Isolation of aquatic yeasts with the ability to neutralize acidic media, from an extremely acidic river near Japan's Kusatsu-Shirane Volcano.

The Yukawa River is an extremely acidic river whose waters on the east foot of the Kusatu-Shirane Volcano (in Gunma Prefecture, Japan) contain sulfate ions. Here we isolated many acid-tolerant yeasts from the Yukawa River, and some of them neutralized an acidic R2A medium containing casamino acid. Candida fluviatilis strain CeA16 had the strongest acid tolerance and neutralizing activity against the acidic medium. To clarify these phenomena, we performed neutralization tests with strain CeA16 using casamino acid, a mixture of amino acids, and 17 single amino acid solutions adjusted to pH 3.0, respectively. Strain CeA16 neutralized not only acidic casamino acid and the mixture of amino acids but also some of the acidic single amino acid solutions. Seven amino acids were strongly decomposed by strain CeA16 and simultaneously released ammonium ions. These results suggest strain CeA16 is a potential yeast as a new tool to neutralize acidic environments.


INTRODUCTION
The various extreme environments on Earth include physical environments (e.g., 2 temperature, radiation and pressure extremes) and geochemical environments 3 (desiccation, salinity, pH, oxygen species and redox potential environments) in which 4 various organisms have adapted and thrived (1). For example, the biodiversity and 5 ecology of eukaryotic organisms living in extreme acidic environments near volcanos 6 have been studied (2). Yeast diversity has been extensively investigated (3). Several 7 types of yeast have been isolated from these extreme acidic environments to date; e.g., 8 Rhodotorula glutinis (4), Candida maltosa (5), and Cryptococcus tepidarius (6). In 9 Saccharomyces cerevisiae, yeast genes involved in responses to acid stress have been 10 studied (7-9). It has also been reported that acidification of the external medium during  In order to remove damage due to the river's acidity for the purpose of river 22 conservancy and to provide water for agricultural, industrial and other uses, the Yukawa 23 River water is neutralized by adding milk of lime (i.e., calcium carbonate) from the 24 Kusatsu neutralization plant (14). The Yukawa River thus has an unusual aquatic 1 environment composed of both the natural aquatic environment and artificial elements.

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In this study, we isolated many acid-tolerant yeasts from both upstream and 3 downstream of the Kusatsu neutralization plant in the Yukawa River. Interestingly, some 4 of these isolated acid-tolerant yeasts possessed a neutralizing ability against an acidic 5 medium. We discuss the mechanisms and the optimum conditions for the neutralization 6 of acidic media by these novel yeasts.

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Collection of environmental samples 10 In June 2010, we collected water samples from the Yukawa River in Agatsuma-gun, 11 Gunma Prefecture, Japan. The samples were transported to the laboratory in sterile 12 bottles in contact with ice. The water temperature was measured at each of the two 13 sampling stations, and the pH value of each sample was measured in our laboratory.   With the colonies thus obtained, we carried out the yeast isolation procedure.    The amounts of ammonium ion were measured by the enzymatic method with F-10 kit ammonium (JK International, Tokyo). The pH value was measured using a LAQUA 11 pH meter ( F-72, Horiba, Kyoto, Japan).  2 Therefore, we selected these 32 isolates as the yeasts capable of the neutralization of 3 acid. Then, among these 32 strains, five (CeA14, CeG17, EeB28, EeC21, GeC45) and 4 two strains (AeA6, CeA16) capable of growing on R2A(iii) medium at pH 1.5 and 1.0 5 respectively were identified on the basis of the DNA sequence analysis included 6 complete sequence of their ITS1 in this study ( Table 1). Six of these seven isolates 7 showed identical sequence with Candida fluviatilis type strain and the other showed 8 identical sequence with Candida palmioleophila type strain. In particular, out of two 9 strains grown at pH 1.0, strain CeA16 of C. fluviatilis showed faster growth than strain 10 AeA6. We therefore focused on strain CeA16 to clarify the mechanism underlying the 11 acid neutralization in this study. Strain CeA16 grew well at 20°-25°C and showed a 12 strong ability to neutralize acid (data not shown). 15 To clarify the mechanism underlying the acid neutralization, we performed a 16 neutralization test with strain CeA16 using casamino acid, which is a component of 17 R2A medium, adjusted to pH 3.0 with sulfuric acid. Strain CeA16 elevated the pH from 18 3.0 to 7.3 in the casamino acid solution after 1 day (Fig. 1A). Strain CeA16 also 19 reduced the total amount of the 17 amino acids from 18.2 mM to 5.3 mM, whereas it 20 increased the ammonium ion from 1.4 mM to 15.9 mM.

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As shown in Figure 1B  After 1 day, >95% of Ser, Arg, Thr and Ala+Pro and 76%-77% of Asp and Glu 2 decreased and were thought to be consumed by strain CeA16 (Fig. 1B). And, 87% of 3 Met and 73% of Lys decreased for two days.  Fig. 2A).

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The results of the HPLC analysis showed that 10 amino acids (Asp, Glu, Ser, Gly, 14 Arg, Thr, Ala, Pro, Cys and Lys) were preferably consumed, whereas most of the 15 hydrophobic amino acids and His were hardly consumed (Fig. 2B). In the experiments 16 with single amino acids, seven (Glu, Ser, Lys, Ala, Pro, Asp and Arg) contributed to an 17 elevation of pH values from 2.9-3.1 to 7.7-8.4, and Ile showed an elevation of pH value 18 from 3.1 to 6.0 (Fig. 3A). Therefore, we monitored the consumption of these seven 19 amino acids (Glu, Ser, Lys, Ala, Pro, Asp and Arg) that contributed largely to an 20 elevation of pH values and the production of ammonium ions. The amounts of the 21 ammonium ions increased as these seven amino acids were consumed by strain CeA16 22 (Fig. 3B). In this study, we isolated strain CeA16, which was capable of growing in a strongly 2 acidic medium and neutralizing the acidic medium by using several amino acids, and 3 identified as Candida fluviatilis. In previous reports, some yeast strains identified as C. 4 fluviatilis (18, 19), C. intermedia (19), and R. glutinis (4) were isolated from the various 5 extreme acidic environments and some of them had the ability to neutralize the acidic 6 medium (pH 2.5-3.0) (4, 19). However, there have been no reports of their mechanisms.

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In the present study, strain CeA16 neutralized oligotrophic medium such as R2A medium, 8 but could not neutralize nutritious YPD medium (2% glucose, 1% polypeptone and 1% 9 yeast extract) adjusted to pH 3.0 (data not shown). These results might indicate that the 10 neutralization ability of strain CeA16 is mainly in oligotrophic environments.

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As shown in Figure 3B, we observed that seven amino acids (Glu, Ser, Lys, Ala, 12 Pro, Asp and Arg) were preferably utilized by strain CeA16 as the single amino acid 13 source was decomposed to release ammonium ion. However, not only these seven 14 amino acids (Glu, Ser, Lys, Ala, Pro, Asp and Arg) but also three amino acids (Gly, Thr 15 and Cys), which did not contribute to an elevation of pH value in the experiments with 16 single amino acids (Fig. 3A), were preferably consumed under the mixture of amino 17 acids (Fig. 2B). In addition, the decrease rates of Met, Gly and Cys under the mixture of 18 amino acids (Fig. 2B) were different from those under casamino acid (Fig. 1B). These 19 differences might be also involved any metabolism related to amino acid in strain 20 CeA16 according to a ratio of amounts of amino acid.

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In bacteria, several common mechanisms of acid tolerance are known, such as the ions in the test tube. Therefore, strain CeA16 also might cope with acid by other 12 mechanisms such as the proton pump until its surrounding pH becomes neutral.

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In conclusion, C. fluviatilis strain CeA16 neutralized strongly acidic solution 14 using not only an amino acid mixture but also a single amino acid of the seven amino