts often inhabit extremely acidic environments: mine drain-ages, hot springs, and even fermented foods. Some of them also possess the ability to neutralize acidic media. However, the examples of these yeasts that are already known were isolated from acidic environments. In this study, the isolation of acid-tolerant yeasts from natural neutral aquatic environments and the identification of yeasts able to neutralize an acidic medium (acid-neutralizing yeast) in Japan were carried out. Various kinds of acid-tolerant and acid-neutralizing yeasts were obtained. In a neutralizing test using an acidic casamino acid solution adjusted to a pH of 4.0 with sulfuric acid, the obtained acid-neutralizing yeasts elevated the pH to approximately 7.0, and their neutralizing abilities were similar to those of previously reported yeasts that had been isolated from acidic environments. These results showed that acid-tolerant yeasts and acid-neutralizing yeasts exist widely in neutral environments, and little difference was found in the neutralizing abilities of yeasts obtained from neutral environments in comparison to those obtained from acidic environments.
Extremophiles are organisms that thrive in extreme environments such as extremely high or low temperature, high pressure, high salinity, and high or low pH [
In this study, we focused on eukaryotes that can inhabit acidic environments. Some previous studies reported that a variety of eukaryotes exist in extreme acidic environments, not only prokaryotes. For instance, Klebsormidium and Dunaliella, which are algae, have been isolated from acidic rivers [
The yeasts already known to be capable of thriving in and neutralizing acidic environments were isolated only from extreme acidic environments. To our knowledge, there has been no report of such yeasts being isolated from a neutral natural environment. In this study, we identified various types of acid-tolerant yeasts that possess the ability to neutralize an acidic medium in neutral aquatic environments and characterized their abilities.
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
Sampling area | Sampling spot | Type of sample | pH | Temperature (˚C) | Isolate numbera | Strainb |
---|---|---|---|---|---|---|
Izumi-no-Mori Park (Yokohama) | Stream | Sediment Water Water Water Water | 6.71 6.68 6.68 6.62 6.62 | 19.3 18.0 18.0 18.9 18.9 | 25 | fi-m10 mi-w16 mi-w17 si-w12 si-w13 |
Izumi River (Yokohama) | Creek | Sediment | - | 18.9 | 31 | h-m7 h-m8 |
Kodomo Shizen Park (Yokohama) | Creek | Water Water | 6.88 6.69 | 20.8 23.3 | 26 | ko-w20 so-w34 |
Kibougaoka Mizu-no-Mori Park (Yokohama) | Stream | Sediment | 6.31 | 19.3 | 38 | m-m25 |
Maioka Park (Yokohama) | Creek | Water | 6.14 | 22.8 | 18 | om-w41 om-w42 om-w44 om-w46 |
Pond | Water | 7.01 | 27.4 | 12 | sm-w37 sm-w38 sm-w39 sm-w40 | |
Nagayamon Park (Yokohama) | Pond | Water | 6.43 | 19.8 | 4 | n-w28 n-w29 n-w33 |
Sagami Mikawa Park (Yokohama) | River | Water | 7.45 | 24.4 | 96 | ks-w31 |
Tokyo Marine Science and Technology (Tokyo) | Pond | Water | 9.74 | - | 2 | mr-w1 |
Tokyo Bay (Tokyo) | Canal | Water | 7.03 | 23.4 | 16 | r-w2 r-w3 r-w4 |
aThe total number of colonies isolated from samples. bObtained strains that were acid-neutralizing yeasts.
Strain | Identification | Identity (%) | Accession number |
---|---|---|---|
fi-m10 | Aureobasidium pullulans | 100 | LC326044 |
mi-w16 | Filobasidium magnum | 100 | LC326047 |
mi-w17 | Hannaella pagnoccae | 100 | LC326048 |
si-w12 | Rhodotorula sp. | 100 | LC326045 |
si-w13 | Leucosporidium golubevii | 100 | LC326046 |
h-m7 | Candida parapsilosis | 100 | LC326042 |
h-m8 | Candida sp. | 100 | LC326043 |
ko-w20 | Cryptococcus sp. | 100 | LC326049 |
so-w34 | Candida cylindracea | 100 | LC326055 |
m-m25 | Cryptococcus flavescens | 100 | LC326050 |
om-w41 | Auriculibuller sp. | 99 | LC326060 |
om-w42 | Cryptococcus sp. | 100 | LC326061 |
om-w44 | Papiliotrema flavescens | 100 | LC326062 |
om-w46 | Candida sp. | 100 | LC326063 |
sm-w37 | Pseudozyma antarctica | 100 | LC326056 |
sm-w38 | Hannaella coprosmae | 100 | LC326057 |
sm-w39 | Pseudozyma tsukubaensis | 100 | LC326058 |
sm-w40 | Microbotryozyma collariae | 100 | LC326059 |
n-w28 | Cryptococcus flavescens | 100 | LC326051 |
n-w29 | Rhodotorula sp. | 100 | LC326052 |
n-w33 | Candida oleophila | 100 | LC326054 |
ks-w31 | Bullera alba | 100 | LC326053 |
mr-w1 | Meyerozyma guilliermondii | 100 | LC326038 |
r-w2 | Aureobasidium pullulans | 100 | LC326039 |
r-w3 | LC326040 | ||
r-w4 | LC326041 |
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
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
In this study, we isolated 26 acid-neutralizing yeasts which exhibited acid tolerance and the ability to neutralize acidic R2A (2) medium from 14 natural neutral aquatic locations in Tokyo and Yokohama, Kanagawa prefecture, Japan. These 26 isolates were identified. Using 10 strains that rapidly changed the color of R2A (2) medium containing bromocresol purple from yellow to purple, we
carried out neutralizing tests using 0.5% (w/v) casamino acid solution adjusted to pH 4.0 with sulfuric acid.
In previous reports, yeast strains identified as C. fluviatilis [
As a result of the neutralizing test, 10 strains (strain m-m25 was from sediment, while the nine other strains were from water) elevated the pH from 4.0 to approximately 8.0 after 72 h (
In conclusion, we isolated 26 strains of neutralizing yeast from neutral water environments. Of these, 10 strains elevated the pH of acidic casamino acid solution from 4.0 to approximately 8.0 in 72 h, and there were no significant differences in neutralizing ability among them. This study indicates that neutralizing yeasts exist widely in the neutral environment. The results presented in this study may further our understanding of the acid tolerance and neutralizing ability of yeasts.
This work was supported in part by a Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Scientific Research (Grant No. 16K07868).
Nagaoka. S., Kobayashi, T., Kajiwara, Y., Okai, M., Ishida, M. and Urano, N. (2017) Characterization of Yeasts Capable of Neutralizing Acidic Media from Natural Neutral Environments. Advances in Microbiology, 7, 887-897. https://doi.org/10.4236/aim.2017.712068