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Research Paper Different effects of sodium chloride preincubation on cadmium tolerance of Pichia kudriavzevii and Saccharomyces cerevisiae Ning Ma, Chunsheng Li, Xiaoyan Dong, Dongfeng Wang and Ying Xu Laboratory of Food Chemistry and Nutrition, College of Food Science and Engineering, Ocean University of China, Qingdao, China
Application of growing microorganisms for cadmium removal is restricted by high cadmium toxicity. The effects of sodium chloride (NaCl) preincubation on the cadmium tolerance and removal ability of Pichia kudriavzevii and Saccharomyces cerevisiae were investigated in this study. NaCl preincubation improved the biomass of P. kudriavzevii under cadmium stress, while no obvious effect was observed in S. cerevisiae. The improved activities of peroxidase (POD) and catalase (CAT) after NaCl preincubation might be an important reason for the decrease of the reactive oxygen species (ROS) accumulation, cell death, and oxidative damage of proteins and lipids induced by cadmium, contributing to the improvement of the yeast growth. The cadmium bioaccumulation capacity of P. kudriavzevii decreased significantly after NaCl preincubation, which played an important role in mitigating the cadmium toxicity to the yeast. The cadmium removal rate of P. kudriavzevii was obviously higher than S. cerevisiae and was significantly enhanced after NaCl preincubation. The results suggested that NaCl preincubation improved the cadmium tolerance and removal ability of P. kudriavzevii. Keywords: Pichia kudriavzevii / Saccharomyces cerevisiae / NaCl preincubation / Cadmium bioaccumulation / Oxidative stress Received: November 6, 2014; accepted: February 10, 2015 DOI 10.1002/jobm.201400847
Introduction Aquatic environment as a result of increased industrial activities deteriorates permanently because of a wide range of environmental factors including heavy metals [1]. Due to biomagnification and accumulation via the food chain, there is an increasing risk of heavy metal contamination in aquatic products, causing significant threats to human health [2]. Since heavy metals are not biodegradable [3], the removal of heavy metals should be taken into consideration first before using the food materials contaminated by heavy metals, e.g., enzymatic hydrolysate of aquatic product. Conventional physiochemical methods for heavy metal removal, such as chemical precipitation, adsorption method, and ion The first two authors contributed equally to this work. Correspondence: Ying Xu, Laboratory of Food Chemistry and Nutrition, College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China E-mail: [email protected] Phone: þ86 0532-82031851 Fax: þ86 0532-82032093 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
exchange, are ineffective in food environment for low concentrations of heavy metals and high concentrations of organic compounds [4, 5]. Therefore, an alternative technique is urgently required for removing heavy metals from food environment. The bioaccumulation of heavy metals via growing microorganisms has received a great deal of attention in the field of heavy metal treatment in recent years [1, 5]. In the process of bioaccumulation, heavy metals are not only bound to the cell surface but also accumulated inside the cell mainly via energy-consuming active transport systems [6]. However, the bioremoval of heavy metals is highly affected by high concentrations of heavy metals which can inhibit both the cell growth and bioaccumulation [1]. It was reported that the increase of intracellular cadmium concentration resulted in the overproduction of reactive oxygen species (ROS) which rapidly attack all biomolecules, such as DNA, lipids, and proteins, eventually leading to cell death [7–10]. Therefore, it is particularly important to improve the heavy metal tolerance of
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microorganisms for employing growing cells in heavy metal removal. Our previous study showed that the combined use of sodium chloride (NaCl) and cadmium reduced the growth-inhibiting effect of cadmium as well as the cadmium-induced ROS production and cell death of Zygosaccharomyces rouxii, indicating that NaCl might display a protective effect on Z. rouxii under cadmium stress [11]. However, little work has been done to study whether NaCl preincubation could improve the cadmium tolerance of yeasts. Pichia kudriavzevii, synonymously known as Issatchenkia orientalis or Candida krusei, has been isolated from a variety of fermented foods and fruit sources [12, 13]. P. kudriavzevii has recently been described as a multistress-tolerant yeast which can tolerate up to 40% glucose, 10% NaCl, pH as low as 2, and temperatures over 42 °C [12, 14–16]. In addition, P. kudriavzevii is more resistant to lactic acid [17] and acetic acid [18] than S. cerevisiae. It provides more possibilities to employ growing P. kudriavzevii biomass for heavy metal removal in complex food environment. In this study, the effect of NaCl preincubation on the growth of P. kudriavzevii under cadmium stress was studied. The oxidative stress and cadmium bioaccumulation capacity in P. kudriavzevii were also studied to preliminarily explore the mechanism of the protective effect of NaCl preincubation against cadmium. The cadmium removal ability of P. kudriavzevii was measured in order to elucidate the possibility of using NaCl preincubation for cadmium removal by growing P. kudriavzevii. S. cerevisiae was used as the reference yeast for comparison.
Materials and methods Strains and preincubation Pichia kudriavzevii A16 was isolated from a high-temperature Chinese liquor starter, and Saccharomyces cerevisiae CICC1211 was purchased from the China Center of Industrial Culture Collection. Both yeasts were maintained in YEPD agar slants (1% yeast extract powder, 2% peptone, 2% glucose, and 2% agar, pH 5.0) at 4 °C. After being refreshed in YEPD agar slant at 28 °C for 24 h, the yeasts were transferred to 250 ml Erlenmeyer flasks containing 50 ml YEPD broth, and incubated at 28 °C and 180 rpm for 24 h. Thereafter, NaCl preincubation was done by transferring 1 ml of culture to 250 ml Erlenmeyer flasks containing 50 ml YEPD broth with different NaCl concentrations (10–60 g L1), and incubating at 28 °C and 180 rpm for 24 h. The yeasts cultured in YEPD broth without NaCl were used as control. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Growth assay The cadmium stock solution (100 mg L1) was prepared by dissolving CdCl22.5H2O (Sinopharm Group, China) in ultrapure water (Millipore, America). The cell concentrations of both yeasts after NaCl preincubation were measured by a hemocytometer. Then, the yeast cells with same inoculum size (4 108 cells) were pelleted and transferred to 50 ml YEPD broth with different concentrations of cadmium (6 and 20 mg L1). After cultivation at 28 °C and 180 rpm for 24 h, the cells were centrifuged, washed twice with ultrapure water, and then weighed after drying to constant weight at 80 °C to determine the dry weight. The biomass was defined as the dry weight of the yeast cells per liter of cultured medium. Determination of ROS in yeast cells 20 ,70 -dichlorofluorescein diacetate (DCFH-DA, Sigma, America) and propidium iodide (PI, Sigma, America) were used for double staining to determine the intracellular ROS level and cell death rate according to Li et al. [11]. Briefly, the yeast cells incubated with different concentrations (6 and 20 mg L1) of cadmium were washed twice with phosphate buffered saline (PBS, pH 7.0, 0.05 mol L1) and diluted to 1 107 cells ml1. After incubation with 100 mmol L1 DCFH-DA at 37 °C and 60 rpm for 50 min in the dark, the samples were rapidly placed on ice, washed twice, resuspended in 1 ml of PBS, and then stained with 10 mg ml1 PI. A flow cytometer (Beckman Coulter Cytomics FC 500 MPL) equipped with an air-cooled 488 nm argon laser was used to record the fluorescence of cell suspensions. Green fluorescence of the cells stained with 20 ,70 dichlorofluorescein (DCF) was collected in the FL1 channel (525 nm 20 nm), and red fluorescence of the cells labeled with PI was collected in the FL3 Channel (625 nm 20 nm). A total of 20,000 events were registered per sample and the obtained data were analyzed with the CXP Analysis 2.1 software. Protein carbonyls and malonyldialdehyde (MDA) assays After being incubated with 6 mg L1 of cadmium for 24 h, the yeast cells were centrifuged and washed twice with PBS. The cells were ground with liquid nitrogen, resuspended in PBS and centrifuged at 4 °C and 12,000 rpm for 15 min. The supernatant was collected to assay the content of protein carbonyls and MDA. The protein carbonylation was detected by a protein carbonyl detection kit (Nanjing Jiancheng Bioengineering Institute, China). Briefly, protein carbonyls were derivatized to 2,4-dinitrophenylhydrazone (DNP) after the addition
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of 2,4-dinitrophenylhydrazine (DNPH). DNP was resuspended in guanidine HCl, and the absorbance was measured at 370 nm. Results were expressed as nmol of carbonyls per mg of protein. A micro-malonaldehyde detection kit (Nanjing Jiancheng Bioengineering Institute, China) was used for MDA determination. Simply, thiobarbituric acid (TBA) reacted with MDA to form red products, and the absorbance was measured at 532 nm. The MDA concentration was expressed as nmol of MDA per mg of protein [19]. Detection of antioxidant enzyme activity The yeast cells were collected and resuspended in PBS as the methods described above. The samples were then centrifuged at 4 °C and 12,000 rpm for 10 min. The supernatant was collected to assay the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) with a commercial reagent kit (Nanjing Jiancheng Bioengineering Institute, China) spectrophotometrically. The activities of SOD, CAT, and POD were expressed as units per milligram protein (U mgprot1). The activity of SOD was assayed at 550 nm using the xanthine and xanthine oxidase system. One unit (U) of SOD activity was defined as the amount of enzyme causing 50% inhibition of xanthine and xanthine oxidase reaction system. The samples were treated with excess of H2O2 at 37 °C, and the absorbance of remaining H2O2 was measured at 405 nm to determine the activity of CAT. One unit (U) of CAT activity was defined based on the decomposition of 1 mmol H2O2 by the enzyme per second [20]. The samples were treated with H2O2 at 37 °C for 30 min. POD activity was determined by detection of changes in the absorbance at 420 nm. One unit (U) of POD activity was defined as the amount of enzyme that catalyzed 1 mg H2O2 min1 at 37 °C. The content of total protein was measured by coomassie blue staining, using bovine serum albumin (BSA, Solarbio, China) as a standard. Cadmium bioaccumulation capacity assay The cells incubated under cadmium stress for 24 h were centrifuged, washed twice with ultrapure water, and then weighed after drying to constant weight at 80 °C to calculate the biomass (M) of the yeasts. The supernatant was thoroughly digested with nitric acid and perchloric acid, and diluted with ultrapure water. The cadmium concentration of the solution was measured by an atomic absorption spectrophotometer (AA-6800, Shimadzu, Japan) [21]. Cadmium removal rate (R) and cadmium bioaccumulation capacity (Q) were calculated by using the following equations: ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
R¼
1 Ct C0
Q ¼
ðC0 Ct Þ M
100%
ð1Þ
ð2Þ
where R is cadmium removal rate in the YEPD medium (%), Ct is final cadmium concentration in YEPD medium (mg L1), C0 is initial cadmium concentration in YEPD medium (mg L1), Q is cadmium bioaccumulation capacities of the yeasts (mg g1), and M is the yeast biomass in the cultured YEPD medium (g L1). Statistical analysis All experiments were performed in triplicate and the data were expressed as mean standard deviation. The statistical analyses were performed with one-way analysis of variance (ANOVA). A multiple comparison Tukey test was used to evaluate if significant differences among treatments existed.
Results Effect of NaCl preincubation on the growth of P. kudriavzevii P. kudriavzevii is an extremophilic yeast that represents an attractive target for applied biotechnological research. In this study, NaCl preincubation showed different effects on the biomass of P. kudriavzevii and S. cerevisiae at 6 and 20 mg L1 of cadmium (Fig. 1). The biomass of P. kudriavzevii after NaCl preincubation was significantly enhanced at both tested cadmium concentrations. When the concentration of NaCl in preincubation was 60 g L1, the biomass of P. kudriavzevii at 6 and 20 mg L1 of cadmium increased 2.6 and 8.3 times compared with control respectively. However, there was no significant effect of NaCl preincubation on the biomass of S. cerevisiae at the tested cadmium concentrations. Effect of NaCl preincubation on ROS production and cell death Cadmium could enhance the intracellular ROS accumulation which is associated with the cell death of yeasts. The ROS production and cell death of P. kudriavzevii and S. cerevisiae pre-cultured by 10 and 60 g L1 of NaCl were determined at 6 and 20 mg L1 of cadmium (Fig. 2). The ROS production of P. kudriavzevii at both tested cadmium concentrations decreased after NaCl preincubation. Differently from P. kudriavzevii, the accumulation of
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Figure 1. Effect of NaCl preincubation on the biomass of P. kudriavzevii and S. cerevisiae incubated in the YEPD medium with the addition of 6 mg L1 (a) and 20 mg L1 (b) of cadmium. Bars with different letters are significantly different at p < 0.05.
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Figure 2. Flow cytometry dot plots of P. kudriavzevii at 6 mg L1 of cadmium (a), S. cerevisiae at 6 mg L1 of cadmium (b), P. kudriavzevii at 20 mg L1 of cadmium (c), and S. cerevisiae at 20 mg L1 of cadmium (d) after double staining with DCFH-DA and PI. Quadrant K1: DCF/PIþ, no (or low) ROS accumulation, dead cells; quadrant K2: DCFþ/PIþ, high ROS accumulation, dead cells; quadrant K3: DCF/PI, no (or low) ROS accumulation, living cells; quadrant K4: DCFþ/PI, high ROS accumulation, living cells. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 1. Effect of NaCl preincubation on protein carbonylation and malonyldialdehyde (MDA) production in P. kudriavzevii and S. cerevisiae at 6 mg L1 of cadmium. Strain P. kudriavzevii S. cerevisiae
NaCl (g L1)
Carbonylated protein (nmol mgprot1)
MDA (nmol mgprot1)
0 10 60 0 10 60
14.02 0.17c 11.09 0.23bc 2.29 0.18a 19.68 2.13d 43.36 2.52e 8.99 0.85b
3.10 0.23d 2.61 0.16cd 2.30 0.26bc 1.76 0.11b 1.95 0.23b 0.84 0.00a
Values in the same row with different letters (a–e) are significantly different at p < 0.05.
ROS in S. cerevisiae was enhanced by 10 g L1 of NaCl and reduced by 60 g L1 of NaCl at 6 mg L1 of cadmium, while the ROS production was inhibited by NaCl preincubation at 20 mg L1 of cadmium. The death rates of P. kudriavzevii at both 6 and 20 mg L1 of cadmium attenuated with the increase of NaCl concentrations, while the death rates of S. cerevisiae were on an increasing trend at 20 mg L1 of cadmium when the NaCl concentrations increased. NaCl preincubation increased the percentage of normal cells (quadrant K3) in P. kudriavzevii under both tested cadmium concentrations. However, the percentage of normal cells in S. cerevisiae was reduced by 10 g L1 of NaCl and promoted by 60 g L1 of NaCl at 6 mg L1 of cadmium, while there was a reducing trend of normal cells in S. cerevisiae at 20 mg L1 of cadmium after NaCl preincubation. Effect of NaCl preincubation on protein carbonylation and MDA production Protein carbonylation and MDA are commonly used markers to assess the oxidative damage of proteins and lipids in cells. The influence of NaCl preincubation on the contents of protein carbonyls and MDA in P. kudriavzevii and S. cerevisiae was studied at 6 mg L1 cadmium concentration and the results are presented in Table 1. Protein carbonylation in P. kudriavzevii under cadmium stress was alleviated by NaCl preincubation,
and 60 g L1 of NaCl showed more significant effect on reducing the contents of protein carbonyls than 10 g L1 of NaCl. An obvious decrease of the carbonyl contents in S. cerevisiae was induced by 60 g L1 of NaCl, while 10 g L1 of NaCl stimulated the level of protein carbonylation in the yeast. The contents of protein carbonyls in P. kudriavzevii and S. cerevisiae decreased from 14.02 and 19.68 nmol mgprot1 to 2.29 and 8.99 nmol mgprot1 respectively, when the yeasts pre-cultured with 60 g L1 of NaCl were compared with those without preincubation. The contents of MDA in P. kudriavzevii at 6 mg L1 of cadmium were reduced by NaCl preincubation, while the MDA production of S. cerevisiae was enhanced by 10 g L1 of NaCl and inhibited by 60 g L1 of NaCl. It could be observed that the contents of MDA in P. kudriavzevii and S. cerevisiae decreased from 3.10 and 1.76 nmol mgprot1 to 2.30 and 0.84 nmol mgprot1, respectively, when the NaCl concentration was raised from 0 to 60 g L1. Preincubation with 60 g L1 of NaCl displayed a significantly protective effect on lipids of both P. kudriavzevii and S. cerevisiae. Effect of NaCl preincubation on the activities of SOD, POD and CAT To mitigate the ROS-induced oxidative damage, yeast cells have developed a complex antioxidative defense system, including non-enzyme scavengers (e.g.,
Table 2. Effect of NaCl preincubation on superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in P. kudriavzevii and S. cerevisiae at 6 mg L1 of cadmium. Strain P. kudriavzevii
S. cerevisiae
NaCl (g L1)
SOD (U mgprot1)
POD (U mgprot1)
CAT (U mgprot1)
0 10 20 40 60 0 10 20 40 60
66.22 2.86a 48.64 3.31a 49.47 3.29a 52.93 0.47a 51.38 2.11a 293.11 16.44c 285.90 4.12c 227.84 8.67b 340.92 22.42d 440.88 11.35e
8.99 0.90ab 11.96 1.96bc 15.90 2.36cde 20.07 3.76e 17.92 0.16de 6.63 1.69a 16.15 0.85cde 14.98 1.00cd 12.23 1.11bc 15.85 0.50cde
13.13 1.23cd 11.44 0.61c 13.92 0.55d 15.35 0.00de 17.09 0.49e 3.55 0.75ab 3.23 0.90ab 2.12 0.96a 4.29 0.32ab 5.37 1.49b
Values in the same row with different letters (a–e) are significantly different at p < 0.05. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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glutathione, uric acid, vitamin C, and vitamin E) and antioxidant enzymes (e.g., SOD, CAT, and POD). In this study, the activities of SOD, POD, and CAT in P. kudriavzevii and S. cerevisiae were determined to evaluate the influence of NaCl preincubation on the oxidative stress tolerance of the yeasts (Table 2). There was no significant difference in the SOD activities of P. kudriavzevii with and without NaCl preincubation, while the SOD activities in S. cerevisiae pre-cultured in 40– 60 g L1 of NaCl increased significantly compared with that without preincubation. The POD activities of both P. kudriavzevii and S. cerevisiae increased after NaCl preincubation and reached a maximum after the yeasts were pre-cultured with 40 and 10 g L1 of NaCl, respectively. The CAT activities of P. kudriavzevii were significantly enhanced at 60 g L1 of NaCl, and the activities of CAT in S. cerevisiae increased at 40–60 g L1 of NaCl. Effect of NaCl preincubation on cadmium bioaccumulation capacity One possible explanation for the observed protective effect of NaCl preincubation on P. kudriavzevii was that NaCl preincubation might inhibit cadmium uptake in the yeast. The cadmium bioaccumulation capacities in P. kudriavzevii and S. cerevisiae showed different trends with the variation of NaCl concentrations at 6 and 20 mg L1 of cadmium (Fig. 3). For P. kudriavzevii, the continuously decreased cadmium bioaccumulation capacity was observed as the concentrations of NaCl in preincubation increased at both 6 and 20 mg L1 of cadmium. However, the cadmium bioaccumulation capacities in S. cerevisiae at both tested cadmium concentrations first increased and then decreased with the increase of NaCl concentrations and reached a maximum of 4.01 and 12.84 mg g1 when the NaCl concentrations were 10 and 20 g L1, respectively. The trends of cadmium bioaccumulation of both yeasts were opposite to that of biomass at both tested cadmium concentrations after NaCl preincubation (Fig. 1). Effect of NaCl preincubation on cadmium removal rate The cadmium removal rate of P. kudriavzevii was analyzed to evaluate the possibility of applying P. kudriavzevii with NaCl preincubation for cadmium removal. The cadmium removal rates of P. kudriavzevii and S. cerevisiae were differently affected by NaCl preincubation (Fig. 4). NaCl preincubation enhanced the cadmium removal rate of P. kudriavzevii at both 6 and 20 mg L1 of cadmium. The cadmium removal rate of P. kudriavzevii at 6 mg L1 of cadmium reached to maximum as 84.7% after the yeast was pre-cultured ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
with 10 g L1 of NaCl (Fig. 4a). In the presence of 20 mg L1 of cadmium, the cadmium removal rate of P. kudriavzevii was continuously improved by the increasing NaCl concentrations (Fig. 4b). However, NaCl preincubation did not show significant effect on the enhancement of cadmium removal rate of S. cerevisiae at the tested cadmium concentrations (Fig. 4). P. kudriavzevii after NaCl preincubation exhibited more powerful cadmium removal ability than S. cerevisiae. The cadmium removal rates of P. kudriavzevii at 6 and 20 mg L1 of cadmium were 77.8 and 37.8% after preincubation with 60 g L1 of NaCl, while the cadmium removal rate of S. cerevisiae were 47.5 and 13.5%, respectively.
Discussion As a nonessential element, cadmium is considered to be one of the most toxic heavy metals to microorganisms, which limits the application of growing cells in cadmium removal [22]. In microorganisms, stress conditions may induce some cross-protection which in turn defends other environmental challenges [23– 25]. In this study, NaCl preincubation displayed a protective effect on P. kudriavzevii against cadmium stress and remarkably enhanced the biomass of the yeast at different concentrations of cadmium (Fig. 1). The salt stress in the yeast cells caused by NaCl preincubation might induce some defenses to protect the yeast against cadmium stress. Similar results were reported where NaCl pretreatment increased the cellular viability of Geotrichum candidum at freezing– thawing challenge [26]. Exposure to heavy metals is an inducer of undesirable ROS accumulation [8, 27], which is associated with cell ageing and apoptosis in yeasts [10]. ROS accumulation and death rate in P. kudriavzevii at both 6 and 20 mg L1 of cadmium were reduced by NaCl preincubation (Fig. 4a, c), and the results were consistent with the variation of biomass in the yeast. It suggested that NaCl preincubation could defend P. kudriavzevii against cadmium stress through inhibiting ROS accumulation. Differently from P. kudriavzevii, the ROS accumulation in S. cerevisiae was inhibited by NaCl preincubation except 10 g L1 of NaCl at 6 mg L1 of cadmium, while the death rate of the yeast increased after NaCl preincubation. High amounts of ROS usually lead to increased accumulation of protein carbonyls and MDA [7], and this fact was also observed in P. kudriavzevii. NaCl preincubation markedly reduced the contents of carbonyls and MDA in P. kudriavzevii
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Figure 3. Effect of NaCl preincubation on cadmium bioaccumulation capacities of P. kudriavzevii and S. cerevisiae incubated in the YEPD medium with the addition of 6 mg L1 (a) and 20 mg L1 (b) of cadmium. Bars with different letters are significantly different at p < 0.05.
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Figure 4. Effect of NaCl preincubation on cadmium removal rate of P. kudriavzevii and S. cerevisiae incubated in the YEPD medium with the addition of 6 mg L1 (a) and 20 mg L1 (b) of cadmium. Bars with different letters are significantly different at p < 0.05.
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(Table 1). For S. cerevisiae, the content of protein carbonyl and MDA decreased when the yeast was precultured with 60 g L1 of NaCl, while 10 g L1 of NaCl enhanced the oxidative damage of proteins and lipids in the yeast (Table 1). The trends of protein carbonylation and MDA accubulation in both P. kudriavzevii and S. cerevisiae were consistent with those of biomass (Fig. 1). Protecting proteins and lipids from oxidative damage might be part of the protection mechanism of NaCl preincubation on P. kudriavzevii under cadmium stress. Similar results have been reported by An et al. [28], who found that exogenous calcium could alleviate cadmium toxicity substantially by reducing the level of ROS and protein carbonylation in Debaryomyces hansenii. The stimulation of antioxidant enzyme activities is one of the important pathways to resist the oxidative stress in yeasts [29]. NaCl preincubation showed no significant effect on the SOD activity in P. kudriavzevii, while the activities of POD and CAT were evidently enhanced by NaCl preincubation (Table 1). In S. cerevisiae, NaCl preincubation enhanced the activities of POD, and activation of SOD was observed after the yeast was precultured with 40–60 g L1 of NaCl (Table 1). The activation of POD and CAT in P. kudriavzevii by NaCl preincubation might play an important role in decreasing the ROS accumulation and alleviating protein and lipid oxidative damage under cadmium stress to improve the viability of yeast cells. It was reported that NaCl could induce an enhancement of SOD and POD activities in Debaryomyces hansenii under oxidative stress caused by cadmium [30]. The toxicity of heavy metals was usually related to the amount of heavy metal uptake into the cells [31]. The cadmium bioaccumulation capacities of P. kudriavzevii were reduced significantly by NaCl preincubation (Fig. 3). The protective effect exerted by NaCl can be explained, at least in part, by the substantial reduction in cadmium uptake produced by the preincubation with NaCl. Li et al. [21] have reported similar results where NaCl showed a protective effect against cadmium stress in both Z. rouxii and S. cerevisiae through inhibiting cadmium uptake in both yeasts. The cadmium removal rate of P. kudriavzevii was significantly enhanced mainly because of the improved biomass of P. kudriavzevii after NaCl preincubation. The results indicated that NaCl preincubation was a practicable way for improving cadmium tolerance and removal ability of P. kudriavzevii. The salt-tolerant P. kudriavzevii that exhibited more powerful cadmium removal ability than S. cerevisiae might be more suitable for cadmium removal. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Acknowledgments The authors would like to thank for the financial support of the National Natural Science Foundation of China (No. 31101330) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1188).
References [1] Chojnacka, K., 2010. Biosorption and bioaccumulation— the prospects for practical applications, Environ. Int., 36, 299–307. [2] Kwok, C.K., Liang, Y., Wang, H., Dong, Y.H. et al. 2014. Bioaccumulation of heavy metals in fish and Ardeid at Pearl River Estuary, China. Ecotoxicol. Environ. Saf., 106, 62–67. [3] Mudhoo, A., Garg, V.K., Wang, S., 2012. Heavy Metals: Toxicity and Removal by Biosorption, In: Lichtfouse, E., Schwarzbauer, J., Robert, D. (eds.) Environmental Chemistry for a Sustainable World. Springer, Netherlands. 379–442. [4] Ghimire, K.N., Kai, H., Inoue, K., Ohto, K. et al. 2008. Heavy metal removal from contaminated scallop waste for feed and fertilizer application, Bioresour. Technol., 99, 2436– 2441. [5] Wang, J., Chen, C., 2009. Biosorbents for heavy metals removal and their future, Biotechnol. Adv., 27, 195–226. [6] Li, C., Jiang, W., Ma, N., Zhu, Y. et al. 2014. Bioaccumulation of cadmium by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae, Bioresour. Technol., 155, 116–121. [7] Avery, S.V., 2011. Molecular targets of oxidative stress, Biochem. J., 434, 201–210. [8] Brennan, R.J., Schiestl, R.H., 1996. Cadmium is an inducer of oxidative stress in yeast, Mutat. Res. Mol. Mech. Mutagen., 356, 171–178. [9] Chakraborty, S., Mukherjee, A., Khuda-Bukhsh, A.R., Das, T.K., 2014. Cadmium-induced oxidative stress tolerance in cadmium resistant Aspergillus foetidus: its possible role in cadmium bioremediation, Ecotoxicol. Environ. Saf., 106, 46–53. [10] Perrone, G.G., Tan, S-X., Dawes, I.W., 2008. Reactive oxygen species and yeast apoptosis, Biochim. Biophys. Acta BBA - Mol. Cell Res., 1783, 1354–1368. [11] Li, C., Xu, Y., Jiang, W., Lv, X. et al. 2014. Effect of sodium chloride and cadmium on the growth, oxidative stress and antioxidant enzyme activities of Zygosaccharomyces rouxii, J. Ocean Univ. China, 13, 460–466. [12] Gallardo, J.C.M., Souza, C.S., Cicarelli, R.M.B., Oliveira, K.F. et al. 2011. Enrichment of a continuous culture of Saccharomyces cerevisiae with the yeast Issatchenkia orientalis in the production of ethanol at increasing temperatures, J. Ind. Microbiol. Biotechnol., 38, 405–414. [13] Chan, G.F., Gan, H.M., Ling, H.L., Rashida, N.A.A., 2012. Genome sequence of Pichia kudriavzevii M12, a potential producer of bioethanol and phytase, Eukaryot. Cell, 11, 1300–1301.
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[14] Isono, N., Hayakawa, H., Usami, A., Mishima, T. et al. 2012. A comparative study of ethanol production by Issatchenkia orientalis strains under stress conditions, J. Biosci. Bioeng., 113, 76–78. [15] Kitagawa, T., Tokuhiro, K., Sugiyama, H., Kohda, K. et al. 2010. Construction of a beta-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis, Appl. Microbiol. Biotechnol., 87, 1841–1853. [16] Oberoi, H.S., Babbar, N., Sandhu, S.K., Dhaliwal, S.S. et al. 2012. Ethanol production from alkali-treated rice straw via simultaneous saccharification and fermentation using newly isolated thermotolerant Pichia kudriavzevii HOP-1, J. Ind. Microbiol. Biotechnol., 39, 557–566. [17] Halm, M., Hornbæk, T., Arneborg, N., Sefa-Dedeh, S. et al. 2004. Lactic acid tolerance determined by measurement of intracellular pH of single cells of Candida krusei and Saccharomyces cerevisiae isolated from fermented maize dough, Int. J. Food Microbiol., 94, 97–103. [18] Casey, G.D., Dobson, A.D., 2003. Molecular detection of Candida krusei contamination in fruit juice using the citrate synthase gene cs1 and a potential role for this gene in the adaptive response to acetic acid, J. Appl. Microbiol., 95, 13–22. [19] Meng, F., Zhang, Y., Liu, F., Guo, X. et al. 2014. Characterization and mutational analysis of omega-class GST (GSTO1) from Apis cerana cerana, a gene involved in response to oxidative stress, PLoS ONE, 9, e93100. [20] Zhang, Y., Lu, H., Yuan, Y., Liu, X. et al. 2009. Neuroprotective effects of combined lead and cadmium, as well as N-acetylcysteine, on cerebral cortical neurons following lipid peroxidation injury, Neural Regen. Res., 4, 907– 911. [21] Li, C., Xu, Y., Jiang, W., Dong, X., et al. 2013. Effect of NaCl on the heavy metal tolerance and bioaccumulation of Zygosaccharomyces rouxii and Saccharomyces cerevisiae, Bioresour. Technol., 143, 46–52.
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[22] Nies, D.H., 1999. Microbial heavy-metal resistance, Appl. Microbiol. Biotechnol., 51, 730–750. [23] Kastbjerg, V.G., Nielsen, D.S., Arneborg, N., Gram, L., 2009. Response of Listeria monocytogenes to disinfection stress at the single-cell and population levels as monitored by intracellular pH measurements and viable-cell counts, Appl. Environ. Microbiol., 75, 4550–4556. [24] Rangel, D.E.N., 2011. Stress induced cross-protection against environmental challenges on prokaryotic and eukaryotic microbes, World J. Microbiol. Biotechnol., 27, 1281–1296. [25] Suga, M., Kusanagi, I., Hatakeyama, T., 2003. High osmotic stress improves electro-transformation efficiency of fission yeast, FEMS Microbiol. Lett., 225, 235–239. [26] Dubernet, S., Panoff, J-M., Thammavongs, B., Gueguen, M., 2002. Nystatin and osmotica as chemical enhancers of the phenotypic adaptation to freeze–thaw stress in Geotrichum candidum ATCC 204307, Int. J. Food Microbiol., 76, 215– 221. [27] Jamieson, D.J., 1998. Oxidative stress responses of the yeast Saccharomyces cerevisiae, Yeast, 14, 1511–1527. [28] An, B., Li, B., Qin, G., Tian, S., 2012. Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein, Curr. Microbiol., 65, 122–127. [29] Moradas-Ferreira, P., Costa, V., Piper, P., Mager, W., 1996. The molecular defences against reactive oxygen species in yeast, Mol. Microbiol., 19, 651–658. [30] Navarrete, C., Siles, A., Martınez, J.L., Calero, F., et al. 2009. Oxidative stress sensitivity in Debaryomyces hansenii, FEMS Yeast Res., 9, 582–590. [31] Jiang, W., Xu, Y., Li, C., Lv, X., Wang, D., 2013. Effect of inorganic salts on the growth and Cd2þ bioaccumulation of Zygosaccharomyces rouxii cultured under Cd2þ stress, Bioresour. Technol., 128, 831–834.
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Research Paper Different effects of sodium chloride preincubation on cadmium tolerance of Pichia kudriavzevii and Saccharomyces cerevisiae Ning Ma, Chunsheng Li, Xiaoyan Dong, Dongfeng Wang and Ying Xu Laboratory of Food Chemistry and Nutrition, College of Food Science and Engineering, Ocean University of China, Qingdao, China
Application of growing microorganisms for cadmium removal is restricted by high cadmium toxicity. The effects of sodium chloride (NaCl) preincubation on the cadmium tolerance and removal ability of Pichia kudriavzevii and Saccharomyces cerevisiae were investigated in this study. NaCl preincubation improved the biomass of P. kudriavzevii under cadmium stress, while no obvious effect was observed in S. cerevisiae. The improved activities of peroxidase (POD) and catalase (CAT) after NaCl preincubation might be an important reason for the decrease of the reactive oxygen species (ROS) accumulation, cell death, and oxidative damage of proteins and lipids induced by cadmium, contributing to the improvement of the yeast growth. The cadmium bioaccumulation capacity of P. kudriavzevii decreased significantly after NaCl preincubation, which played an important role in mitigating the cadmium toxicity to the yeast. The cadmium removal rate of P. kudriavzevii was obviously higher than S. cerevisiae and was significantly enhanced after NaCl preincubation. The results suggested that NaCl preincubation improved the cadmium tolerance and removal ability of P. kudriavzevii. Keywords: Pichia kudriavzevii / Saccharomyces cerevisiae / NaCl preincubation / Cadmium bioaccumulation / Oxidative stress Received: November 6, 2014; accepted: February 10, 2015 DOI 10.1002/jobm.201400847
Introduction Aquatic environment as a result of increased industrial activities deteriorates permanently because of a wide range of environmental factors including heavy metals [1]. Due to biomagnification and accumulation via the food chain, there is an increasing risk of heavy metal contamination in aquatic products, causing significant threats to human health [2]. Since heavy metals are not biodegradable [3], the removal of heavy metals should be taken into consideration first before using the food materials contaminated by heavy metals, e.g., enzymatic hydrolysate of aquatic product. Conventional physiochemical methods for heavy metal removal, such as chemical precipitation, adsorption method, and ion The first two authors contributed equally to this work. Correspondence: Ying Xu, Laboratory of Food Chemistry and Nutrition, College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China E-mail: [email protected] Phone: þ86 0532-82031851 Fax: þ86 0532-82032093 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
exchange, are ineffective in food environment for low concentrations of heavy metals and high concentrations of organic compounds [4, 5]. Therefore, an alternative technique is urgently required for removing heavy metals from food environment. The bioaccumulation of heavy metals via growing microorganisms has received a great deal of attention in the field of heavy metal treatment in recent years [1, 5]. In the process of bioaccumulation, heavy metals are not only bound to the cell surface but also accumulated inside the cell mainly via energy-consuming active transport systems [6]. However, the bioremoval of heavy metals is highly affected by high concentrations of heavy metals which can inhibit both the cell growth and bioaccumulation [1]. It was reported that the increase of intracellular cadmium concentration resulted in the overproduction of reactive oxygen species (ROS) which rapidly attack all biomolecules, such as DNA, lipids, and proteins, eventually leading to cell death [7–10]. Therefore, it is particularly important to improve the heavy metal tolerance of
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microorganisms for employing growing cells in heavy metal removal. Our previous study showed that the combined use of sodium chloride (NaCl) and cadmium reduced the growth-inhibiting effect of cadmium as well as the cadmium-induced ROS production and cell death of Zygosaccharomyces rouxii, indicating that NaCl might display a protective effect on Z. rouxii under cadmium stress [11]. However, little work has been done to study whether NaCl preincubation could improve the cadmium tolerance of yeasts. Pichia kudriavzevii, synonymously known as Issatchenkia orientalis or Candida krusei, has been isolated from a variety of fermented foods and fruit sources [12, 13]. P. kudriavzevii has recently been described as a multistress-tolerant yeast which can tolerate up to 40% glucose, 10% NaCl, pH as low as 2, and temperatures over 42 °C [12, 14–16]. In addition, P. kudriavzevii is more resistant to lactic acid [17] and acetic acid [18] than S. cerevisiae. It provides more possibilities to employ growing P. kudriavzevii biomass for heavy metal removal in complex food environment. In this study, the effect of NaCl preincubation on the growth of P. kudriavzevii under cadmium stress was studied. The oxidative stress and cadmium bioaccumulation capacity in P. kudriavzevii were also studied to preliminarily explore the mechanism of the protective effect of NaCl preincubation against cadmium. The cadmium removal ability of P. kudriavzevii was measured in order to elucidate the possibility of using NaCl preincubation for cadmium removal by growing P. kudriavzevii. S. cerevisiae was used as the reference yeast for comparison.
Materials and methods Strains and preincubation Pichia kudriavzevii A16 was isolated from a high-temperature Chinese liquor starter, and Saccharomyces cerevisiae CICC1211 was purchased from the China Center of Industrial Culture Collection. Both yeasts were maintained in YEPD agar slants (1% yeast extract powder, 2% peptone, 2% glucose, and 2% agar, pH 5.0) at 4 °C. After being refreshed in YEPD agar slant at 28 °C for 24 h, the yeasts were transferred to 250 ml Erlenmeyer flasks containing 50 ml YEPD broth, and incubated at 28 °C and 180 rpm for 24 h. Thereafter, NaCl preincubation was done by transferring 1 ml of culture to 250 ml Erlenmeyer flasks containing 50 ml YEPD broth with different NaCl concentrations (10–60 g L1), and incubating at 28 °C and 180 rpm for 24 h. The yeasts cultured in YEPD broth without NaCl were used as control. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Growth assay The cadmium stock solution (100 mg L1) was prepared by dissolving CdCl22.5H2O (Sinopharm Group, China) in ultrapure water (Millipore, America). The cell concentrations of both yeasts after NaCl preincubation were measured by a hemocytometer. Then, the yeast cells with same inoculum size (4 108 cells) were pelleted and transferred to 50 ml YEPD broth with different concentrations of cadmium (6 and 20 mg L1). After cultivation at 28 °C and 180 rpm for 24 h, the cells were centrifuged, washed twice with ultrapure water, and then weighed after drying to constant weight at 80 °C to determine the dry weight. The biomass was defined as the dry weight of the yeast cells per liter of cultured medium. Determination of ROS in yeast cells 20 ,70 -dichlorofluorescein diacetate (DCFH-DA, Sigma, America) and propidium iodide (PI, Sigma, America) were used for double staining to determine the intracellular ROS level and cell death rate according to Li et al. [11]. Briefly, the yeast cells incubated with different concentrations (6 and 20 mg L1) of cadmium were washed twice with phosphate buffered saline (PBS, pH 7.0, 0.05 mol L1) and diluted to 1 107 cells ml1. After incubation with 100 mmol L1 DCFH-DA at 37 °C and 60 rpm for 50 min in the dark, the samples were rapidly placed on ice, washed twice, resuspended in 1 ml of PBS, and then stained with 10 mg ml1 PI. A flow cytometer (Beckman Coulter Cytomics FC 500 MPL) equipped with an air-cooled 488 nm argon laser was used to record the fluorescence of cell suspensions. Green fluorescence of the cells stained with 20 ,70 dichlorofluorescein (DCF) was collected in the FL1 channel (525 nm 20 nm), and red fluorescence of the cells labeled with PI was collected in the FL3 Channel (625 nm 20 nm). A total of 20,000 events were registered per sample and the obtained data were analyzed with the CXP Analysis 2.1 software. Protein carbonyls and malonyldialdehyde (MDA) assays After being incubated with 6 mg L1 of cadmium for 24 h, the yeast cells were centrifuged and washed twice with PBS. The cells were ground with liquid nitrogen, resuspended in PBS and centrifuged at 4 °C and 12,000 rpm for 15 min. The supernatant was collected to assay the content of protein carbonyls and MDA. The protein carbonylation was detected by a protein carbonyl detection kit (Nanjing Jiancheng Bioengineering Institute, China). Briefly, protein carbonyls were derivatized to 2,4-dinitrophenylhydrazone (DNP) after the addition
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of 2,4-dinitrophenylhydrazine (DNPH). DNP was resuspended in guanidine HCl, and the absorbance was measured at 370 nm. Results were expressed as nmol of carbonyls per mg of protein. A micro-malonaldehyde detection kit (Nanjing Jiancheng Bioengineering Institute, China) was used for MDA determination. Simply, thiobarbituric acid (TBA) reacted with MDA to form red products, and the absorbance was measured at 532 nm. The MDA concentration was expressed as nmol of MDA per mg of protein [19]. Detection of antioxidant enzyme activity The yeast cells were collected and resuspended in PBS as the methods described above. The samples were then centrifuged at 4 °C and 12,000 rpm for 10 min. The supernatant was collected to assay the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) with a commercial reagent kit (Nanjing Jiancheng Bioengineering Institute, China) spectrophotometrically. The activities of SOD, CAT, and POD were expressed as units per milligram protein (U mgprot1). The activity of SOD was assayed at 550 nm using the xanthine and xanthine oxidase system. One unit (U) of SOD activity was defined as the amount of enzyme causing 50% inhibition of xanthine and xanthine oxidase reaction system. The samples were treated with excess of H2O2 at 37 °C, and the absorbance of remaining H2O2 was measured at 405 nm to determine the activity of CAT. One unit (U) of CAT activity was defined based on the decomposition of 1 mmol H2O2 by the enzyme per second [20]. The samples were treated with H2O2 at 37 °C for 30 min. POD activity was determined by detection of changes in the absorbance at 420 nm. One unit (U) of POD activity was defined as the amount of enzyme that catalyzed 1 mg H2O2 min1 at 37 °C. The content of total protein was measured by coomassie blue staining, using bovine serum albumin (BSA, Solarbio, China) as a standard. Cadmium bioaccumulation capacity assay The cells incubated under cadmium stress for 24 h were centrifuged, washed twice with ultrapure water, and then weighed after drying to constant weight at 80 °C to calculate the biomass (M) of the yeasts. The supernatant was thoroughly digested with nitric acid and perchloric acid, and diluted with ultrapure water. The cadmium concentration of the solution was measured by an atomic absorption spectrophotometer (AA-6800, Shimadzu, Japan) [21]. Cadmium removal rate (R) and cadmium bioaccumulation capacity (Q) were calculated by using the following equations: ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
R¼
1 Ct C0
Q ¼
ðC0 Ct Þ M
100%
ð1Þ
ð2Þ
where R is cadmium removal rate in the YEPD medium (%), Ct is final cadmium concentration in YEPD medium (mg L1), C0 is initial cadmium concentration in YEPD medium (mg L1), Q is cadmium bioaccumulation capacities of the yeasts (mg g1), and M is the yeast biomass in the cultured YEPD medium (g L1). Statistical analysis All experiments were performed in triplicate and the data were expressed as mean standard deviation. The statistical analyses were performed with one-way analysis of variance (ANOVA). A multiple comparison Tukey test was used to evaluate if significant differences among treatments existed.
Results Effect of NaCl preincubation on the growth of P. kudriavzevii P. kudriavzevii is an extremophilic yeast that represents an attractive target for applied biotechnological research. In this study, NaCl preincubation showed different effects on the biomass of P. kudriavzevii and S. cerevisiae at 6 and 20 mg L1 of cadmium (Fig. 1). The biomass of P. kudriavzevii after NaCl preincubation was significantly enhanced at both tested cadmium concentrations. When the concentration of NaCl in preincubation was 60 g L1, the biomass of P. kudriavzevii at 6 and 20 mg L1 of cadmium increased 2.6 and 8.3 times compared with control respectively. However, there was no significant effect of NaCl preincubation on the biomass of S. cerevisiae at the tested cadmium concentrations. Effect of NaCl preincubation on ROS production and cell death Cadmium could enhance the intracellular ROS accumulation which is associated with the cell death of yeasts. The ROS production and cell death of P. kudriavzevii and S. cerevisiae pre-cultured by 10 and 60 g L1 of NaCl were determined at 6 and 20 mg L1 of cadmium (Fig. 2). The ROS production of P. kudriavzevii at both tested cadmium concentrations decreased after NaCl preincubation. Differently from P. kudriavzevii, the accumulation of
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Figure 1. Effect of NaCl preincubation on the biomass of P. kudriavzevii and S. cerevisiae incubated in the YEPD medium with the addition of 6 mg L1 (a) and 20 mg L1 (b) of cadmium. Bars with different letters are significantly different at p < 0.05.
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Figure 2. Flow cytometry dot plots of P. kudriavzevii at 6 mg L1 of cadmium (a), S. cerevisiae at 6 mg L1 of cadmium (b), P. kudriavzevii at 20 mg L1 of cadmium (c), and S. cerevisiae at 20 mg L1 of cadmium (d) after double staining with DCFH-DA and PI. Quadrant K1: DCF/PIþ, no (or low) ROS accumulation, dead cells; quadrant K2: DCFþ/PIþ, high ROS accumulation, dead cells; quadrant K3: DCF/PI, no (or low) ROS accumulation, living cells; quadrant K4: DCFþ/PI, high ROS accumulation, living cells. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 1. Effect of NaCl preincubation on protein carbonylation and malonyldialdehyde (MDA) production in P. kudriavzevii and S. cerevisiae at 6 mg L1 of cadmium. Strain P. kudriavzevii S. cerevisiae
NaCl (g L1)
Carbonylated protein (nmol mgprot1)
MDA (nmol mgprot1)
0 10 60 0 10 60
14.02 0.17c 11.09 0.23bc 2.29 0.18a 19.68 2.13d 43.36 2.52e 8.99 0.85b
3.10 0.23d 2.61 0.16cd 2.30 0.26bc 1.76 0.11b 1.95 0.23b 0.84 0.00a
Values in the same row with different letters (a–e) are significantly different at p < 0.05.
ROS in S. cerevisiae was enhanced by 10 g L1 of NaCl and reduced by 60 g L1 of NaCl at 6 mg L1 of cadmium, while the ROS production was inhibited by NaCl preincubation at 20 mg L1 of cadmium. The death rates of P. kudriavzevii at both 6 and 20 mg L1 of cadmium attenuated with the increase of NaCl concentrations, while the death rates of S. cerevisiae were on an increasing trend at 20 mg L1 of cadmium when the NaCl concentrations increased. NaCl preincubation increased the percentage of normal cells (quadrant K3) in P. kudriavzevii under both tested cadmium concentrations. However, the percentage of normal cells in S. cerevisiae was reduced by 10 g L1 of NaCl and promoted by 60 g L1 of NaCl at 6 mg L1 of cadmium, while there was a reducing trend of normal cells in S. cerevisiae at 20 mg L1 of cadmium after NaCl preincubation. Effect of NaCl preincubation on protein carbonylation and MDA production Protein carbonylation and MDA are commonly used markers to assess the oxidative damage of proteins and lipids in cells. The influence of NaCl preincubation on the contents of protein carbonyls and MDA in P. kudriavzevii and S. cerevisiae was studied at 6 mg L1 cadmium concentration and the results are presented in Table 1. Protein carbonylation in P. kudriavzevii under cadmium stress was alleviated by NaCl preincubation,
and 60 g L1 of NaCl showed more significant effect on reducing the contents of protein carbonyls than 10 g L1 of NaCl. An obvious decrease of the carbonyl contents in S. cerevisiae was induced by 60 g L1 of NaCl, while 10 g L1 of NaCl stimulated the level of protein carbonylation in the yeast. The contents of protein carbonyls in P. kudriavzevii and S. cerevisiae decreased from 14.02 and 19.68 nmol mgprot1 to 2.29 and 8.99 nmol mgprot1 respectively, when the yeasts pre-cultured with 60 g L1 of NaCl were compared with those without preincubation. The contents of MDA in P. kudriavzevii at 6 mg L1 of cadmium were reduced by NaCl preincubation, while the MDA production of S. cerevisiae was enhanced by 10 g L1 of NaCl and inhibited by 60 g L1 of NaCl. It could be observed that the contents of MDA in P. kudriavzevii and S. cerevisiae decreased from 3.10 and 1.76 nmol mgprot1 to 2.30 and 0.84 nmol mgprot1, respectively, when the NaCl concentration was raised from 0 to 60 g L1. Preincubation with 60 g L1 of NaCl displayed a significantly protective effect on lipids of both P. kudriavzevii and S. cerevisiae. Effect of NaCl preincubation on the activities of SOD, POD and CAT To mitigate the ROS-induced oxidative damage, yeast cells have developed a complex antioxidative defense system, including non-enzyme scavengers (e.g.,
Table 2. Effect of NaCl preincubation on superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in P. kudriavzevii and S. cerevisiae at 6 mg L1 of cadmium. Strain P. kudriavzevii
S. cerevisiae
NaCl (g L1)
SOD (U mgprot1)
POD (U mgprot1)
CAT (U mgprot1)
0 10 20 40 60 0 10 20 40 60
66.22 2.86a 48.64 3.31a 49.47 3.29a 52.93 0.47a 51.38 2.11a 293.11 16.44c 285.90 4.12c 227.84 8.67b 340.92 22.42d 440.88 11.35e
8.99 0.90ab 11.96 1.96bc 15.90 2.36cde 20.07 3.76e 17.92 0.16de 6.63 1.69a 16.15 0.85cde 14.98 1.00cd 12.23 1.11bc 15.85 0.50cde
13.13 1.23cd 11.44 0.61c 13.92 0.55d 15.35 0.00de 17.09 0.49e 3.55 0.75ab 3.23 0.90ab 2.12 0.96a 4.29 0.32ab 5.37 1.49b
Values in the same row with different letters (a–e) are significantly different at p < 0.05. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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glutathione, uric acid, vitamin C, and vitamin E) and antioxidant enzymes (e.g., SOD, CAT, and POD). In this study, the activities of SOD, POD, and CAT in P. kudriavzevii and S. cerevisiae were determined to evaluate the influence of NaCl preincubation on the oxidative stress tolerance of the yeasts (Table 2). There was no significant difference in the SOD activities of P. kudriavzevii with and without NaCl preincubation, while the SOD activities in S. cerevisiae pre-cultured in 40– 60 g L1 of NaCl increased significantly compared with that without preincubation. The POD activities of both P. kudriavzevii and S. cerevisiae increased after NaCl preincubation and reached a maximum after the yeasts were pre-cultured with 40 and 10 g L1 of NaCl, respectively. The CAT activities of P. kudriavzevii were significantly enhanced at 60 g L1 of NaCl, and the activities of CAT in S. cerevisiae increased at 40–60 g L1 of NaCl. Effect of NaCl preincubation on cadmium bioaccumulation capacity One possible explanation for the observed protective effect of NaCl preincubation on P. kudriavzevii was that NaCl preincubation might inhibit cadmium uptake in the yeast. The cadmium bioaccumulation capacities in P. kudriavzevii and S. cerevisiae showed different trends with the variation of NaCl concentrations at 6 and 20 mg L1 of cadmium (Fig. 3). For P. kudriavzevii, the continuously decreased cadmium bioaccumulation capacity was observed as the concentrations of NaCl in preincubation increased at both 6 and 20 mg L1 of cadmium. However, the cadmium bioaccumulation capacities in S. cerevisiae at both tested cadmium concentrations first increased and then decreased with the increase of NaCl concentrations and reached a maximum of 4.01 and 12.84 mg g1 when the NaCl concentrations were 10 and 20 g L1, respectively. The trends of cadmium bioaccumulation of both yeasts were opposite to that of biomass at both tested cadmium concentrations after NaCl preincubation (Fig. 1). Effect of NaCl preincubation on cadmium removal rate The cadmium removal rate of P. kudriavzevii was analyzed to evaluate the possibility of applying P. kudriavzevii with NaCl preincubation for cadmium removal. The cadmium removal rates of P. kudriavzevii and S. cerevisiae were differently affected by NaCl preincubation (Fig. 4). NaCl preincubation enhanced the cadmium removal rate of P. kudriavzevii at both 6 and 20 mg L1 of cadmium. The cadmium removal rate of P. kudriavzevii at 6 mg L1 of cadmium reached to maximum as 84.7% after the yeast was pre-cultured ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
with 10 g L1 of NaCl (Fig. 4a). In the presence of 20 mg L1 of cadmium, the cadmium removal rate of P. kudriavzevii was continuously improved by the increasing NaCl concentrations (Fig. 4b). However, NaCl preincubation did not show significant effect on the enhancement of cadmium removal rate of S. cerevisiae at the tested cadmium concentrations (Fig. 4). P. kudriavzevii after NaCl preincubation exhibited more powerful cadmium removal ability than S. cerevisiae. The cadmium removal rates of P. kudriavzevii at 6 and 20 mg L1 of cadmium were 77.8 and 37.8% after preincubation with 60 g L1 of NaCl, while the cadmium removal rate of S. cerevisiae were 47.5 and 13.5%, respectively.
Discussion As a nonessential element, cadmium is considered to be one of the most toxic heavy metals to microorganisms, which limits the application of growing cells in cadmium removal [22]. In microorganisms, stress conditions may induce some cross-protection which in turn defends other environmental challenges [23– 25]. In this study, NaCl preincubation displayed a protective effect on P. kudriavzevii against cadmium stress and remarkably enhanced the biomass of the yeast at different concentrations of cadmium (Fig. 1). The salt stress in the yeast cells caused by NaCl preincubation might induce some defenses to protect the yeast against cadmium stress. Similar results were reported where NaCl pretreatment increased the cellular viability of Geotrichum candidum at freezing– thawing challenge [26]. Exposure to heavy metals is an inducer of undesirable ROS accumulation [8, 27], which is associated with cell ageing and apoptosis in yeasts [10]. ROS accumulation and death rate in P. kudriavzevii at both 6 and 20 mg L1 of cadmium were reduced by NaCl preincubation (Fig. 4a, c), and the results were consistent with the variation of biomass in the yeast. It suggested that NaCl preincubation could defend P. kudriavzevii against cadmium stress through inhibiting ROS accumulation. Differently from P. kudriavzevii, the ROS accumulation in S. cerevisiae was inhibited by NaCl preincubation except 10 g L1 of NaCl at 6 mg L1 of cadmium, while the death rate of the yeast increased after NaCl preincubation. High amounts of ROS usually lead to increased accumulation of protein carbonyls and MDA [7], and this fact was also observed in P. kudriavzevii. NaCl preincubation markedly reduced the contents of carbonyls and MDA in P. kudriavzevii
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Figure 3. Effect of NaCl preincubation on cadmium bioaccumulation capacities of P. kudriavzevii and S. cerevisiae incubated in the YEPD medium with the addition of 6 mg L1 (a) and 20 mg L1 (b) of cadmium. Bars with different letters are significantly different at p < 0.05.
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Figure 4. Effect of NaCl preincubation on cadmium removal rate of P. kudriavzevii and S. cerevisiae incubated in the YEPD medium with the addition of 6 mg L1 (a) and 20 mg L1 (b) of cadmium. Bars with different letters are significantly different at p < 0.05.
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(Table 1). For S. cerevisiae, the content of protein carbonyl and MDA decreased when the yeast was precultured with 60 g L1 of NaCl, while 10 g L1 of NaCl enhanced the oxidative damage of proteins and lipids in the yeast (Table 1). The trends of protein carbonylation and MDA accubulation in both P. kudriavzevii and S. cerevisiae were consistent with those of biomass (Fig. 1). Protecting proteins and lipids from oxidative damage might be part of the protection mechanism of NaCl preincubation on P. kudriavzevii under cadmium stress. Similar results have been reported by An et al. [28], who found that exogenous calcium could alleviate cadmium toxicity substantially by reducing the level of ROS and protein carbonylation in Debaryomyces hansenii. The stimulation of antioxidant enzyme activities is one of the important pathways to resist the oxidative stress in yeasts [29]. NaCl preincubation showed no significant effect on the SOD activity in P. kudriavzevii, while the activities of POD and CAT were evidently enhanced by NaCl preincubation (Table 1). In S. cerevisiae, NaCl preincubation enhanced the activities of POD, and activation of SOD was observed after the yeast was precultured with 40–60 g L1 of NaCl (Table 1). The activation of POD and CAT in P. kudriavzevii by NaCl preincubation might play an important role in decreasing the ROS accumulation and alleviating protein and lipid oxidative damage under cadmium stress to improve the viability of yeast cells. It was reported that NaCl could induce an enhancement of SOD and POD activities in Debaryomyces hansenii under oxidative stress caused by cadmium [30]. The toxicity of heavy metals was usually related to the amount of heavy metal uptake into the cells [31]. The cadmium bioaccumulation capacities of P. kudriavzevii were reduced significantly by NaCl preincubation (Fig. 3). The protective effect exerted by NaCl can be explained, at least in part, by the substantial reduction in cadmium uptake produced by the preincubation with NaCl. Li et al. [21] have reported similar results where NaCl showed a protective effect against cadmium stress in both Z. rouxii and S. cerevisiae through inhibiting cadmium uptake in both yeasts. The cadmium removal rate of P. kudriavzevii was significantly enhanced mainly because of the improved biomass of P. kudriavzevii after NaCl preincubation. The results indicated that NaCl preincubation was a practicable way for improving cadmium tolerance and removal ability of P. kudriavzevii. The salt-tolerant P. kudriavzevii that exhibited more powerful cadmium removal ability than S. cerevisiae might be more suitable for cadmium removal. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Acknowledgments The authors would like to thank for the financial support of the National Natural Science Foundation of China (No. 31101330) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1188).
References [1] Chojnacka, K., 2010. Biosorption and bioaccumulation— the prospects for practical applications, Environ. Int., 36, 299–307. [2] Kwok, C.K., Liang, Y., Wang, H., Dong, Y.H. et al. 2014. Bioaccumulation of heavy metals in fish and Ardeid at Pearl River Estuary, China. Ecotoxicol. Environ. Saf., 106, 62–67. [3] Mudhoo, A., Garg, V.K., Wang, S., 2012. Heavy Metals: Toxicity and Removal by Biosorption, In: Lichtfouse, E., Schwarzbauer, J., Robert, D. (eds.) Environmental Chemistry for a Sustainable World. Springer, Netherlands. 379–442. [4] Ghimire, K.N., Kai, H., Inoue, K., Ohto, K. et al. 2008. Heavy metal removal from contaminated scallop waste for feed and fertilizer application, Bioresour. Technol., 99, 2436– 2441. [5] Wang, J., Chen, C., 2009. Biosorbents for heavy metals removal and their future, Biotechnol. Adv., 27, 195–226. [6] Li, C., Jiang, W., Ma, N., Zhu, Y. et al. 2014. Bioaccumulation of cadmium by growing Zygosaccharomyces rouxii and Saccharomyces cerevisiae, Bioresour. Technol., 155, 116–121. [7] Avery, S.V., 2011. Molecular targets of oxidative stress, Biochem. J., 434, 201–210. [8] Brennan, R.J., Schiestl, R.H., 1996. Cadmium is an inducer of oxidative stress in yeast, Mutat. Res. Mol. Mech. Mutagen., 356, 171–178. [9] Chakraborty, S., Mukherjee, A., Khuda-Bukhsh, A.R., Das, T.K., 2014. Cadmium-induced oxidative stress tolerance in cadmium resistant Aspergillus foetidus: its possible role in cadmium bioremediation, Ecotoxicol. Environ. Saf., 106, 46–53. [10] Perrone, G.G., Tan, S-X., Dawes, I.W., 2008. Reactive oxygen species and yeast apoptosis, Biochim. Biophys. Acta BBA - Mol. Cell Res., 1783, 1354–1368. [11] Li, C., Xu, Y., Jiang, W., Lv, X. et al. 2014. Effect of sodium chloride and cadmium on the growth, oxidative stress and antioxidant enzyme activities of Zygosaccharomyces rouxii, J. Ocean Univ. China, 13, 460–466. [12] Gallardo, J.C.M., Souza, C.S., Cicarelli, R.M.B., Oliveira, K.F. et al. 2011. Enrichment of a continuous culture of Saccharomyces cerevisiae with the yeast Issatchenkia orientalis in the production of ethanol at increasing temperatures, J. Ind. Microbiol. Biotechnol., 38, 405–414. [13] Chan, G.F., Gan, H.M., Ling, H.L., Rashida, N.A.A., 2012. Genome sequence of Pichia kudriavzevii M12, a potential producer of bioethanol and phytase, Eukaryot. Cell, 11, 1300–1301.
www.jbm-journal.com
J. Basic Microbiol. 2015, 55, 1002–1012
1012
Ning Ma et al.
[14] Isono, N., Hayakawa, H., Usami, A., Mishima, T. et al. 2012. A comparative study of ethanol production by Issatchenkia orientalis strains under stress conditions, J. Biosci. Bioeng., 113, 76–78. [15] Kitagawa, T., Tokuhiro, K., Sugiyama, H., Kohda, K. et al. 2010. Construction of a beta-glucosidase expression system using the multistress-tolerant yeast Issatchenkia orientalis, Appl. Microbiol. Biotechnol., 87, 1841–1853. [16] Oberoi, H.S., Babbar, N., Sandhu, S.K., Dhaliwal, S.S. et al. 2012. Ethanol production from alkali-treated rice straw via simultaneous saccharification and fermentation using newly isolated thermotolerant Pichia kudriavzevii HOP-1, J. Ind. Microbiol. Biotechnol., 39, 557–566. [17] Halm, M., Hornbæk, T., Arneborg, N., Sefa-Dedeh, S. et al. 2004. Lactic acid tolerance determined by measurement of intracellular pH of single cells of Candida krusei and Saccharomyces cerevisiae isolated from fermented maize dough, Int. J. Food Microbiol., 94, 97–103. [18] Casey, G.D., Dobson, A.D., 2003. Molecular detection of Candida krusei contamination in fruit juice using the citrate synthase gene cs1 and a potential role for this gene in the adaptive response to acetic acid, J. Appl. Microbiol., 95, 13–22. [19] Meng, F., Zhang, Y., Liu, F., Guo, X. et al. 2014. Characterization and mutational analysis of omega-class GST (GSTO1) from Apis cerana cerana, a gene involved in response to oxidative stress, PLoS ONE, 9, e93100. [20] Zhang, Y., Lu, H., Yuan, Y., Liu, X. et al. 2009. Neuroprotective effects of combined lead and cadmium, as well as N-acetylcysteine, on cerebral cortical neurons following lipid peroxidation injury, Neural Regen. Res., 4, 907– 911. [21] Li, C., Xu, Y., Jiang, W., Dong, X., et al. 2013. Effect of NaCl on the heavy metal tolerance and bioaccumulation of Zygosaccharomyces rouxii and Saccharomyces cerevisiae, Bioresour. Technol., 143, 46–52.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
[22] Nies, D.H., 1999. Microbial heavy-metal resistance, Appl. Microbiol. Biotechnol., 51, 730–750. [23] Kastbjerg, V.G., Nielsen, D.S., Arneborg, N., Gram, L., 2009. Response of Listeria monocytogenes to disinfection stress at the single-cell and population levels as monitored by intracellular pH measurements and viable-cell counts, Appl. Environ. Microbiol., 75, 4550–4556. [24] Rangel, D.E.N., 2011. Stress induced cross-protection against environmental challenges on prokaryotic and eukaryotic microbes, World J. Microbiol. Biotechnol., 27, 1281–1296. [25] Suga, M., Kusanagi, I., Hatakeyama, T., 2003. High osmotic stress improves electro-transformation efficiency of fission yeast, FEMS Microbiol. Lett., 225, 235–239. [26] Dubernet, S., Panoff, J-M., Thammavongs, B., Gueguen, M., 2002. Nystatin and osmotica as chemical enhancers of the phenotypic adaptation to freeze–thaw stress in Geotrichum candidum ATCC 204307, Int. J. Food Microbiol., 76, 215– 221. [27] Jamieson, D.J., 1998. Oxidative stress responses of the yeast Saccharomyces cerevisiae, Yeast, 14, 1511–1527. [28] An, B., Li, B., Qin, G., Tian, S., 2012. Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein, Curr. Microbiol., 65, 122–127. [29] Moradas-Ferreira, P., Costa, V., Piper, P., Mager, W., 1996. The molecular defences against reactive oxygen species in yeast, Mol. Microbiol., 19, 651–658. [30] Navarrete, C., Siles, A., Martınez, J.L., Calero, F., et al. 2009. Oxidative stress sensitivity in Debaryomyces hansenii, FEMS Yeast Res., 9, 582–590. [31] Jiang, W., Xu, Y., Li, C., Lv, X., Wang, D., 2013. Effect of inorganic salts on the growth and Cd2þ bioaccumulation of Zygosaccharomyces rouxii cultured under Cd2þ stress, Bioresour. Technol., 128, 831–834.
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J. Basic Microbiol. 2015, 55, 1002–1012
