Effect of inorganic salt stress on the thermotolerance and ...

ORIGINAL ARTICLE

Effect of inorganic salt stress on the thermotolerance and ethanolproduction at high temperature of Pichia kudriavzevii

Chunsheng Li1 & Laihao Li1 & Xianqing Yang1 & Yanyan Wu1& Yongqiang Zhao1 &

Yueqi Wang1

Received: 15 May 2017 /Accepted: 7 November 2017 /Published online: 10 April 2018# Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Abstract Application of cross-protection is expected to im-prove the thermotolerance of yeasts to enhance their ethanolproduction at high temperature. In this study, the effects ofeight kinds of inorganic salts on the thermotolerance and eth-anol production at high temperature in Pichia kudriavzeviiwere investigated. P. kudriavzevii showed strong thermotoler-ance and the ability to produce ethanol at high temperature,and higher ethanol production of P. kudriavzeviiwas observedat high temperature (37–42 °C) compared with that at 30 °C.Inorganic salt stresses induced obvious cross-protection ofthermotolerance in P. kudriavzevii. The presence of 0.1 mol/L KNO3 or Na2SO4 or 0.2 mol/L NaCl, KCl, NaNO3, K2SO4

or MgCl2 increased the yeast biomass in YEPD medium at44 °C to 2.72–3.46 g/L, obviously higher than that in theabsence of salt stress (2.17 g/L). The addition of NaCl, KCl,NaNO3, KNO3, Na2SO4, K2SO4, CaCl2 and MgCl2 signifi-cantly increased the ethanol production of P. kudriavzevii inYEPD fermentation medium at 44 °C by 37–58%. KCl andMgCl2 exhibited the best performance on improving the ther-motolerance and ethanol production, respectively, ofP. kudriavzevii. A highly significant correlation (P < 0.01)was obtained among ethanol production, biomass and glucoseconsumpt ion , sugges t ing the impor tan t ro le of

thermotolerance and glucose consumption in enhanced etha-nol production. The combination of NaCl, KCl and MgCl2had a synergistic effect on the improvement of thermotoler-ance and ethanol production at high temperature inP. kudriavzevii. This study provides some important cluesfor improving ethanol production of thermotolerant yeasts athigh temperature.

Keywords Pichia kudriavzevii . Inorganic salt .

Thermotolerance . Ethanol production . Cross-protection

Introduction

Ethanol, as a clean and renewable fuel, is expected to replacetraditional fossil energy sources to alleviate the increasingenergy crisis and environmental pollution (Ko et al. 2016;Travaini et al. 2016). Yeasts are the microorganisms mostcommonly used in industrial production of ethanol. Ethanolproduction using thermotolerant yeasts shows obvious advan-tages, and has received a great deal of attention in recent years.Thermotolerant yeasts can help increase ethanol productivity,reduce operating costs of maintaining growth temperature inlarge-scale systems, and decrease risk of bacterial contamina-tion (Dhaliwal et al. 2011). Moreover, thermotolerant yeastscan adapt to the high reaction temperature in the simultaneoussaccharification and fermentation process for converting cel-lulosic biomass to ethanol (Olofsson et al. 2008; Isono et al.2012).

Recently, a great deal of attention has been paid toethanol production at high temperature using Pichiak u d r i a v z e v i i b e c a u s e o f i t s t h e rmo t o l e r a n t ,ethanol-tolerant and acid-tolerant characteristics (Sandhuet al. 2011; Isono et al. 2012; Koutinas et al. 2016; Lie t a l . 2016a ) . P. kudr iav zev i i showed h ighe r

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s13213-018-1339-x) contains supplementarymaterial, which is available to authorized users.

* Xianqing [email protected]; [email protected]

1 Key Laboratory of Aquatic Product Processing, Ministry ofAgriculture, Guangdong Provincial Key Laboratory of FisheryEcology Environment, South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences, Guangzhou 510300, China

Ann Microbiol (2018) 68:305–312https://doi.org/10.1007/s13213-018-1339-x

thermotolerance and ethanol production at high tempera-ture than most strains of Saccharomyces cerevisiae(Gallardo et al. 2011; Yuangsaard et al. 2013). ManyP. kudriavzevii strains exhibited a good ability to growand produce ethanol over 40 °C (Dhaliwal et al. 2011;Sandhu et al. 2011; Yuangsaard et al. 2013). However,growth of P. kudriavzevii was significantly inhibitedwhen the fermentation temperature increased to over44 °C, leading to a significant decrease in ethanol pro-duction (Isono et al. 2012). It is particularly important toexplore ways of improving the thermotolerance ofP. kudriavzevii when applied to ethanol production athigh temperature.

Microorganisms exposed to one stress can develop toler-ance not only to higher doses of the same stress, but also tostress caused by other agents. This phenomenon, known ascross-protection, suggests the existence of an integratingmechanism that senses and causes responses to different formsof stress (Rangel et al. 2008; Rangel 2011; Bergholz et al.2012; Isohanni et al. 2013). The responses to different stressesthat induce cross-protection against high temperature havebeen studied extensively in microorganisms. Adaptation tosodium lactate or sodium acetate significantly enhanced thethermotolerance of Salmonella typhimurium (Yuan et al.2012). Osmotic stress induced cross-protection of thermotol-erance was found in Metarhizium anisopliae conidia (Rangelet al. 2008). However, there is a lack of information about theeffect of salt stress on the thermotolerance of yeasts. In thepresent study, eight kinds of inorganic salts, including NaCl,KCl, NaNO3, KNO3, Na2SO4, K2SO4, CaCl2 and MgCl2,were used to test whether salt stress could inducecross-protection against high temperature in P. kudriavzevii.Ethanol production by P. kudriavzevii at high temperature un-der various inorganic salt stresses was also measured. Thisstudy is expected to provide an effective way to improve thethermotolerance of P. kudriavzevii and its ethanol productionat high temperature.

Materials and methods

Yeast strain and pre-culture

Pichia kudriavzevii A16 was isolated from a high temperatureChinese liquor starter (Li et al. 2016b). The yeast was main-tained in YEPD agar slant (1% yeast extract powder, 2% pep-tone, 2% glucose and 2% agar; pH 5.0) at 4 °C.

P. kudriavzevii was pre-cultured as follows. The yeast wasfirst transferred to a fresh YEPD agar slant and incubated at30 °C for 24 h. Thereafter, the yeast were transferred to250 mL Erlenmeyer flasks containing 50 mL liquid YEPDmedium (1% yeast extract powder, 2% peptone and 2%

glucose; pH 5.0) and incubated for 24 h at 30 °C and180 rpm.

Thermotolerance and ethanol production at hightemperature

The thermotolerance and ethanol production at high tempera-ture of P. kudriavzeviiwere determined in 250 mL Erlenmeyerflasks containing 50 mL YEPD medium and YEPD fermen-tation medium (1% yeast extract powder, 2% peptone and10% glucose; pH 5.0), respectively. After pre-culture,P. kudriavzevii was cultivated at different temperatures (30–45 °C). The initial biomass of P. kudriavzevii was adjusted to0.1 g dry weight/L. After cultivation at 180 rpm in a shakingwater bath for 24 h, the yeast culture was centrifuged. Thesupernatant was used for analysis of glucose, ethanol andglycerol, while the pellet was used for determination of yeastgrowth.

Effect of inorganic salts on thermotolerance

After pre-culture, the yeast was cultivated in YEPD mediumwith, respectively, 0–0.8 mol/L NaCl, KCl, NaNO3 or KNO3,or 0–0.4 mol/L Na2SO4, K2SO4, CaCl2 or MgCl2. The initialbiomass ofP. kudriavzevii in the mediumwas adjusted to 0.1 gdry weight/L. After cultivation at 44 °C and 180 rpm in ashaking water bath for 24 h, the yeast culture was centrifugedand the biomass (g/L) was used to determine the effect ofinorganic salts on the thermotolerance of P. kudriavzevii.

Effect of inorganic salts on ethanol production at hightemperature

The effect of inorganic salts on ethanol production ofP. kudriavzevii at high temperature was studied in YEPD fer-mentation medium. After pre-culture, the yeast was cultivatedin the medium with, respectively, 0–0.8 mol/L NaCl, KCl,NaNO3 or KNO3, or 0–0.4 mol/L Na2SO4, K2SO4, CaCl2 orMgCl2. The initial biomass of P. kudriavzevii in the mediumwas adjusted to 0.1 g dry weight/L. After cultivation at 44 °Cand 180 rpm in a shaking water bath for 24 h, the sampleswere drawn and centrifuged. The supernatant was used foranalysis of glucose, ethanol and glycerol, while the pelletwas used for measuring yeast growth.

Effect of inorganic salt interaction on thermotoleranceand ethanol production at high temperature

NaCl, KCl and MgCl2 were chosen to study on the effect ofinorganic salt interaction on thermotolerance and ethanol pro-duction at high temperature of P. kudriavzevii. Afterpre-culture, the yeast was cultivated in YEPD medium andYEPD fermentation medium with, respectively, (1) 0.1 mol/

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L NaCl +0.1 mol/L KCl, (2) 0.1 mol/L NaCl +0.05 mol/LMgCl2, (3) 0.1 mol/L KCl + 0.05 mol/L MgCl2, or (4)0.0667 mol/L NaCl +0.0667 mol/L KCl + 0.0333 mol/LMgCl2. The initial biomass of P. kudriavzevii in the mediumwas adjusted to 0.1 g dry weight/L. After cultivation at 44 °Cand 180 rpm in a shaking water bath for 24 h, the sampleswere drawn and centrifuged. The supernatant was used foranalysis of glucose, ethanol and glycerol, while the pelletwas used for measuring yeast growth.

Analytical methods

The growth of P. kudriavzevii was determined as describedpreviously (Li et al. 2014). Briefly, the yeast culture was cen-trifuged at 6000 rpm and 4 °C for 5 min. The pellet waswashed twice with ultrapure water and then weighed afterdrying to constant weight at 80 °C to calculate the yeast bio-mass (g/L). The glucose, ethanol and glycerol were analyzedby UPLC (ACQUITY UPLC H-Class System; Waters,Milford, MA) using an organic acid analysis column (ICSepCoregel 87H3 column, 300 × 7.8 mm; Transgenomic ,Omaha, NE). Degassed H2SO4 solution (5 mmol/L) was usedas a mobile phase at a flow rate of 0.6 mL/min. The columnoven and refractive index (RI) detector were maintained at35 °C. The culture supernatant was filtered through 0.22 μmPTFE membranes (ANPEL Laboratory Technologies Inc.,Shanghai, China). Peaks were detected by the RI detectorand quantified on the basis of retention time and area of thestandards.

Statistical analysis

All experiments were performed in triplicate, and the datawere expressed as mean ± standard deviation (SD).Statistical analyses were performed with one-way analysis ofvariance (ANOVA) and multiple comparison Tukey test.Kruskal-Wallis nonparametric tests were performed to com-pare the different effects of inorganic salts on the thermotol-erance and ethanol production of P. kudriavzevii. Pearson cor-relation analysis was used to evaluate correlation among thefermentation parameters of P. kudriavzevii under various inor-ganic salt stresses.

Results and discussion

Thermotolerance and ethanol production at hightemperature

The growth and ethanol production of P. kudriavzevii at hightemperature are shown in Table 1. The biomass ofP. kudriavzevii in YEPD medium medium decreased signifi-cantly with increased temperature. P. kudriavzevii has recently

been described as a thermotolerant yeast (Isono et al. 2012;Toivari et al. 2013). Similar results were found in this studythat the biomass of P. kudriavzevii reached to 1.05 g/L afterincubation in theYEPDmedium at 45 °C for 24 h. The biomasswas significantly improved when P. kudriavzeviiwas cultivatedin YEPD fermentation medium. This is probably because thereis more glucose used as a carbon source for yeast growth.

Glucose consumption clearly increased at 37–42 °C but de-creased at 44–45 °C compared with that at 30 °C, and no glu-cose was left in YEPD fermentation medium after 24 h cultiva-tion at 37–42 °C. P. kudriavzevii exhibited good ability of eth-anol production at 30–42 °C in YEPD fermentation medium.Although yeast growth was inhibited with increased tempera-ture, ethanol production by P. kudriavzevii was improved sig-nificantly by high temperatures (37–42 °C), probably due to theenhanced glucose consumption. The maximum ethanol con-centration (36.78 g/L) was observed at 40 °C, and was clearlyhigher than that at 30 °C (30.48 g/L). Several studies havereported similar results, i.e., that high temperature could in-crease the ethanol production of thermotolerant yeasts (Isonoet al. 2012; Charoensopharat et al. 2015; Koutinas et al. 2016).However, ethanol production was significantly inhibited whenthe fermentation temperatures increased to over 44 °C. Oneimportant reason was that the growth of P. kudriavzevii wasclearly inhibited under high temperature. Improving the ther-motolerance of P. kudriavzevii may be an effective way to en-hance its ethanol production at high temperature.

During yeast osmoadaptation against high sugar concentra-tions in fermentation, the biosynthesis of glycerol is promotedin order to balance osmotic pressure across the yeast plasmamembrane (Gibson et al. 2007). In addition, glycerol is alsoinvolved in protecting yeast cells against high temperature(Siderius et al. 2000; Kitichantaropas et al. 2016). To examinethe response to environmental stresses in P. kudriavzevii, theglycerol concentration in the medium were measured afterstress exposure. Similarly, a high concentration of glycerol(1.94 g/L) was produced when P. kudriavzevii was exposedto high glucose concentration in YEPD fermentation medium.Moreover, high temperatures (37–45 °C) significantly pro-moted the biosynthesis of glycerol in YEPD fermentation me-dium compared with that at 30 °C. The maximum glycerolconcentration reached (3.49 g/L) was observed at 42 °C.

Effect of inorganic salts on thermotolerance

In order to improve the thermotolerance of P. kudriavzevii,eight kinds of inorganic salts including NaCl, KCl, NaNO3,KNO3, Na2SO4, K2SO4, CaCl2 and MgCl2, were used fortesting whether salt stress could induce cross-protectionagainst high temperature in P. kudriavzevii. As shown inFig. 1A, the growth of P. kudriavzevii at 44 °C in YEPDmedium first increased and then decreased with the increas-ing concentration of NaCl, KCl, NaNO3, KNO3, Na2SO4,

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K2SO4 and MgCl2, while the yeast biomass significantlydecreased with the increase of CaCl2 concentration.Interestingly, except CaCl2, high concentrations of NaCl,KCl, NaNO3, KNO3, Na2SO4, K2SO4 and MgCl2 could

all induce cross-protection against high temperature inP. kudriavzevii. The thermotolerance of P. kudriavzeviiwas significantly enhanced by high concentrations ofNaCl (0.1–0.6 mol/L), KCl (0.1–0.8 mol/L), NaNO3 (0.1–

Fig. 1. Effect of various concentrations of NaCl, KCl, NaNO3, KNO3,Na2SO4, K2SO4, CaCl2 and MgCl2 on the thermotolerance of Pichiakudriavzevii. A Biomass of P. kudriavzevii cultivated for 24 h at 44 °Cin YEPD medium with various concentrations of inorganic salts. Barslabeled with different letters are statistically different (P < 0.05) tested by

one-way ANOVA and multiple comparison Tukey test. B Comparison ofthe thermotolerance of P. kudriavzevii under different inorganic saltstresses. Values above the bars are the mean ranks of the biomass atdifferent concentrations of inorganic salts tested by Kruskal-Wallis non-parametric test

Table 1 Growth and ethanolproduction of Pichia kudriavzeviiat different temperatures. Valuesin the same column with differentsuperscript letters are statisticallydifferent (P < 0.05) tested by one-way ANOVA and multiple com-parison Tukey test.

Temperature (°C) YEPD medium YEPD fermentation medium

Biomass (g/L) Glucose (g/L) Ethanol (g/L) Biomass (g/L) Glycerol (g/L)

30 7.11 ± 0.05 f 5.76 ± 0.11 a 32.48 ± 0.01 c 8.47 ± 0.03 f 1.94 ± 0.01 a

37 6.11 ± 0.03 e NDa 35.19 ± 0.07 d 7.55 ± 0.03 e 2.64 ± 0.08 b

40 5.60 ± 0.06 d ND 36.78 ± 0.13 e 5.75 ± 0.06 d 2.92 ± 0.01 c

42 3.75 ± 0.03 c ND 36.73 ± 0.24 e 4.00 ± 0.01 c 3.49 ± 0.12 d

44 2.17 ± 0.17 b 33.98 ± 2.24 b 21.62 ± 0.23 b 2.70 ± 0.11 b 2.91 ± 0.04 c

45 1.05 ± 0.03 a 64.60 ± 3.02 c 10.38 ± 0.02 a 2.27 ± 0.02 a 2.49 ± 0.11 b

aNot detected

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0.2 mol/L), KNO3 (0.1–0.6 mol/L), Na2SO4 (0.05–0.1 mol/L), K2SO4 (0.05–0.4 mol/L) and MgCl2 (0.1–0.4 mol/L).The maximum biomass of P. kudriavzevii at 44 °C wasobserved in the presence of 0.1 mol/L KNO3 or Na2SO4

or 0.2 mol/L NaCl, KCl, NaNO3, K2SO4 or MgCl2, respec-tively, reaching 2.72–3.46 g/L, clearly higher than that inthe absence of inorganic salts (2.17 g/L). No improvementin thermotolerance was observed in the tested CaCl2 con-centrations (0.05–0.4 mol/L).

Kruskal-Wallis nonparametric tests were performed tocompare the effects of different inorganic salts on the thermo-tolerance of P. kudriavzevii. According to the mean rank ofeach group shown in Fig. 1B, KCl exhibited the best perfor-mance in improving the thermotolerance of P. kudriavzevii inthe tested inorganic salts; in the same concentrations of Cl−,the order was KCl, NaCl, MgCl2 and CaCl2; in the sameconcentrations of Na+, the order was NaCl, NaNO3 andNa2SO4; in the same concentrations of K+, the order wasKCl, K2SO4 and KNO3. Similar results were reported byRangel et al. (2008), who found that high concentrations ofKCl (0.4–1.0 mol/L) and NaCl (0.4–0.6 mol/L) could inducecross-protection of thermotolerance in Metarhiziumanisopliae conidia, and the thermotolerance was higher forconidia produced on PDAY with KCl than with NaCl. Theimproved thermotolerance after pre-culture with high concen-trations of NaCl, was also found in Bacillus species. (denBesten et al. 2006, 2010). Similar to inorganic salts, organicacid salts were previously reported in the protective effect onmicroorganisms against thermal destruction (Murphy et al.2004; Yuan et al. 2012). It was demonstrated that some pro-teins induced at high temperature were also induced under saltstress, and this could have contributed to the salt-inducedcross-protective effects toward high temperature (den Bestenet al. 2010). In addition, the protective effect might result froma reduction in water activity of microorganism cells by salts,which decreased the heat penetration, thereby leading to en-hanced thermotolerance (Juneja 2003).

In order to elucidate whether the improved thermotolerancedue to inorganic salts could contribute to ethanol production athigh temperature, glucose consumption and ethanol produc-tion in YEPD medium were analyzed after yeast cultivation.As shown in Fig. S1, glucose consumption and ethanol pro-duction of P. kudriavzevii cultivated at 44 °C in YEPD medi-um increased significantly under inorganic salt stress. Theenhanced yeast biomass and glucose consumption might beimportant reasons for the increase of ethanol production inYEPD medium.

Effect of inorganic salts on ethanol production at hightemperature

High concentrations of inorganic salts could improve the ther-motolerance of P. kudriavzevii and its ethanol production at

high temperature in YEPD medium. However, much higherconcentrations of carbon source were used for ethanol produc-tion in practical application. Therefore, in this study, YEPDfermentation medium was used to examine whether inorganicsalts could improve the growth and ethanol production ofP. kudriavzevii at high temperature in high glucoseenvironment.

As shown in Fig. 2A, different from the improved biomassin a wide range of salt concentrations in YEPD medium, thegrowth of P. kudriavzevii in YEPD fermentation medium wasenhanced by inorganic salts only at low concentrations. Apossible reason was that P. kudriavzevii cultivated in YEPDfermentation medium faced osmotic pressure not only frominorganic salts but also from high concentrations of glucose,causing inhibitory effects on yeast growth. Inhibition of yeastgrowth after addition of CaCl2 (0.05–0.4 mol/L) was alsoobserved in YEPD fermentation medium. With the increaseof inorganic salt concentrations, both glucose consumptionand ethanol production first increased and then decreased.The ethanol production of P. kudriavzevii cultivated at 44 °Cwas significantly improved after addition of high concentra-tions of NaCl (0.1–0.4 mol/L), KCl (0.1–0.6 mol/L), NaNO3

(0.1–0.4 mol/L), KNO3 (0.1–0.2 mol/L), Na2SO4 (0.05–0.1 mol/L), K2SO4 (0.05–0.3 mol/L), CaCl2 (0.05–0.1 mol/L) and MgCl2 (0.05–0.4 mol/L) (P < 0.05). The inorganicsalts could improve the ethanol production of P. kudriavzeviiat 44 °C by 37–58%. The maximum ethanol yield was ob-served at 0.05 mol/L K2SO4 or CaCl2, or 0.1 mol/L NaCl,KCl, NaNO3 or Na2SO4, or 0.2 mol/L KNO3 or MgCl2,reaching to 72.61%–77.46% of the theoretical ethanol yield,obviously higher than that in the absence of salt stress(70.62%) (Fig. S2). As one of the main thermoprotectantsagainst high temperature, glycerol was induced by high con-centrations of inorganic salts in the YEPD fermentation me-dium. In this study, the glycerol concentration significantlyincreased after the YEPD fermentation medium containedhigh concentrations of inorganic salts. The promotion of glyc-erol synthesis might play an important role in the improve-ment of the growth of P. kudriavzevii.

The effects of high stress conditions on the ethanol produc-tion of yeasts have been studied. Isono et al. (2012) reportedthat ethanol production by Issatchenkia orientalis MF-121strain incubated at 30 °C in acidic medium was enhanced afteraddition of 50 g/L Na2SO4, while for other I. orientalis strainsthe ethanol production was inhibited under these conditions.Both NaCl and Na2SO4 significantly decreased ethanol pro-duction of Zymomonas mobilis at 30 °C (Vriesekoop et al.2002). However, there has been a total lack of informationin the literature regarding the effect of salts on the ethanolproduction of yeasts at high temperature. In this study,Kruskal-Wallis nonparametric tests were used to comparethe improvement in ethanol production by P. kudriavzevii un-der different inorganic salt stresses, and the results are shown

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in Fig. 2B. According to the mean rank of each group, MgCl2exhibited the best performance in improving the ethanol pro-duction of P. kudriavzevii in the tested inorganic salts; in thesame concentrations of Cl−, the order was MgCl2, KCl, NaCland CaCl2; in the same concentrations of Na+, the order wasNaCl, NaNO3 and Na2SO4; in the same concentrations of K+,the order was K2SO4, KCl and KNO3.

Ethanol production is reported to be usually related to yeastgrowth, and ethanol production at high temperature could beenhanced by improving yeast thermotolerance (Shi et al.2009). Similar results were found in this study, i.e., a highlysignificant correlation (P < 0.01) was noted between biomassand ethanol production (r = 0.921) (Table 2). The ethanol pro-duction increased with the improvement in yeast growth anddecreased with inhibition of yeast growth (Fig. 2).Furthermore, higher ethanol production was observed inYEPD fermentation medium with added high concentrationsof MgCl2, K2SO4, KCl and KNO3, which were better able toimprove the growth of P. kudriavzevii than other inorganicsalts, like NaCl, NaNO3, Na2SO4 and CaCl2 (Fig. 2).

Similar to biomass, glucose consumption also showed a high-ly significant correlation (P < 0.01) with ethanol production (r= 0.993) (Table 2). These results suggested that improvedgrowth and glucose consumption of P. kudriavzevii by inor-ganic salt stresses might play an important role in the increaseof ethanol production at high temperature. In addition, highlysignificant correlations (P < 0.01) with glucose consumptionwas obtained for biomass (r = 0.921), while no fermentationparameters showed a significant correlation with glycerolproduction.

Effect of inorganic salt interaction on thermotoleranceand ethanol production at high temperature

In order to test whether different inorganic salts caused syner-gies in improving the yeast thermotolerance and ethanol pro-duction at high temperature, the biomass and ethanol produc-tion of P. kudriavzevii cultivated with different combinationsof NaCl, KCl and MgCl2 were compared with that with thesingle salt (0.2 mol/L NaCl, 0.2 mol/L KCl or 0.1 mol/L

Fig. 2 Effect of various concentrations of NaCl, KCl, NaNO3, KNO3,Na2SO4, K2SO4, CaCl2 and MgCl2 on the ethanol production ofP. kudriavzevii. A Glucose consumption, ethanol production, biomassand glycerol production of P. kudriavzevii cultivated for 24 h at 44 °Cin YEPD fermentation medium with various concentrations of inorganic

salts. B Comparison of the ethanol production of P. kudriavzevii underdifferent inorganic salt stresses. Values above the bars are the mean ranksof the ethanol production at different concentrations of inorganic saltstested by Kruskal-Wallis nonparametric test

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MgCl2). As shown in Fig. 3, similar to the single inorganicsalt, all combinations of NaCl, KCl and MgCl2 could improvethe thermotolerance of P. kudriavzevii. In the same concentra-tions of Cl−, there was no significant difference in the thermo-tolerance of P. kudriavzevii among the group of NaCl and KClin combination and group of either NaCl or KCl; KCl andMgCl2 in combination expressed a more notable protectingeffect on the thermotolerance of P. kudriavzevii than MgCl2;NaCl, KCl and MgCl2 in combination showed the bestprotecting effect on the thermotolerance of P. kudriavzevii.In YEPD fermentation medium, all combinations of NaCl,KCl and MgCl2 could improve yeast biomass, glucose con-sumption, ethanol production and glycerol production ofP. kudriavzevii at high temperature. Similarly, in the same

concentrations of Cl−, NaCl, KCl and MgCl2 in combinationshowed the most positive effect on biomass and ethanol pro-duction of P. kudriavzevii. In this study, it was shown thatNaCl, KCl and MgCl2 in combination had a synergistic effecton the improvement of thermotolerance and ethanol produc-tion at high temperature in P. kudriavzevii.

The multi-stress-tolerant P. kudriavzevii is considered a po-tential candidate for use in ethanol production at high temper-ature. Similar results were confirmed in this study, i.e., thatP. kudriavzevii showed strong thermotolerance and ethanolproduction ability at high temperature. Much more work isrequired to study ethanol production in practicalapplicationconditions and to explain the improved thermotol-erance and ethanol production of P. kudriavzevii in the subse-quent research. However, this is the first work to report im-proved thermotolerance and ethanol production at high tem-perature of P. kudriavzevii by inorganic salt stresses. Thisstudy provides some important clues to enhance the thermo-tolerance of yeasts and their ethanol production at hightemperature.

Conclusion

The presence of 0.1 mol/L KNO3 or Na2SO4 or 0.2 mol/LNaCl, KCl, NaNO3, K2SO4 or MgCl2 in YEPD medium sig-nificantly increased yeast biomass at 44 °C, from 2.17 g/L to2.72–3.46 g/L. The addition of NaCl, KCl, NaNO3, KNO3,Na2SO4, K2SO4, CaCl2 and MgCl2 significantly increasedethanol production by P. kudriavzevii in YEPD fermentationmedium at 44 °C by 37–58%. KCl and MgCl2 exhibited thebest performance on improving the thermotolerance and eth-anol production, respectively, of P. kudriavzevii. A highly sig-nificant (P < 0.01) correlation was noted among ethanol pro-duction, biomass and glucose consumption. NaCl, KCl andMgCl2 in combination had a synergistic effect on the improve-ment of thermotolerance and ethanol production at high tem-perature in P. kudriavzevii.

Acknowledgments The authors would like to thank the NationalNatural Science Foundation of China (No. 31301454), the CentralPublic-interest Scientific Institution Basal Research Fund, South ChinaSea Fisheries Research Institute, CAFS (No. 2015TS23), and the Scienceand Technology Program of Guangzhou of China (No. 201707010300)for financial support .

Fig. 3 Effect of different combinations of NaCl, KCl and MgCl2 onthermotolerance and ethanol production at high temperature ofP. kudriavzevii. Bars labeled with different letters are statisticallydifferent (P < 0.05) tested by one-way ANOVA and multiple comparisonTukey test

Table 2 Correlation (Pearson)matrix for fermentation parame-ters of P. kudriavzevii at 44 °C inYEPD fermentation medium un-der inorganic salt stresses

Glucose consumption Biomass Ethanol Glycerol

Glucose consumption 1

Biomass 0.921a 1

Ethanol 0.993a 0.921a 1

Glycerol 0.146 0.039 0.056 1

aDenotes statistical significance at P < 0.01

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Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict ofinterest.

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