Characterization of Low pH and Inhibitor Tolerance Capacity of Candida krusei Strains

Abstract

Interest in the production of bioethanol from inedible biomass is growing worldwide because of its sustainable supply and lack of competition with food supplies. Candida krusei (also known as Pichia kudriavzevii or Issatchenkia orientalis) is one of the most suitable thermotolerant yeasts used in the simultaneous saccharification and fermentation process for bioethanol production. In the production of bioethanol from lignocellulosic biomass as a feedstock, various environmental conditions occur, and the stress tolerance capacity of C. krusei, especially its low pH and tolerance to inhibitors, limits its practical application. In this study, to select a suitable second-generation bioethanol-producing strain, the tolerance capacity of five available C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) was characterized. Spot assay and growth experiment results showed that among the five C. krusei strains, C. krusei NBRC1664 showed superior tolerance capacity for low pH and inhibitors. Furthermore, this strain efficiently produced ethanol from glucose under low pH conditions with or without sulfate. A comparative analysis of the draft genome sequences suggested that Opy2, Sln1 and Cdc24 in the HOG pathway are conserved only in C. krusei NBRC1664, which may contribute to its superior tolerance to low pH levels. Moreover, amino acid sequence alignment showed that aldehyde dehydrogenase family proteins, which catalyze the degradation of cyclic aldehydes, are commonly conserved in C. krusei. In addition, the increased transcription levels in C. krusei NBRC1664 could play a role in its higher tolerance to inhibitors. These results suggest that C. krusei NBRC1664 is a more suitable strain for application in industrial processes for second-generation bioethanol production.

1. Introduction

Global greenhouse gas emissions from fossil fuel combustion continue to increase with the rise in economic activity, and emissions are predicted to peak around 2025 [1]. At present, increased greenhouse gas emissions are leading to global warming, rising sea levels, urban pollution and a loss of biodiversity, posing a threat to the global environment [2]. Against this background, the Paris Climate Agreement, signed at the United Nations Climate Change Conference in 2015, aims to limit the increase in global temperature to below 2 °C above pre-industrial levels and strives to limit the increase to 1.5 °C [3]. There are several efforts underway to reduce greenhouse gas emissions, and one of the most effective is the shift from fossil fuels to renewable fuels.

Bioethanol is one of the most well-known examples of renewable fuels. Based on differences in the source materials or the production method, bioethanol can be classified as first, second or third generation. First-generation bioethanol is produced from crops such as corn, sugarcane and potato, and this fuel is used for blending into gasoline, mainly in the United States and Brazil. From 2008 to 2018, when the demand for bioethanol expanded, global bioethanol production increased approximately 16.5-fold, from 6.7 billion liters to 110.4 billion liters [4]. However, the production of first-generation biofuels has been criticized because it competes with the food and feed supply in the face of global food-related challenges such as hunger. In the context of these challenges, interest in second-generation bioethanol production from lignocellulosic biomass, which does not compete with the food supply, has increased. Microbial production of second-generation bioethanol necessitates three steps: pretreatment, enzymatic hydrolysis and fermentation [5]. During the pretreatment step, lignocellulosic biomass particles are hydrolyzed into cellulose, hemicellulose and lignin by hydrothermal treatment or the addition of inorganic acids, such as sulfuric acid, ensuring that saccharification enzymes are accessed easily during enzymatic hydrolysis. Subsequently, at the enzymatic hydrolysis step, monosaccharides such as glucose and xylose are extracted from cellulose and hemicellulose by saccharification enzymes, and the resultant solution is used as the hydrolysate. Finally, at the fermentation step, the hydrolysate is used as a carbon source for bioethanol production. Using this method, bioethanol production from bagasse [6], corn stover [7] and seaweed [8] has been reported. However, this method has not been put to practical use because the costs of second-generation bioethanol production are higher than those of first-generation bioethanol.

To develop a cost-effective method for second-generation bioethanol production, we previously performed simultaneous saccharification and fermentation (SSF) using the thermotolerant yeast strains Issatchenkia orientalis NBRC1279 and Candida krusei NBRC1664 [9]. C. krusei is the asexual form (anamorph) of a species whose sexual form (teleomorph) is Pichia kudriavzevii, also known as I. orientalis [10,11]. When particles prepared from Japanese cedar or eucalyptus are used as raw materials for SSF, both strains produce more than 21 g/L of ethanol and C. krusei NBRC1664 shows the highest concentration (24 g/L). SSF offers several advantages including a simpler and easier-to-perform production process and reduced energy input, since enzymatic hydrolysis and fermentation are performed simultaneously in the same vessel, leading to lower production costs [5]. Our method demonstrated that the use of I. orientalis and C. krusei strains is essential for bioethanol production via the SSF process. The activity of commercial saccharification enzymes differs depending on the type, but those enzymes exhibit maximum activity at approximately pH 5.0–6.0 and a temperature of 40–50 °C. Both strains can produce ethanol under low pH and high-temperature conditions; thus, enzymatic saccharification and fermentation are performed simultaneously by using both thermotolerant yeast strains. In other words, S. cerevisiae shows an excellent ethanol yield in the production of first-generation bioethanol, but because its optimal growth temperature is around 30 °C, this species cannot be used for SSF.

When ethanol is produced using SSF, yeast cells are exposed to various types of stress, such as acids, sulfate and other chemicals used in the pretreatment or secondary products (overdecomposition products) derived from lignocellulosic biomass, which can reduce the ethanol productivity of the yeast. To select useful yeast for the production of second-generation bioethanol, we previously investigated the stress tolerance capacity of various yeast strains, and reported that when I. orientalis NBRC1279 and C. krusei NBRC1664 are subjected to osmotic stress, the high osmolarity glycerol (HOG) pathway is up-regulated, facilitating the production of the osmotic agent glycerol and allowing both strains to survive [12]. Moreover, C. krusei NBRC1664 shows a superior tolerance to inhibitors such as furfural, 5-hydroxymethylfurfural (HMF) and vanillin, which are degradation products generated by lignocellulose hydrolysis, compared to I. orientalis NBRC1279 and S. cerevisiae BY4742 [13]. These results demonstrated that C. krusei NBRC1664 is more suitable for second-generation bioethanol production than other strains. Thus, a more detailed investigation of the growth performance of various C. krusei strains (including strain NBRC1664) under multiple stresses is vital in the selection of efficient second-generation bioethanol producers. Here, we characterized the growth capacity of five available C. krusei strains under different stressors, including a low pH, sulfate and various inhibitors, to select the optimal yeast strain for second-generation bioethanol production. Subsequently, based on a comparative analysis of the draft genome sequences and amino acid sequence alignment of the aldehyde dehydrogenase family proteins (ADHFs), the low pH and inhibitor tolerance capacity was elucidated.

2. Materials and Methods

2.1. Yeast Strains and Media

C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) and S. cerevisiae strain NBRC10217 were purchased from the NITE Biological Resource Center (NBRC, Chiba, Japan). The yeast strains were grown in yeast peptone dextrose (YPD) broth or on agar plates (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) unless otherwise noted.

For the spot assay and aerobic growth experiments, a synthetic complete (SC) minimal medium (6.7 g/L yeast nitrogen base without amino acids) supplemented with the appropriate amino acids (0.2 g/L L-threonine, 0.15 g/L L-valine, 0.1 g/L L-leucine, 0.05 g/L L-phenylalanine, 0.03 g/L each of L-isoleucine, L-lysine and L-tyrosine, 0.02 g/L each of L-arginine, L-histidine, L-methionine and L-tryptophan), nucleic acids (0.02 g/L each of adenine and uracil), 20 g/L glucose and 18 g/L agar (SCD plate) and SCD medium (medium from the SCD plate without 18 g/L agar) were used, respectively.

In anaerobic fermentation experiments using C. krusei and S. cerevisiae strains, glucose (40 g/L) was added to the SC medium (SCD2 medium). The pH of YPD and SCD media was adjusted to pH 5.8 with HCl and NaOH, respectively, unless otherwise specified. The pH values of the SC-based media (SCD and SCD2) were adjusted to 2.2, 2.0, 1.7 and 1.4 by the addition of HCl. For salt stress experiments, appropriate amounts (2.5 or 7.5%) of sodium sulfate (Na₂SO₄) were added to the SC-based media prior to pH adjustment, and then the pH of the media was adjusted with HCl. Unless specified, the yeast strains were grown at 30 °C either on agar plates or in liquid medium.

2.2. Spot Assay

C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) were cultivated aerobically in SCD medium at 30 °C with shaking until the early stationary growth phase was reached. The cells were then washed with sterile water and adjusted to an optical density at 600 nm (A₆₀₀) of 10. Cell growth (A₆₀₀) was measured by a V-630iRM spectrophotometer (JASCO, Tokyo, Japan). Subsequently, aliquots (2 µL) of 10-fold serial dilutions of the cultures were spotted onto SCD plates without salt at pH 2.2, 1.7 and 1.4, and onto SCD plates containing 7.5% sodium sulfate (Na₂SO₄) at pH 2.2, 2.0 and 1.7, and onto SCD plates containing different inhibitors (15% ethanol, 30 mM furfural, 35 mM HMF and 10 mM vanillin) and different inorganic acids (30 mM sulfuric acid and 60 mM hydrochloric acid). These plates were photographed after 2 to 3 days of incubation at 30 °C.

2.3. Aerobic Growth Experiments

C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) were pre-cultivated aerobically in YPD medium (pH 5.8) for 16 h at 30 °C with shaking. Subsequently, each yeast culture was washed with sterile water and inoculated into SCD media without salt at each of the indicated pH values (pH 5.8, 2.0 or 1.7) and into SCD medium supplemented with 2.5% Na₂SO₄ at pH 1.7. The initial optical density at 600 nm (A₆₀₀) in all cases was around 0.2. During cultivation, cell growth was monitored by A₆₀₀ measurements using a bio-microplate reader (HiTS, Scinics Corporation, Tokyo, Japan) as described previously [12]. All cultivations in 96-well microplates were carried out at 30 °C with mild agitation (150 rpm) using the HiTS microplate reader. Cultivation was repeated three times and standard deviations were less than 10%.

2.4. Anaerobic Fermentation Experiments

For the batch fermentation experiments, C. krusei strains (NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664) and S. cerevisiae strain NBRC10217 were first cultivated aerobically in 5 mL of YPD medium (pH 5.8) for 16 h at 30 °C with shaking. Then, the culture was centrifuged at 6000× g for 5 min at room temperature, and the pelleted cells were washed and resuspended in distilled water. These cells were inoculated into 20 mL of fermentation medium [SCD2 medium without salt at pH 2.0 or with salt (7.5% Na₂SO₄) at pH 2.2] in 50 mL closed bottles, in which enough headspace was provided for foaming and expansion during fermentation. For all strains, the initial cell density in the fermentation medium was adjusted to approximately 2.5 at 600 nm (A₆₀₀). Cell growth (A₆₀₀) was measured with a V-630iRM spectrophotometer (JASCO). Anaerobic batch fermentations were performed at 30 °C in sterile, closed 50 mL bottles with magnetic stirring. The amount of ethanol produced during fermentation was determined as a theoretical value of ethanol by measuring the amount of CO₂ gas emitted. All experiments were performed in triplicate.

2.5. Pathway Analysis to Elucidate Low pH Tolerance Capacity

Based on draft genome sequences of C. krusei NBRC1395 and NBRC1664, pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server ver. 2.1.

2.6. Comparative Sequence Analysis to Elucidate Inhibitor Tolerance Capacity

The BLAST program was used for comparative amino acid sequence analysis conducted on human aldehyde dehydrogenase 1A3 (hADH1A3) and ADHFs from C. krusei NBRC1395 and NBRC1664, which are available in the GenBank/EMBL/DDBJ databases. The sequences were aligned using ClustalW ver. 2.1 [14] and ESpript ver. 3.0 [15].

3. Results and Discussion

3.1. Effect of Low pH, Acids and Inhibitors on Growth

During the pretreatment step, inorganic acids such as sulfuric acid and hydrochloric acid are used to hydrolyze lignocellulosic biomass particles; however, the residual acids inhibit yeast cell growth and ethanol production. Therefore, a neutralization step, in which the residual acid is neutralized with alkalis to form salts such as Na₂SO₄, is required prior to downstream enzymatic hydrolysis and fermentation; however, such salts are known to have significant inhibitory effects on the fermentation ability of yeasts. In addition, several inhibitors such furfural, HMF and vanillin are also generated during saccharification enzyme action. Thus, to enhance the productivity of second-generation bioethanol even in the presence of acids, salts and inhibitors, the selection of multiple stress-tolerant C. krusei strains is desirable. In this study, we used C. krusei strains NBRC0584, NBRC0841, NBRC1162, NBRC1395 and NBRC1664, obtained from NBRC as described in the Materials and Methods.

To investigate the multiple stress-tolerant capacity of C. krusei strains, a spot assay was performed (Figure 1). Under varying low pH stress conditions, C. krusei strains grew at pH 1.4–2.2, with C. krusei NBRC1664 showing slightly superior growth at pH 1.4. Moreover, all strains, except C. krusei NBRC1162, showed increased growth and were able to grow well under low pH conditions (pH 1.7–2.2) even when high concentrations (7.5%) of Na₂SO₄ were added. Among the strains, only C. krusei NBRC1664 grew well under hydrochlorate and sulfate stress, while C. krusei NBRC1162 and NBRC1395 grew poorly under these stresses. In contrast, C. krusei strains could grow in the presence of 15% ethanol, 30 mM furfural, 35 mM HMF and 10 mM vanillin, although the growth of C. krusei NBRC0841 and NBRC1395 was slightly decreased with the addition of 30 mM furfural. These results demonstrated that, among the strains used in this study, C. krusei NBRC1664 has a multiple stress-tolerant capacity, as this strain was able to tolerate low pH as well as acid and inhibitor stresses.

To further investigate the tolerance of the strains to a low pH and sulfate, C. krusei strains were grown in SCD medium at various pHs with or without sulfate (Figure 2 and

Table 1. Growth rate (h⁻¹) of C. krusei strains at the logarithmic growth phase when cultured in SCD medium at pH 5.8, pH 2.0, pH 1.7 and pH 1.7 with 2.5% Na₂SO₄. The values are the average of three independent experiments ± standard deviation.

Strain	pH 5.8	pH 2.0	pH 1.7	pH 1.7 with 2.5% Na2SO4
NBRC0584	0.147 ± 0.001	0.153 ± 0.002	0.0655 ± 0.0032	0.0258 ± 0.0018
NBRC0841	0.144 ± 0.003	0.156 ± 0.001	0.101 ± 0.002	0.0406 ± 0.0050
NBRC1162	0.139 ± 0.002	0.152 ± 0.002	0.0230 ± 0.0021	0.0145 ± 0.0010
NBRC1395	0.155 ± 0.011	0.150 ± 0.003	0.0646 ± 0.0044	0.0249 ± 0.0047
NBRC1664	0.154 ± 0.002	0.158 ± 0.002	0.114 ± 0.002	0.0456 ± 0.0051

). Although all C. krusei strains grew at pH 1.7–5.8, the time required to reach the stationary phase of C. krusei NBRC1162 increased at pH 1.7 (Figure 2A–C). Moreover, in all strains, the increase in the time required to reach the stationary phase at pH 1.7 was more pronounced with the addition of 2.5% Na₂SO₄ (Figure 2D). At pH 5.8, the growth rates of C. krusei strains were not significantly different, ranging from 0.139 ± 0.0020 to 155 ± 0.011 h⁻¹. At pH 2.0, a decrease in the growth rates of C. krusei strains was observed, ranging from 0.150 ± 0.0030 to 158 ± 0.0020 h⁻¹. Moreover, the growth rates of C. krusei strains at pH 1.7 were low compared with those at pH 5.8 and 2.0, and the growth rate was reduced by more than half by the addition of 2.5% Na₂SO₄ (Table 1). On the other hand, at pH 1.7 with 2.5% Na₂SO₄, the growth rate of C. krusei NBRC1664 was highest and the value was more than 3.1-fold higher than that of C. krusei NBRC1162, which showed the lowest growth rate (Table 1); these rates for C. krusei NBRC1664 were significantly different from those for C. krusei NBRC0584 (p-value = 0.019), C. krusei NBRC0841 (p-value = 0.008), C. krusei NBRC1162 (p-value = 0.003) and C. krusei NBRC1395 (p-value = 0.025).

Based on the growth phenotypes (Figure 1), growth curves (Figure 2) and growth rates (Table 1) observed under various stress conditions, C. krusei NBRC1664 has a multiple stress-tolerant capacity compared to other C. krusei strains. To obtain higher ethanol productivity, a strain that is tolerance to residual sulfuric acid and inhibitors following biomass pretreatment is necessary. Thus, among C. krusei strains used in this study, C. krusei NBRC1664 was found to be a superior second-generation bioethanol producer.

3.2. Ethanol Production Under Low pH Stress Conditions

For the practical application of a production method for second-generation bioethanol, several methods using C. krusei strains have been investigated. For example, when C. krusei strains are used, coconut waste [16], corncob [17] and wheat straw [18] have been demonstrated to be useful feedstocks for ethanol production. Moreover, to enhance ethanol productivity, the effects of culture temperature [19], inhibitors [13,20] and salt stress [21] have been investigated. However, the effect of another important factor, lowering the pH of the medium with or without salt, has only been investigated in a few studies. Therefore, the effect of lowering the pH of the medium with or without salt was investigated to better understand the production of ethanol under these stress conditions.

To investigate the effect of lowering the pH of the medium with or without salt in more detail, the ethanol production of five C. krusei strains and S. cerevisiae strain NBRC10217 (as a negative control strain) was evaluated at pH 2.0 with 7.5% Na₂SO₄, which showed growth differences in the spot assay. At pH 2.0, without the addition of 7.5% Na₂SO₄, all C. krusei strains produced ethanol by consuming glucose (Figure 3A,B). In particular, C. krusei NBRC1664 showed the maximum ethanol concentration (21.6 g/L). By contrast, when S. cerevisiae was used, the glucose consumption and ethanol production were significantly decreased under both low pH conditions, regardless of the presence or absence of sulfate, which indicated that growth was considerably inhibited by low pH stress. Subsequently, when more intense stress was applied (7.5% Na₂SO₄ addition), C. krusei NBRC1664 produced 22.8 g/L of ethanol without a decrease in its production concentration (Figure 3C,D). These results suggest that C. krusei strains including NBRC1664 may be able to produce ethanol without being affected by the residual acids produced after pretreatment in an SSF process performed using commercial saccharification enzymes, directly resulting in decreased costs.

3.3. Elucidation of the Low pH and Inhibitor Tolerance Capacity of C. krusei NBRC1664

Of the C. krusei NBRC strains used in this study, draft genome sequences have been sequenced for two strains, NBRC1395 and NBRC1664 [9]. In this study, the NBRC1664 strain showed a multiple stress-tolerant capacity, while the stress-tolerant capacity of the NBRC1395 strain was narrow. Thus, to clarify the tolerance capacity of the strains to a low pH and inhibitors, a comparative analysis of the draft genome sequences of both strains was performed.

The HOG pathway is the prototypical mitogen-activated protein kinase (MAPK) signaling system. This pathway is well conserved among eukaryotic organisms, and is responsible for the response of strains to high osmolarity, with some differences among species [22,23]. In our previous study, we demonstrated that C. krusei NBRC1664 adapted to acid stress by upregulating the HOG pathway [12]. When gene clusters in the HOG pathway of both strains were compared, Opy2, Sln1 and Cdc24 were not contained in the draft genome sequence of C. krusei NBRC1395 (Figure 4). In S. cerevisiae, Opy2 and SLN1 are transmembrane proteins, which act as osmosensors and activate the MAPK cascade following osmotic stress [24]. On the other hand, the guanine nucleotide exchange factor Cdc24 is essential for Cdc42 activity, and this complex regulates cell division in response to stress [25]. Moreover, a Cdc42 mutant showed weak activity in the MAPK cascade of the HOG pathway, resulting in growth inhibition [26]. Thus, C. krusei NBRC1664 shows superior tolerance compared to C. krusei NBRC1395 due to the conservation of Opy2, Sln1 and Cdc24, which function under low pH stress.

Our previous study suggested that ADHFs are important for the inhibitor tolerance of C. krusei NBRC1664 [13]. When C. krusei NBRC1664 is stressed by HMF, the ADHF1 and ADH2 gene transcription levels are increased compared to the housekeeping gene. Moreover, compared to ADHF2, the substrate binding site of ADHF1 has a large and wide-open tunnel formed by Ser125, Thr303 and Glu461, which may enable the recognition of cyclic aldehydes such as HMF and furfural. In the draft genome sequence of C. krusei NBRC1395, similar to strain NBRC1664, two kinds of ADHFs are conserved. To elucidate the tolerance of C. krusei NBRC1395 to inhibitors, the amino acid sequence alignment of ADHFs from C. krusei NBRC1395 and NBRC1664, as well as hADH1A3 was conducted to determine the substrate and coenzyme recognition sites (Figure 5). The amino acid residues proposed to play a role in substrate and coenzyme recognition in ADHF1 from C. krusei NBRC1664 were completely conserved in ADHF2 from C. krusei NBRC1395. Moreover, except for Gly483, Ser485 and Ile487 in ADHF2 from C. krusei NBRC1664, the amino acid residues involved in the substrate and coenzyme recognition site matched between ADHF2 from C. krusei NBRC1664 and ADHF1 from C. krusei NBRC1395. In other words, ADHFs are conserved in both strains, but the transcription levels of these enzymes are higher in C. krusei NBRC1664, which may lead to a superior tolerance to cyclic aldehydes. To analyze the mechanisms involved in the tolerance of C. krusei NBRC1664 to a low pH and inhibitors in more detail, we are planning to perform a transcriptome analysis. The results will be described elsewhere in the future.

4. Conclusions

In this study, the multiple stress-tolerant capacity of five C. krusei strains was investigated using various acids, inhibitors and sulfate as potential sources of stress during second-generation bioethanol production. The spot assay revealed that C. krusei NBRC1664 grew well under all stress conditions. When C. krusei NBRC1664 was incubated in SCD2 medium with 7.5% Na₂SO₄ at pH 2.0, it showed the fastest growth rate and an ethanol concentration comparable (not decreased) to the other strains. Based on the observed growth phenotypes, growth curves, growth rates and ethanol productivity, C. krusei NBRC1664 was a more suitable second-generation bioethanol producer. To elucidate the tolerance capacity to a low pH, a comparative analysis was carried out using the genomes of C. krusei NBRC1395 and NBRC1664, and the results revealed that Opy 2, Sln1 and Cdc24, which are involved in the HOG pathway, function under low pH stress, and may be conserved in C. krusei NBRC1664 only. The amino acid sequence alignment suggested that ADHF1, which catalyzes the degradation of cyclic aldehydes, is conserved in both strains, but C. krusei NBRC1664 exhibits a higher tolerance to inhibitors due to increased transcription levels.