Rhizome is vital for carbon and nitrogen metabolism of the whole plant. However, the effect of carbon and nitrogen in the rhizome on rhizome expansion remains unclear.Poa pratensis L.) germplasms with different rhizome expansion capacity  were planted in the field and the rhizomes number, tiller number, rhizome dry weight, physiological indicators and enzyme activity associated carbon and nitrogen metabolisms were measured. Liquid chromatography coupled to mass spectrometry (LC-MS) was utilized to analyze the metabolomic of the rhizomes. The results showed that the rhizome and tiller numbers of the YZ were 3.26 and 2.69-fold of that of the AD, respectively. The aboveground dry weight of the YZ was the greatest among all three germplasms. Contents of soluble sugar, starch, sucrose, NO3−-N, and free amino acid were significantly higher in rhizomes of the YZ than those of the WY and AD (P < 0.05). The activities of glutamine synthetase (GS), glutamate dehydrogenase (GDH) and sucrose phosphate synthase (SPS) of the YZ were the highest among all three germplasm, with values of 17.73 A·g− 1 h− 1, 5.96 µmol·g− 1 min− 1, and 11.35 mg·g− 1 h− 1, respectively. Metabolomics analyses revealed that a total of 28 differentially expressed metabolites (DEMs) were up-regulated, and 25 DEMs were down-regulated in both comparison groups (AD vs. YZ group and WY vs. YZ group). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that metabolites related to histidine metabolism, tyrosine metabolism, tryptophan metabolism, and phenylalanine metabolism were associated with rhizomes carbon and nitrogen metabolism.Three wild Kentucky bluegrass (3−-N, and free amino acid in rhizome are important to and promote rhizome expansion in Kentucky bluegrass, while tryptamine, 3-methylhistidine, 3-indoleacetonitrile, indole, and histamine may be key metabolites in promoting carbon and nitrogen metabolism of rhizome.Overall, the results suggest that soluble sugar, starch, sucrose, NOThe online version contains supplementary material available at 10.1186/s12870-023-04230-x. Poa pratensis L.), are easier to establish as a turf, have greater resistance to stress [Rhizome is a modified, horizontal underground stem of plant. It is a vital organ for nutrient storage, ramet generation, information and material exchange between cloned ramets and vegetative propagation for the rhizomatous plants –3. Rhizoo stress , and cano stress , 9. TherOryza longistaminata) [tin) and rice (moc 2), reduction in sucrose content led to inhibition of tiller growth [Oryza sativa) branching, tiller number positively correlated with plant total nitrogen accumulation [Arabidopsis) [The development of rhizome of turfgrass is affected by various factors, including carbohydrate and nitrogen levels in plant, environment and phytohormones. Plant carbohydrates and nitrogen status affect plant overwintering, rhizome bud germination, and community reproduction , 11. Theaminata) . Higher aminata) . In the r growth , 16. Furr growth  found thr growth . Similarr growth , 19. Newmulation , and higmulation . Furthermulation . The balidopsis) . These rPoa pratensis L.) is a perennial rhizomatous plant that is widely distributed in cool and humid regions of temperate zones and is an important plant species of grassland in such areas [P. sibiricum rhizomes. Masson et al. [Iris rhizomes, and tentatively identified 32 metabolite discriminating markers to distinguish origins of these plants. Kentucky bluegrass varies greatly in its rhizome expansion capacities [Kentucky bluegrass  investigate the relationship between carbon and nitrogen metabolism and Kentucky bluegrass rhizome expansion capacity; (ii) investigate the relationship between enzymes, metabolism pathways, and metabolites and Kentucky bluegrass rhizome extension.P < 0.05). All four parameters ranked YZ > WY > AD. The rhizome number of the YZ were 1.81 and 3.26-fold of that of the WY and AD, respectively . The soluble sugar content of the YZ was 147.66% and 178.63% higher than that of the WY and AD, respectively, whereas the latter two had a similar content ; however, no differences were found between values of the WY and AD  , while for SS, the activities ranked AD > WY > YZ (P < 0.05). The rhizome GS, GDH and SPS activities of the YZ were 25.9–92.4% greater than those of the WY and AD.Enzyme activities of the three Kentucky bluegrass germplasms are indicated in Table 3−-N, free amino acid, GDH, and GS (nitrogen metabolism) showed a high positive contribution.Morphological and physiological indicators accounted for 81.2% of the total variation  between AD and YZ  transformants increase growth rate by increasing sucrose transporter proteins in sucrose medium. Spaying with sucrose increased the sucrose content and enzyme (SPS and SS) activity of apple plants, which keeps the N metabolism in balance and promotes plant growth [Rhizome is a form shoot branching. The structural features of rhizomes are similar to those of tillers . Developr number . Sucroser number , which mtaminata . Cai et taminata  demonstrt growth . SPS is t growth  also demt growth –48, this3−-N might facilitate the growth and development of rhizome, by acting not only as a main form of inorganic nitrogen but also perhaps as a signaling molecule [15 N isotope labeling method, finding that much nitrogen accumulates in the rhizome, which helps it expand across the belowground space. In the present study, GS and NR activity showed the same trend as NO3−-N content, although differences in NR activity were not significant . Furthermore, amino acid and carbohydrate metabolites  exhibited an enhanced accumulation in strong rhizome expansion. Tryptophan metabolism and phenylalanine metabolism may be the main metabolic pathway for enhancing rhizome expansion. These results reveal pronounced differences in carbon and nitrogen metabolism in the rhizomes of different materials, which can provide new insights for the molecular breeding of rhizomatous plants.Overall, noticeable variation was present across the rhizomes of different Kentucky bluegrass materials. The Kentucky bluegrass with strong rhizome expansion exhibited the highest rhizome number and tiller number and the greatest carbon  and nitrogen metabolism . Lanzhou has a temperate continental climate, with a mean annual temperature of 10 °C, and a mean annual rainfall of 362 mm. The soil in the farm belongs to loess loam. In the 0–40 cm soil layer, the organic matter, alkali-hydrolyzable nitrogen, available phosphorus and available potassium were 2.34%, 40.37 mg·kg-1, 45.62 mg·kg-1 and 155.68 mg·kg-1, respectively. The pH was 7.5.Each Kentucky bluegrass had 12 plots in the field work, thus there were 36 plots (each 0.5 × 0.5 m) with a spacing of 1 × 1 m. In June 2019, Kentucky bluegrass seeds were sterilized and randomly assigned to the plots. Twenty-one days after germination, plants in each plot were thinned to 12. Plants grew under natural sunlight and temperature. The plants were harvested in October 2020, with all the plants in each plot harvested for analysis. Whole plants were separated into rhizome and aboveground and rinsed sequentially with tap water and deionized water for three times. A portion of these rhizome samples were kept for morphology measurements and the remaining samples used for physiological parameter and metabolite determinations (stored at -80 °C). The metabolomics analysis was conducted with six replicates. The other measurements in this study were three replicates.The numbers of tillers and rhizomes of each plant were counted, dry weights were determined after the samples being dried at 80 °C for 48 h. The dried samples were used for subsequent analysis.3− content was measured with salicylic acid colorimetry test according to Cataldo et al. [4+ content was determined by the Berthelot color reaction method according to Gordon et al. [Fresh plant samples were extracted in boiling water bath, then the extractant NOo et al. , NH4+ con et al. , while fn et al.  .Dry rhizome samples were extracted with ethanol at 80℃, then the extractant was used for soluble sugar  and sucrFrozen rhizome samples were used for enzyme activity analysis. Sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities were determined according to the method of Sicher et al. . NitrateUntargeted metabolomics was performed using liquid chromatography-mass spectrometry (LC-MS). Rhizome sample preparation and sequencing were performed at Novogene . Briefly, 100 mg of each rhizome sample was first ground with liquid nitrogen and then homogenate added to a 500-µl aqueous solution of 80% methanol. After centrifugation, the ensuing supernatant was collected and injected into the LC-MS system for analysis .− 1. The mobile phase of the positive polarity mode was eluent A (0.1% FA in Water) and eluent B (Methanol). The mobile phase of the negative polarity mode was eluent A  and eluent B (methanol). The operating solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 12.0 min; 100% B, 14.0 min; 100–2% B, 14.1 min; 2% B, 17 min. The spray voltage was 3.2 kV, the capillary temperature was 320 °C, the sheath gas flow rate was 40 arb, and the aux gas flow rate was 10 arb.Samples were injected onto a Hypesil Gold column , using a 17-min linear gradient at a flow rate of 0.2 mLminhttps://www.mzcloud.org/), mzVaul, and Masslist database. Statistical analyses were performed using R v4.0.2 software.Raw files were processed in Compound Discoverer 3.1 software , which performed the peak alignment, peak picking, and quantitation for each metabolite. Next, the molecular formulas were aligned to the mzCloud (https://www.genome.jp/kegg/pathway.html) [https://hmdb.ca/ metabolites), and LIPID Maps (http://www.lipidmaps.org) databases. A partial least squares discriminant analysis (PLS-DA) of the identified metabolites implemented using was metaX [P-value < 0.05.The detected metabolites were annotated using the KEGG (ay.html) , 79, HMDas metaX . We applP < 0.05.Morphological and physiological data of different Kentucky bluegrass were processed with SPSS  using one-way ANOVA with Tukey’s test, the graphs were generated with Origin Pro . The metabolomics analysis was run in R Software . Significance was set at Below is the link to the electronic supplementary material.Supplementary Material 1


Burkina, a traditional fermented dairy product, is consumed in most parts of West Africa, including Ghana. Studies on the microflora of Burkina have indicated the presence of Escherichia coli and other pathogenic organisms. Thus, predicting the survival of E. coli in the product will inform the best manufacturing and handling practices. This study investigated the combined effect of storage temperature and time on the survival of acid-adapted and acid-non-adapted E. coli O157:H7 in Burkina. Samples were pasteurised and inoculated with acid-adapted or acid-non-adapted E. coli O157:H7. They were stored at 5, 15, and 30°C for 0, 2, 4, 6, 8, 10, 12, 14, 18, and 48 h, and the bacteria colonies were enumerated. Growth rate  models were developed using MATLAB software. Observed data were fitted to the Baranyi model using the DMFit curve fitting software. The E. coli O157:H7 strain appeared inherently tolerant to acid, with storage time having the most significant effect on the response parameter, survival (log CFU/mL). A negative correlation was observed for the primary models , which accounted for 79-97% of the relationship (p < 0.05). Although E. coli survived, its growth was inhibited over time regardless of acid adaptation. The growth potential of microorganisms in food has recently been evaluated and predicted by applying mathematical models that serve as promising tools for risk assessment , 2. SuchBurkina is a nutritious fermented milk product composed of steam-cooked agglomerates of millet and spontaneously fermented milk, also known as nunu in Ghana. It is similar in consistency and consumption to yoghurt [ yoghurt .Burkina [Enterobacter, Klebsiella, Escherichia coli, Proteus vulgaris, Staphylococcus, and Shigella in Burkina. Although not all foodborne microorganisms are harmful, Escherichia coli O157:H7 in food can cause medium to gastrointestinal illness in humans when consumed. Studies carried out between 2000 and 2006 by the Foodborne Diseases Active Surveillance Network of the United States of America reported 3464 E. coli O157:H7 infections, among which 218 persons developed haemolytic uremic syndrome and 33 people had died [Although widely consumed, the product has no standardised processing protocols with controlled hygienic practices and critical control points. This limits the potential target market for the product since it does not appeal to many potential consumers who avoid the product due to the perception of poor handling practices during processing and sales . In addiBurkina –7. Thesehad died .Escherichia coli (EHEC) [The World Health Organisation (WHO) provided global estimates of the burden of foodborne diseases for thirty-one (31) food hazards, which cause two diseases . Six huni (EHEC) . For thii (EHEC) .Burkina, there is a need to predict storage temperature and time conditions to prevent contamination after processing. Considering this, the study developed mathematical models that predict the survival of Escherichia coli O157:H7 in Burkina as a function of temperature and time.With the increasing consumer demand for E. coli O157:H7 were obtained from the Microbiology Research Laboratory of the Department of Nutrition and Food Science, University of Ghana. The bacterium was previously isolated from wagashi . The bacteria culture was activated twice on nutrient agar, then sterile Tryptic Soy Broth (TSB) at 37°C for 24 h.Unless specified, all reagents used in this study were ordered from an Oxoid supplier (UK). Pure strains of E. coli, resulting in a drop in pH. The same procedure was followed except for glucose addition for the nonadaptation process. Approximately 1 mL of 24-hour culture broth was transferred into TSB and Tryptic Soy Broth +1% Glucose (TSBG) for nonadaptation and acid adaptation, respectively. The cultures were incubated at 37°C for 18 h to reach the exponential phase. To confirm acid adaptation, 1 mL of acid-adapted and non-acid-adapted cells was separately cultured in 9 mL of sterile TSB acidified with lactic acid to pH 4.0 and incubated at 30°C for 24 h. Cell viability was tested on prepoured MacConkey agar plates at 0, 2, 4, 6, and 24 h of incubation.Acid adaptation was carried out according to the method described by Ryu et al. . BrieflyBurkina was obtained from a local University of Ghana Legon campus vendor. The samples were transported to the laboratory on ice at 4°C. The samples were homogenised using a high-speed blender for 5min. Aliquots (100 mL) of the samples were dispensed into sterile screw-cup containers and pasteurised at 63°C for 30min using a water bath set at 70°C, then cooled.Burkina to achieve a final concentration of 10.49 and 10.42 log CFU/mL, respectively.An aliquot (1 mL) of either acid-adapted or non-acid-adapted culture was added to the pasteurised Burkina samples inoculated with acid-adapted (AA) and non-acid-adapted (NA) E. coli O157:H7 were thoroughly mixed and stored at 5, 15, and 30°C for 48 h. Storage temperatures were selected to mimic refrigeration and likely temperature abuse during street hawking and consumption.At each selected sampling time , 1 mL of each inoculated sample was added to 9 mL of 0.1% sterile peptone water in a test tube and vortexed for 2 min using a vortex mixer.μL of the suspensions was spread on MacConkey agar and incubated at 37°C for 24-48 h before enumeration.The homogenates (1 mL) were serially diluted in 9 mL of 0.1% sterile peptone water. After that, 100 The pH was measured for samples at the selected temperatures and sampling times  using the Mettler Toledo pH meter.y is the survival (log CFU/mL), x is the incubation time, and α, β1, and β2 are the estimated coefficients derived from the model.One hundred twenty data points were collected at the selected storage temperatures  for the duplicated experiments. Primary models were developed using linear regression in MATLAB software 2018 using Equations (N(t)) is the log of cell concentration at time t (h) (CFU/mL), ln(No) is the log of initial cell concentration (CFU/mL), μmax is the exponential growth rate (log/CFU/mL/h), and ln(Nmax) is the log of maximum cell concentration.The Baranyi model was used to fit the observed data points using the DMFit Excel add-in (version 3.5) to obtain the maximum growth rate fromn is the number of observations, μ is the empirical values, and μ∗ is the predicted values.Root mean squared error (RMSE) values were used to estimate the mean difference between the observed and predicted data points to measure the bias factor. RSME was calculated usingα = 0.05.All means and standard deviations were tabulated for observation using Microsoft Excel. Variance analysis (ANOVA) was conducted using the Minitab software version 17. Tukey's pairwise comparison and Duncan's multiple range tests were used to analyse the significance between means at Burkina samples before acid adaptation was 6.71 ± 0.01 and 6.98 ± 0.00 for TSBG and TSB, respectively . Deng et al. [E. coli O157:H7 in reconstituted infant rice cereal. This similar growth pattern observed for the nonadapted E. coli O157:H7 to the adapted could have been because the strain had already adapted to the acidic environment of the fermented dairy product from which it was isolated, proving its tolerance to acid. Moreover, the strain could have been inherently resistant to low acids and may not have required acid adaption for survival. Foster [E. coli is one of the organisms with the best systems. E. coli has well-developed glutamate and arginine decarboxylases whose metabolites enhance acid tolerance [E. coli in the sample. Various studies have reported the tolerance and survivability of E. coli O157:H7 in yoghurt. Guruya et al. [E. coli O157:H7 (NAEC) in traditional yoghurt survived for up to 35 days, and Hudson et al. [E. coli in plain yoghurt for up to 17 days. Although enumeration of the bacteria in Burkina did not continue beyond 48 h, the high concentrations observed after 48 h of storage at 5 and 15°C, as presented in E. coli inoculated into the Burkina samples.Acid adaptation of n et al.  reportedg et al.  also rep. Foster  stated tolerance , 19, anda et al.  reportedn et al.  also repp = 0.948). However, there was a statistical difference between storage temperatures of 15°C and 30°C (p = 0.01) and 5°C and 30°C (p = 0.01), irrespective of acid adaptation.AAEC and NAEC in samples stored at 5°C and 15°C survived better than those stored at 30°C . There wBurkina stored at 30°C reduced by about 5 log CFU/mL within 48 h of storage at pH 4.03 and 4.02, respectively. At 5°C, there was a reduction of about 3 and 2 logs for AAEC and NAEC, respectively, compared to about 2 and 3 log reduction for AAEC and NAEC at 15°C. The lower reduction in counts observed for AAEC and NAEC in samples stored at 5 and 15°C could have been because the low temperatures protected the organisms much better than 30°C. At lower temperatures, vegetative cells are less metabolically active, so most stop growing to survive, making them less vulnerable to environmental stresses. Beales [There was a significant reduction in the counts of AAEC and NAEC in samples stored at 30°C compared to AAEC and NAEC in samples stored at 5°C and 15°C . AAEC an. Beales  reportedR2 value of 0.97 was observed for AAEC in Burkina stored at 5°C for the primary models (p < 0.05). Though the R2 values for AAEC in Burkina at 15 and 30°C and NAEC in the sample stored at 30°C ranged from 0.744 to 0.79, the models obtained were statistically significant (p < 0.05). R2 is an index used to measure the output of a model, although other factors like p value and RMSE are used together to arrive at a good decision [R2 as R2 > 0.75 for higher values of R2. All R2 values obtained were above 0.75 except the model developed for AAEC in Burkina stored at 30°C. Even though it was 0.744 which was less than 0.75, it was still close to 0.75 and can be considered high. Meanwhile, a low R2 does not mean a model is insignificant without assessing the corresponding p value [Burkina at 30°C, the corresponding p value was 0.00133, making the model statistically significant. All x terms for primary models developed for the survival of AAEC and NAEC in Burkina stored at 5, 15, and 30°C  to predict its survival in Burkina.Models explained 79% to 98% of the primary and secondary models developed from the RSME obtained. As time increases, the bacterium's survival decreases, decreasing faster at 30°C > 15°C > 5°C. The Baranyi model and the primary model developed explained the organism's survival, requiring the input parameter time (E. coli O157:H7 in the product, although concentrations decreased. From the models obtained, the concentration of E. coli O157:H7 in the product reduced as the storage time and temperature increased. Whereas storage at a higher temperature would not be advisable, an initial pasteurisation step to reduce microbial load is encouraged.Lower temperatures yielded higher counts of


E)-N′-(2-nitrobenzylidene)dodecanehydrazide (NBDH), (E)-N′-dodecanehydrazide (NMDH), and (E)-N′-(4-fluorobenzylidene)dodecanehydrazide (FBDH), were synthesized and characterized using spectroscopic techniques. The newly synthesized lauric acid-based hydrazones were screened for their anticancer and antioxidant potential. The antioxidants showed their activity by inhibiting the oxidative chain reactions that produce reactive oxygen species. The antioxidant activity showed that NBDH exhibited the maximum DPPH inhibitory activity when compared with that of NMDH and FBDH, whereas the anticancer activity showed that FBDH exhibited maximum percent viability when compared to that of NBDH and NMDH. The reactivity and biological needs of the synthesized compounds NBDH, NMDH, and FBDH were met by performing geometrical, FT-IR vibrational, UV-visible, global reactivity parameters (GRP), MEP, FMO, NBO, ELF, LOL, and nonlinear optical (NLO) analysis at the DFT/B3LYP/6-311+G level. NBO analysis confirmed the existence of extended conjugation and intramolecular charge transfer among NBDH, NMDH, and FBDH, which have the lowest gap in π → π*, which are in line with the FMO results where successful charge transfer occurred from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). GRP analysis confirmed the potential of NBDH, NMDH, and FBDH for biological, electronic, and NLO applications. It is clear from the comparative analysis of the urea molecule that NBDH, NMDH, and FBDH all comprise fine NLO properties.The modification of natural products is one of the key areas of synthetic organic chemistry for obtaining valuable chemical building blocks that have medicinal significance. In this study, lauric acid-based hydrazones, namely ( The modification of natural products is one of the key areas of synthetic organic chemistry for obtaining valuable chemical building blocks that have medicinal significance. It is important to mention that patients are more susceptible to microbial infections after receiving cancer chemotherapy as the immune system is weakened due to the emergence of multidrug resistance.3,4 Hence, the synthesis of multitarget potent molecules with both anticancer and antioxidant potential are highly appreciated area for the synthetic organic community. In this scenario, intensive efforts are required to discover new anticancer molecules having resilient safety and low toxicity profiles accompanied by improved biological activity.5 In this regard, hydrazones are considered to be a fascinated class of organic compounds for researchers owing to their multiple medicinal applications as it has necessary features (binding cites) that are vital for medicinal applications   behavior.20–24 In this scenario, herein, we are presenting our findings regarding the synthesis of lauric acid-based hydrazones, their anticancer and antioxidant behavior, as well as computational investigation.The overlap of interdisciplinary studies are getting consideration to employ computational studies to the newly manufactured organic molecules to investigate their optoelectronic properties. The computational tool has been employed for freshly synthesized organic molecules for the exploration of their noncovalent interactions,22.1Novel hydrazones have been synthesized from natural lauric acid in a stepwise manner. All the reagents used were analytical grade and solvents were used without further purification. The reaction progress was monitored by TLC cards covered with silica gel (0.25 mm thickness). The NMR spectra were recorded using a Bruker-Avance 400 MHz spectrometer, while FTIR analysis was performed using the Shimadzu IR Prestige-21.2.2A mixture of dodecanehydrazide (0.48 mmol) and substituted aromatic aldehydes (0.50 mmol) was stirred in dry ethanol, followed by 3 h reflux. After completion (monitored by TLC), the reaction mixture was concentrated using a rotary evaporator. After the workup, the final products were purified using column chromatographic techniques.2.2.1νmax (cm−1) KBr: 1578  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N, iminic), 1688 , 3072 ; UV λmax (nm) 245; 1H NMR  δ 8.59 , 8.28 , 8.04 , 7.72 , 2.32 , 1.28 , 0.88 . 13C NMR  δ 173.26, 144.37, 140.54, 134.59, 134.24, 131.83, 129.81, 125.63, 35.53, 33.46, 33.04, 30.69, 30.58, 30.44, 30.37, 30.34, 30.26, 26.63, 26.04.IR 2.2.2νmax (cm−1) KBr: 1589 , 1648 , 3072 ; UV λmax (nm) 285; 1H NMR  δ 8.83 , 8.65 , 8.04 , 7.94–7.91 , 7.56–7.51 , 4.55 , 2.35 , 1.75 , 1.29 , 0.85 . 13C NMR  δ 173.83, 148.24, 145.53, 135.35, 132.31, 131.74, 130.71, 129.84, 128.87, 126.41, 125.17, 124.79, 35.69, 33.05, 33.02, 30.71, 30.59, 30.46, 30.41, 30.36, 30.26, 30.31, 26.79.IR 2.2.3νmax (cm−1) KBr: 1550 , 1680 , 3072 ; UV λmax (nm) 235; 1H NMR  δ 8.07 , 7.82 , 7.70 , 7.15 , 2.29 , 1.28 , 0.88 . 13C NMR  δ 172.91, 166.43, 147.49, 144.28, 130.78, 116.82, 35.56, 33.44, 33.04, 30.70, 30.58, 30.44, 30.39, 30.35, 30.28, 26.75, 26.09.IR 2.3−1 to 500 μg mL−1. Next, 20 μL of each test sample was mixed with 180 μL of a solution containing 9.2 mg 2,2-diphenyl-1-picrylhydrazyl (DPPH) dissolved in 100 mL methanol. Ascorbic acid and DMSO were used as positive and negative controls, respectively. The reaction mixtures were incubated in the dark at 37 °C for 60 min, and the absorbance of the samples was measured at 517 nm. The percentage of inhibition or scavenging was determined by measuring the discoloration of the DPPH purple color.The potential antioxidant capacity of derivatized compounds  was determined by preparing the test samples in DMSO at concentrations ranging from 15.625 μg mL2.4−1) and incubated for 4 h at 37 °C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using a microplate reader. Human liver cancer cells (HepG2 cells) were gifted by Cell and Molecular Laboratory, Department of Zoology, Government College University Faisalabad, Pakistan.Human liver cancer cells (HepG2 cells) were cultured in a 96-well plate, and their viability was assessed using the MTT assay. The cells were treated with derivatized compounds  of different concentrations for 48 h, followed by the addition of MTT reagent , 1689 , and 3072 cm−1  in the case of (E)-N′-(2-nitrobenzylidene)dodecanehydrazide (NBDH), 1589 , 1648 , and 3072 cm−1  in the case of (E)-N′-dodecanehydrazide (NMDH), and 1550 , 1680 , and 3072 cm−1  in the case of compound (E)-N′-(4-fluorobenzylidene)dodecanehydrazide (FBDH). In 1H-NMR, new peaks were observed in the aromatic area of the spectrum, which authenticated the formation of hydrazone via the coupling of dodecanehydrazide and substituted aromatic aldehydes.In the current research work, lauric acid has been used as a starting material for the stepwise synthesis of hydrazones. The preparation of these hydrazones were analyzed by various spectroscopic methods. In the IR spectrum, the –OH peak of lauric acid disappeared and a signal of the aliphatic ester was observed, which supports the synthesis of targeted ethyl dodecanoate. Ethyl dodecanoate was converted to dodecanehydrazide, which is supported the by the appearance of two broad peaks at 3288 cm3.1P < 0.05) at all concentrations when compared with the antioxidant potential of other two compounds (NMDH and FBDH) at all their corresponding concentrations. Similarly, the compound NMDH exhibited the maximum antioxidant potential at 250 μg and 500 μg when compared with that of FBDH at its same doses, respectively  of NBDH, NMDH, and FBDH compounds, the percent viability of HeG2 cells was observed. We observed that all the three compounds exhibited moderate to excellent antiproliferative activity, but the difference in the percent viability of the three compounds was due to the aromatic substitution on dodecanehydrazide. Compound FBDH showed maximum percent viability at 100 μg mL−1 and 200 μg mL−1 when compared with that of the other two compounds, i.e., NBDH and NMDH, respectively  method, the optimized structural parameters (bond length and bond angles) were calculated; the results are given in Tables S1 and S2 ESI. The len4.2E values for NBDH, NMDH, and FBDH.HOMO and LUMO are the two main orbitals that make up frontier molecular orbitals (FMOs). To describe the chemical reactivity and interaction of the molecule under study with other species, the FMOs are crucial quantum chemistry parameters. Scientists use FMOs to describe a wide range of electronic and optical characteristics, including the most reactive position in π-electron systems, reaction types in conjugated systems, and UV-vis spectra. DFT calculations at the B3LYP level of theory with 6311+G basis set predicted the electronic properties of NBDH, NMDH, and FBDH. 2), slightly deactivating fluorine (F) group, and moderately activating benzene (–C6H4) substitution affect the FMOs of the investigated molecules. The value of the energy gap (ΔE) in FBDH was determined to be 7.28 eV, and this was found to be the highest value of band gap among all the investigated molecules. When compared with FBDH, NMDH has an energy gap that briefly decreases to 6.43 eV, which indicates the effect of slightly activating the benzene substitution in NMDH. The effect of the deactivating group can be seen quite clearly in the NBDH molecule, which demonstrates a narrowing of the energy gap to 3.87 eV, which is 3.41 eV less than that of the FBDH molecule. It is possible that the highly deactivating (NO2) groups are accountable for this reduction in the ΔE value when compared to the fluorine group. This decrease in the ΔE value demonstrates that the presence of highly electron-withdrawing groups brings about a decrease in the ΔE value. In general, the energy gap is found to decrease as one moves from FBDH to NMDH to NBDH. It is clear from this discussion that the band gap value is inversely proportional to the number of electron deactivating groups and that these groups increase as follows: –F < –C6H4 < –NO2. Auxiliary descriptors such as EHOMO, ELUMO, and ΔE are used to portray the reactivity and stability of NBDH, NMDH, and FBDH by predicting the global reactivity descriptors.The pictorial displays of FMOs are presented in 4.3A) and ionization potential (I) were determined by means of Electron affinity , was reported by Parr et al.31–34 asThe formula for determining the electrophilicity index, denoted by the symbol (η) value (3.64). However, FBDH has the lowest softness (σ) value, leading to greater reactivity and better stability, while NBDH has the highest, indicating better reactivity and less stability. Overall, the global reactivity parameters show that FBDH has higher values for energy gap, ionization potential, and chemical hardness, which means it can donate more, is more stable, and accepts less than the other studied compounds (NBDH and NMDH). On the other hand, NBDH has higher electron affinity, electrophilicity index, and softness values than NMDH and FBDH. This means that NBDH is a compound that accepts electrons, reacts more, is less stable, and gives electrons away less than NMDH and FBDH. Also, all investigated compounds can play important roles in optoelectronic technologies.A closer look at 4.4The density of states, also known as DOS, is the ratio of distinct forms that electrons are permitted to experience at a particular energy level. It can also refer to the frequencies of electronic excitation that occur per unit of volume and unit of energy. In contrast to estimating the gap between vibrational frequencies in semiconductors, the DOS simulations make it possible to compute the Gaussian probability of various stages as a function of energy .λmax, excitation, and total electron scattering. The presence of various types of electron-activating and deactivating groups caused a shift in the distribution of electrical properties across the HOMOs and LUMOs. The electrical configuration of LUMOs was represented by positive quantities along the x-axis, whereas the conducting channel at HOMO was represented by negative quantities. Both of these configurations can be seen in the image below. The gap that existed between these levels was utilized as the representation for the respective energy shortfall. In the NBDH molecule, HOMO contribution originates mainly from the DH fragment (64.4%) and NB fragment (35.6%), while the in LUMO, a large contribution is given by NB (87.2%), and DH contributes about 12.8%. Due to the substitution of a slightly activating group (–C6H4) in NMDH instead of a strongly deactivating group (–NO2), the contribution of the DH fragment reduced to 30.4% while that of the NM fragment increases to 69.6%. The minute effect is also observed in LUMO where the DH and NM fragment contribute about 34.2% and 65.8%, respectively. The effect of a weakly deactivating group is also noted, where HOMO composition is contributed by the DH fragment (52.8%), followed by the FB fragment (47.2%). Similarly, in the LUMO, the chief contribution (56.1%) is given by DH, followed by FB is 43.9%. From this discussion, we predict that NBDH has good composition as compared to NMDH and FBDH.It is necessary to have an understanding of the probability of states that are attainable per unit volume per unit of energy to measure a wide variety of electronic activities in materials with innovative designs. Some of these activities include 4.5Time-dependent DFT (TDDFT) was used at the B3LYP/6311+G level of theory to determine the ultraviolet spectral values. The analysis of NBDH, NMDH, and FBDH using UV-visible light was completed, and the results are shown in 4.6−1). Electrophilic reactive sites are shown in red, and nucleophilic reactive sites are shown in blue  denotes the diagonal, and j and i are the off-diagonal NBO Fock matrix elements. The significant magnitude of the perturbation stabilization energy value, denoted by the symbol E(2), hints at the existence of a more robust association between bonding and antibonding orbitals. NBO research on NBDH has been carried out with the B3LYP/6-311+G level of theory, and the findings of some representative interactions have been compiled in Table S3  → π*(N52–O53) and σ(C44–C47) → σ*(C42–C44) transitions are the most likely to occur in NBDH. Conjugation in the NBDH molecule is strongly suggested by the existence of interactions (π → π*). It has been determined that the stabilization energies for the following transitions in NBDH are 7.14, 13.58, 19.86, and 21.68 kJ mol−1 for π(N52–O53) → π*(N52–O53), π(C47–C48) → π*(C42–C44), π(C42–C44) → π*(N39–C40), and π(C43–C45) → π*(C42–C44), respectively. Resonance transitions such as LP(2)O53 → σ*(C44–N52) and LP(3)O54 → π*(N52–O53) resulted in stabilization energy values of 13.73 and 160.86 kJ mol−1, respectively. The most likely transitions that take place in NMDH are π(C7–C8) → π*(C9–C10) and σ(C14–C16) → σ*(N12–N14), with respective stabilization energy values of 19.09 and 5.26 kJ mol−1. Both these transitions have a high probability of occurring. It was discovered that the stabilization energies in NMDH are 7.57 kJ mol−1 and 6.06 kJ mol−1 for the transitions π(C11–N12) → π*(C9–C10) and σ(C16–H37) → σ*(C14–O15), respectively. In the case of resonance, the transitions such as π(C11–N12) → π*(C9–C10) and σ(C16–H37) → σ*(C14–O15) led to stabilization energy values of 11.40, and 25.24 kJ mol−1, respectively. FBDH's most likely transitions are π(C43–C45) → π*(C47–C49) with a stabilization energy of 20.94 kJ mol−1. In resonance, the transitions LP(1)N39 → σ*(C40–C41), LP(1)N1 → π*(C2–O3), and LP(2)O3 → (N1–C2) have stabilization energy values of 10.13, 47.17, and 28.81 kJ mol−1. However, transitions such as σ(C49–F52) → σ*(C43–C45) and σ(C49–F52) → σ*(C44–C47) have energies of 1.35 and 1.4 kJ mol−1, respectively. Conjugation in the FBDH molecules aids intramolecular charge transfer, as shown in Table S3 N39 → σ*(C40–C41), LP(1)N1 → π*(C2–O3), and LP(2)O3 → (N1–C2) led to the stabilization energy values 10.13, 47.17, and 28.81 kJ mol−1, respectively. In contrast, transitions such as σ(C49–F52) → σ*(C43–C45) and σ(C49–F52) → σ*(C44–C47) have 1.35 and 1.4 kJ mol−1 energies, respectively. Finally, it can be deduced on the basis of findings that conjugation is present in FBDH molecules, which ease intramolecular charge transfer. On the basis of the findings, it is possible to draw the conclusion that conjugation is present in the title molecules, which makes the process of charge transfer within molecules easier. Therefore, the powerful intramolecular conjugation present in NBDH, NMDH, and FBDH are the fundamental reasons that contribute to this system's increased degree of stability.The highest probable transitions that take place in FBDH are π(C43–C45) → π*(C47–C49), having stabilization energy value of 20.94 kJ mol4.7Electron localization functions (ELFs) and localized orbit locators (LOLs) were used extensively to illustrate the connections between molecular geometry and electronic properties and to facilitate a deeper understanding of the bonding dynamics of molecules' primary active sites. Using the critical points of the density function, the localized orbital locator (LOL) places emphasis on topological parameters such as the kinetic energy density. For this research, we used the computer program Multiwfn to calculate them. In this case, as shown in 4.837–39 vibrational frequencies of NBDH, NMDH, and FBDH with proposed assignments are placed in Tables S4–S6 . The theoretical spectrum is recorded at the DFT/B3LYP/6-311G level of theory, and the results are shown in S4–S6 ESI.4.9−1. The benzene unit wavenumbers computed at 908.23 cm−1 correspond to twisting vibrations. The scissoring stretching modes are responsible for the vibrational band that can be seen at 708.11 cm−1. The calculated C–H vibrational bands were detected in NBDH at a frequency of 3127.52 cm−1. In the benzene unit, computed C–H symmetric stretching vibration can be found at 3224.67 and 3208.35 cm−1, whereas at 3193.76 cm−1, both asymmetric and symmetric stretching vibration can be found in the C–H of the benzene unit. At a frequency of 732.61 cm−1, NMDH exhibits rocking vibrations that were computed using DFT. There is evidence of a wagging mode of vibration at a frequency of 562.42 cm−1. At a frequency of 3049.85 cm−1, stretching C–H vibrational bands were detected. In the benzene unit, the computed C–H symmetric stretching vibration can be found at 3199.97 and 3198.75 cm−1, and the C–H asymmetric stretching vibration can be found at 3184.94 and 3163.01 cm−1. However, FBDH assigned the DFT frequencies of 942.11 and 827.11 cm−1 in the benzene unit to twisting and wagging vibrations, respectively. The calculated C–H vibrational bands in FBDH were found at 1026 cm−1. The C–H symmetric vibrational bands were measured at 3205.95 cm−1, and the C–H asymmetric vibrational bands were measured at 3193.01 cm−1, both in agreement with DFT calculations Tables S4–S6 .4.11−1, respectively.It is only possible in NBDH to observe the vibrational mode of N–O scissoring, which has stretching frequencies of 853.3 and 708.11 cm4.12−1 in the studied molecule FBDH. This is mostly because of the deactivating group attached to the benzene unit.C–F vibrations are seen at 804.57 cm4.13−1. Asymmetric vibrations, as calculated by DFT, are also present at 3083.93, 3084.01, and 3079.30 cm−1 in NMDH and FBDH, respectively. Both NMDH and FBDH exhibit symmetric vibrations at 3018.39 cm−1 and a wagging mode of vibration at 896.77 cm−1 .4.14−1. Twisting vibrations are given a wavenumber of 881.28 cm−1. The stretching rocking modes account for the vibratory band detected at 826.85 cm−1. DFT calculations show that vibrations at 622.50 cm−1 are present in NMDH, indicating the presence of rocking motions. The frequencies of 3081.83 and 3024.17 cm−1 are associated with asymmetric and symmetric vibrations, respectively. Asymmetric C–H vibrations are accounted for by the DFT frequencies of 3081.85, 3079.30, 3055.12, and 3041.88 cm−1 calculated for FBDH. Bands of vibrational energy are detected at 938.42 and 880.68 cm−1 because of the twisting and scissoring stretching modes, respectively. The ethyl group exhibits a rocking mode of vibrations with wavenumbers of 732.25 and 634.17 cm−1 .4.15−1 were observed to be associated with vibrational bands that are caused by N–H in NBDH, NMDH, and FBDH.The stretching frequencies of 3489.05, 3486.67, and 3491.28 cm4.16βtot. Calculations show that the dipole moments of NBDH, NMDH, and FBDH are 0.589, 2.766, and 3.332 times greater than those of the urea molecule, respectively. x, y, and z-directions as well as the total linear polarizability (α). According to the findings, the value of the dipole polarizability tensors of FBDH is discovered to be greater in all the three directions when compared to the values of the polarizability tensors of the other compounds that were investigated. The computed value of dipole polarizability is found to be the highest in NMDH at 308.48 (a.u.), while the value of dipole polarizability is observed to be the lowest in FBDH at 256.55 (a.u.). NMDH > NBDH > FBDH is the observed decreasing order of computed dipole polarizability observed in the compounds that were investigated. Because of their extended conjugation, NMDH molecules have the highest dipole polarizability. βtot) as well as its contributing tensors. While the dominant transitions 1488.73 and 298.55 (a.u.) can be seen along the x-axis with a positive direction in NBDH and FBDH, the dominant transitions 134.15 (a.u.) can be seen along the x-axis with a negative direction in NMDH. In NBDH and NMDH, the highest and lowest values for tot that could be computed were 1587.43 (a.u.) and 131.02 (a.u.), respectively. NBDH > FBDH > NMDH is the decreasing order of the total observed in the molecules that have been studied. The fundamental structures of NBDH conceal the secret that explains why it has a higher NLO activity level compared to other compounds that have been investigated. In comparison to NMDH, which has a benzene group that only has a very slight activating effect, NBDH has highly deactivating groups (–NO2), which results in an increase in resonance as well as charge transfer. In addition, FBDH has a greater NLO response in comparison to NMDH because of the slightly deactivating group (F).Nonlinear optical (NLO) properties are exhibited by organic materials used in optical interconnections. We use DFT to draw conclusions about the NLO activities of NBDH, NMDH, and FBDH. Urea is the molecule that is most frequently used when conducting research on the NLO properties of the compound. The results of the calculations for the significant NLO parameters of NBDH, NMDH, and FBDH are presented in 5E)-N′-(2-nitrobenzylidene)dodecanehydrazide (NBDH), (E)-N′-dodecanehydrazide (NMDH), and (E)-N′-(4-fluorobenzylidene)dodecanehydrazide (FBDH) in a stepwise manner and were characterized using spectroscopic techniques. The anticancer and antioxidant potential of these hydrazones have been tested. Accordingly, antioxidant agents showed that their antioxidant activity against oxidative stress by inhibiting the oxidative chain reactions that produced reactive oxygen species, and this can be accomplished by inhibiting or limiting the supply of nutrients (such as proteins and lipids) that are necessary for catalyzing these chain reactions. Anticancer agents showed that their anticancer activity in inhibiting the progression of cancer by different pathways such as inhibiting the cell division by cell cycle arrest, apoptosis by intrinsic and extrinsic pathway, and alteration in the expression of genes involved in cell proliferation. In addition, the novel hydrazones NBDH, NMDH, and FBDH were synthesized and characterized using experimental as well as computational FT-IR vibrational, UV-visible, global reactivity parameters (GRP), MEP, FMO, NBO, ELF, LOL, and nonlinear optical (NLO) analysis. The energy gap order NBDH (3.87 eV) < NMDH (6.43 eV) < FBDH (7.28 eV) is found to be inversely proportional to the number of electron deactivating groups as follows: –F < –C6H4 < –NO2. DOS analysis showed that HOMO contribution in NBDH, NMDH, and FBDH originates mainly from DH (64.4%), NM (69.6%), and DH (52.8%) fragments and LUMO from NB (87.2%), NM (65.8%), and DH (56.1%) respectively. NBO results confirmed that the hyperconjugative interactions among the bonds due to the delocalization of electrons on whole systems provide stability to the investigated compounds. Global reactivity results indicate that NBDH is a soft compound that accepts electrons, reacts more, is less stable, and gives electrons away less than NMDH and FBDH due to their larger energy gap. GR parameters, MEP surfaces, and ELF, LOL analysis reveal that the molecule might be bioactive. The first hyper polarizability is found to be 1654.10, 229.06, and 486.38 (a.u.) for NBDH, NMDH, and FBDH, respectively. The greater value of NBDH as compared to the other investigated molecule may be due to the strongly deactivating group effect. Findings show the evidence of promising NLO performance and may have potential NLO applications.Natural products modification for the search of new chemical architectures with potential medicinal importance is one of the key area of synthetic organic chemistry. In the current research, natural lauric acid have been synthetically modified to hydrazones (There are no conflicts to declare.RA-013-D3RA02433D-s001


P. kudriavzevii via the adaptive laboratory evolution (ALE) technique. The parental strain of P. kudriavzevii was subjected to repetitive long-term cultivation in medium supplemented with a gradually increasing concentration of acetic acid, the major weak acid liberated during the lignocellulosic pretreatment process. Three evolved P. kudriavzevii strains, namely, PkAC-7, PkAC-8, and PkAC-9, obtained in this study exhibited significantly higher resistance toward multiple stressors, including heat, ethanol, osmotic stress, acetic acid, formic acid, furfural, 5-(hydroxymethyl) furfural (5-HMF), and vanillin. The fermentation efficiency of the evolved strains was also improved, yielding a higher ethanol concentration, productivity, and yield than the parental strain, using undetoxified sugarcane bagasse hydrolysate as feedstock. These findings provide evidence that ALE is a practical approach for increasing the multistress tolerance of P. kudriavzevii for stable and efficient second-generation bioethanol production from lignocellulosic biomass.Second-generation bioethanol production using lignocellulosic biomass as feedstock requires a highly efficient multistress-tolerant yeast. This study aimed to develop a robust yeast strain of  Approximately 95% of bioethanol utilized currently is produced via the biological fermentation process, and the primary feedstocks for bioethanol production in Thailand are sugarcane molasses and cassava. Since these feedstocks are also utilized in other industries, such as food, feed, and fine chemical production, competition of feedstocks for producing these products may occur, particularly when the feedstock demand is increased. The supply of feedstocks may need to be increased, but the feedstock price may also be high, which may affect the overall living cost and the country's overall economic growth. Thus, alternative feedstocks with high potential that are not being used in the food, feed, and fine chemical production industries can solve those problems.Ethanol is one of the biorenewable energies determined by the Ministry of Energy, Thailand, as part of the 15-year renewable energy development strategy (2008–2022). The target goal of bioethanol production and utilization is approximately 20% of the country's final energy consumption by 20223. Since these raw materials are in large quantity, inexpensive, and readily available in the country, they are considered high-potential feedstocks for domestic ethanol production.Several economic crops are grown in Thailand, such as rice, corn, cassava, sugarcane, and pineapple, that generate a large number of byproducts, such as rice straw, corncobs, sugarcane bagasse, cassava pulp, and pineapple peel, respectively, after the manufacturing process. These byproducts are considered lignocellulosic materials, which can be used as feedstocks for second-generation bioethanol production. Based on chemical composition analysis, these raw materials comprise a large amount of cellulose and hemicellulose, accounting for approximately 30–50% and 20–30% by weight, respectively. For instance, rice straw contains approximately 30–40% cellulose and 20–30% hemicellulose, and corn cobs contain 40% cellulose and 20% hemicellulose on a dry basis4, liquid hot water, dilute-acid hydrolysis5, the organosolv process6 and ammonia fiber explosion (AFEX)7.The drawbacks of using lignocellulosic materials for bioethanol production include the complex pretreatment process, high-cost operation, and the generation of lignocellulosic inhibitors, such as acetic acid, furfural, 5-(hydroxymethyl) furfural (5-HMF), and phenolic acid, that inhibit the growth and metabolic activity of ethanol-producing microorganisms. To overcome these circumstances, several high-efficiency pretreatment methodologies that can reduce operation costs and the formation of inhibitors have been reported, such as steam explosion3COOH) is the most abundant weak acid, accounting for 5–10 g/L, depending on the type of feedstock and pretreatment process10. High concentrations of acetic acid inhibit the growth and ethanol production of several ethanologenic microorganisms, e.g., Candida shehatae, Pichia stipitis, Saccharomyces cerevisiae11, Kluyveromyces marxianus12 and Zymomonas mobilis14. Several strategies have been applied to minimize the inhibitory effect of acetic acid on microbial growth and ethanol production, for instance, the removal of acetic acid using sodium borohydride15 and activated carbon16, the application of dried air stripping17, or genetic engineering of microbial cells harboring the genes involved in acetic acid tolerance, such as FPS118, PHO1319, and HAA120.Among lignocellulosic inhibitors, acetic acid  is one of the most promising strategies for improving microbial phenotypes or physiological characteristics through long-term cultivation42. Thus, the present study aims to improve the multistress tolerance capability of P. kudriavzevii toward lignocellulosic inhibitors through the ALE strategy using acetic acid as a selective pressure. The growth performance of the selected evolved strains under various stress conditions was determined. Furthermore, the ethanol fermentation efficiency of the selected evolved strains using sugarcane bagasse hydrolysate (SBH) without a detoxifying process was also evaluated.It has previously been demonstrated that the damage induced by thermal or other stresses, such as ethanol, acetic acid, formic acid, phenolic acid, and furfural, shares specific common characteristics, e.g., leakage of the cell membrane, accumulation of reactive oxygen species (ROS), and denaturation of macromolecules, such as proteins, DNA or RNA, which subsequently trigger stress-responsive mechanisms to protect yeast cells from severe stress conditions. Furthermore, yeast cells that exhibit high resistance to one stress may also possess the ability to withstand other stress conditionsThe bacteriological grade of yeast extract, malt extract, and peptone was procured from TM Media . Acetic acid, furfural, 5-HMF , vanillin, isopropanol, and ethanol (HPLC grade) were purchased from Sigma‒Aldrich, St. Louis, MO, USA. Glucose  was obtained from KemAusTM, New South Wales, Australia.Yeast extract malt extract (YM) medium  was used to cultivate yeast cells. After preparation, the medium was autoclaved at 121 °C and 15 psi for 15 min.The sugarcane bagasse used in the present study was kindly provided by the sugar factory in Khon Kaen province, Thailand, with the permission of the Chief Executive Officer. After drying, the bagasse was pulverized into small pieces and kept in a plastic bag at room temperature at the Department of Biotechnology, Faculty of Technology, Khon Kaen University, with the code number KKUDB-SB-2022-01. All plant preparation methods followed the relevant guidelines in the Methods section.43.The chemical compositions of the sugarcane bagasse, including cellulose, hemicellulose, and lignin, were determined using the standard method of the Association of Official Analytical ChemistsPichia kudriavzevii, a thermotolerant yeast isolated from soil39, was used in this study. It was cultured in YM agar and maintained at 4 °C by subculturing every two months.5 cells/mL and subsequently incubated under the abovementioned conditions. After 12 h of incubation, the growing yeast cells were used as a starter culture or inoculum for subsequent experiments.Yeast inoculum was prepared by transferring a loopful of yeast cells into 100 mL YM broth and incubated in a controlled incubator shaker  at 35 °C with a shaking speed of 150 rpm for 24 h. Then, the activated yeast cells were transferred into fresh YM broth with an initial cell concentration of approximately 1 × 10P. kudriavzevii since less than 10% of yeast cells survived under long-term cultivation in YM medium containing 7 g/L acetic acid at 35 °C. Thus, the ALE of P. kudriavzevii was commenced using acetic acid at 7 g/L, and the adaptation process was performed using a protocol described by Samappito et al.13 with some modifications. Three steps of the cultivation process were employed in this study. In the first step, a starter culture of yeast cells (1 × 106 cells/mL) was inoculated into 50 mL YM broth containing 7 g/L acetic acid and incubated at 35°C and 150 rpm. After 48 h of incubation, the cells were transferred into fresh YM medium containing acetic acid at 7 g/L and repeatedly cultivated under the same conditions until the growth profile of the yeast was stable (cell number reached 1 × 108 cells/mL). A total of 24 cycles was performed in the first step, and the resulting evolved yeast strain, designated PkAC-7, was obtained. In the second step, cells of PkAC-7 (1 × 106 cells/mL) were inoculated into 50 mL YM broth containing 8 g/L acetic acid and incubated at 35°C and 150 rpm for 72 h. Then, cells were transferred into fresh YM medium containing 8 g/L acetic acid, and the cultivation process was repeated under the same conditions for 45 cycles. The evolved strain of yeast designated PkAC-8 was obtained in this step. Finally, the same experimental procedure as the second step was conducted using the evolved PkAC-8 as a starter culture and YM medium containing 9 g/L acetic acid as the selective pressure. The cultivation process was repeated for 53 cycles, and the evolved strain designated PkAC-9 was obtained. The parental and selected evolved strains of P. kudriavzevii were subjected to further studies.Based on the preliminary study, acetic acid at a concentration of 7 g/L was a critical point for the growth of P. kudriavzevii PkAC-7, PkAC-8, and PkAC-9, respectively. To test the stability of all P. kudriavzevii evolved strains to withstand a high concentration of acetic acid, starter cultures of the yeast cells were inoculated into 100 mL YM medium containing acetic acid as mentioned earlier with an initial yeast cell concentration of 1 × 106 cells/mL. After incubation at 35 °C and 150 rpm for 48 h (PkAC-7) or 72 h (PkAC-8 and PkAC-9), cells were transferred into fresh YM medium without acetic acid supplementation and subsequently incubated as previously mentioned. The cultivation process was repeated at least 20 cycles for each strain of yeasts, and the yeast cell viability was determined.The YM media containing 7, 8, and 9 g/L acetic acid were prepared and used to cultivate P. kudriavzevii wild-type and evolved strains was determined by culturing yeast cells in YM medium containing 7, 8, and 9 g/L acetic acid and incubating at 35 °C and 150 rpm. The YM medium without acetic acid supplementation was used as a control condition. During cultivation, cells were randomly collected at certain time intervals, and the growth of yeast cells was determined using a haemacytometer  with methylene blue staining.The growth profile of P. kudriavzevii parental and evolved strains were grown in YM broth at 35 °C and 150 rpm for 24 h. The cells were collected by filtration using 0.45 μm filter paper and dried at 60 °C for 24 h. The resulting cells were coated with gold by ion sputter , and the morphology of the yeast cells was visualized using SEM .The P. kudriavzevii parental and evolved strains were determined using the procedure described by Pilap et al.42 and Gurdo et al.44, with some modifications. An initial cell concentration of 1 × 106 cells/mL was used throughout this experiment. For heat stress, starter cultures of yeast cells were transferred into 100 mL YM medium and incubated at 35, 37, and 40 °C and 150 rpm for 48 h. Ethanol stress was evaluated by transferring yeast cells into 100 mL YM medium containing 0, 7, 10, and 13% (v/v) ethanol and incubated at 35 °C and 150 rpm for 48 h. Osmotic stress was assayed by inoculating yeast cells into 100 mL YM medium containing 0, 0.6, and 1.2 M sorbitol and incubated at 35 °C and 150 rpm for 12 h. For lignocellulosic inhibitor stress, different concentrations of acetic acid , formic acid , furfural , 5-HMF , and vanillin  were tested. The yeast cells were inoculated into 100 mL YM containing each inhibitor at different concentrations and incubated at 35 °C and 150 rpm for 48 h. The effect of cocktail inhibitors, including 9 g/L acetic acid, 2 g/L formic acid, 5 g/L furfural, 3 g/L 5-HMF, and 2 g/L vanillin, on the growth of yeast cells was also determined. After transferring yeast cells into 100 mL YM medium containing a cocktail inhibitor at different loadings , the cultures were incubated at 35 °C and 150 rpm for 48 h. The viability of the yeast cells after the stress treatments was measured using a haemacytometer  with methylene blue staining.The inhibitory effects of heat, ethanol, osmotic, and lignocellulosic inhibitors, including acetic acid, formic acid, furfural, 5-HMF, and vanillin, on the growth of the 10. Briefly, dried sugarcane bagasse (10% w/v solid loading) was transferred into 3% (v/v) sulfuric acid solution and soaked at room temperature overnight. The mixture was heated at 121 °C and 15 psi for 60 min. After pretreatment, the liquid fraction (or acid hydrolysate) was collected by filtration through a muslin cloth and directly used as feedstock for ethanol production without a detoxifying process. The solid fraction after acid pretreatment was collected after washing with running tap water, dried at 60 °C until constant weight, and then subjected to enzymatic hydrolysis. A sample of the acid-pretreated solid fraction (5% w/v solid loading) was soaked in 50 mM sodium citrate buffer (pH 4.8), and cellulase (Cellic® CTec2) at a concentration of 15 filter paper units (FPU)/g dry solid was added. The suspension mixture was incubated at 50 °C and 200 rpm for 24 h. The enzymatic reaction was stopped by incubating the sample mixture in boiling water for 5 min. After centrifugation at 8000 rpm for 10 min, the resulting enzymatic hydrolysate was collected and supplemented with acid hydrolysate for ethanol production. The concentrations of sugars and lignocellulosic inhibitors in the acid and enzymatic hydrolysates were determined using high-performance liquid chromatography (HPLC).SBH was prepared following the method described by Sritrakul et al.P. kudriavzevii parental and evolved strains was performed in a 250-mL Erlenmeyer flask. A starter culture of yeast cells was inoculated into 100 mL SBH supplemented with 1.5 g/L yeast extract and 3.0 g/L peptone with an initial cell concentration of approximately 1 × 107 cells/mL. The flasks were incubated in a controlled incubator shaker  at 35 °C and 150 rpm. Samples were randomly withdrawn at specific time intervals, and the ethanol and sugar concentrations in the fermentation broth were analyzed using gas chromatography (GC) and phenol–sulfuric acid method, respectively.Batch ethanol fermentation from SBH by 45 using glucose as a standard. The ethanol concentration  was analyzed by GC  using a protocol of Laopaiboon et al.46. The volumetric ethanol productivity  and ethanol yield  were calculated as described by Nuanpeng et al.47. All experiments were performed twice, each with two replications, and the results are expressed as the means ± standard deviations (SDs). Duncan’s multiple-range test (DMRT) was applied to analyze the mean differences between each treatment at a probability of p ≤ 0.05 using the SPSS program for Windows.The yeast growth as viable cells was determined using a hemocytometer  with methylene blue staining. Total sugar was measured by the phenol–sulfuric acid methodP. kudriavzevii, designated PkAC-7, PkAC-8, and PkAC-9, were obtained after long-term cultivation in YM medium containing acetic acid as a selective pressure. The growth profile of yeast cells during the ALE process is illustrated in Fig. 8 cells/mL of yeast cells were reached at each cultivation cycle. Similarly, a constant number of yeast cells (approximately 108 cells/mL) was also detected using YM medium containing 8 g/L acetic acid. However, a dramatic decrease in yeast cell numbers was found when the acetic acid concentration in the cultivation medium was shifted from 8 to 9 g/L, specifically during the first cultivation period. After long-term repeated cultivation for 53 cycles, a slight increase in yeast cell number and a constant value of approximately 108 cells/mL was observed at the end of the cultivation period, suggesting that the evolved strains adapted and acquired tolerance capability to high concentrations of acetic acid. The results in the present study align with those reported in the literature. For instance, González-Ramos et al.48 and Gurdo et al.44 successfully developed acetic acid-tolerant strains of S. cerevisiae for ethanol production using ALE in medium supplemented with acetic acid. Another study by Zhang et al.37 also obtained S. cerevisiae yeast with very high thermotolerance after long-term adaptation in wheat straw hydrolysate at 35–42 °C. To the best of our knowledge, this is the first report to demonstrate the application of ALE using acetic acid as a selective pressure for selecting acetic acid-tolerant P. kudriavzevii strains for ethanol production.Three evolved strains of P. kudriavzevii evolved strains to withstand a high concentration of acetic acid was assessed by repeat cultivation in YM medium with and without acetic acid supplementation for 20 cycles at 35 °C, as described in the Materials and methods. The results revealed that all selected evolved strains exhibited high cell viability (more than 95%) under acetic acid stress. A comparative analysis of the growth of the parental and evolved strains was carried out using YM medium supplemented with acetic acid at different concentrations at 35 °C, and the growth profiles of the yeasts are illustrated in Supplementary Fig. S1. In YM medium without acetic acid supplementation (control treatment), the evolved strains, particularly PkAC-9 and PkAC-8, but not PkAC-7, exhibited significantly higher specific growth rates than the parental strain. The differences in the specific growth rates of the yeast strains were significantly distinguished when the medium was supplemented with 7, 8, and 9 g/L acetic acid. Among the evolved strains, P. kudriavzevii PkAC-9 displayed the highest specific growth rate under all stress conditions  and a smoother cell surface than the evolved strains. Increasing the concentrations of acetic acid in the culture medium led to a reduced yeast cell size. The evolved strain PkAC-9 displayed the smallest cell size (3.33 μm) compared to the others, i.e., 4.29 μm for PkAC-7 and 4.00 μm for PkAC-8. Furthermore, PkAC-9 also showed a rough cell surface compared to the others. The alteration of cell morphology in the P. kudriavzevii evolved strains, particularly cell size and cell surface, was possibly due to the adverse effect of high acetic acid concentrations during repetitive long-term cultivation. One of the cellular mechanisms proposed to be involved in the adaptation and tolerance to acetic acid is the remodeling of the yeast cell envelope49. High concentrations of acetic acid alter the molecular compositions and physical properties of the microbial plasma membrane and cell wall, causing a reduction in cell envelope permeability52, which is proposed to be essential for reducing acetic acid diffusion from the outside into the inside cellular structure. Ribeiro et al.52 demonstrated that adapting S. cerevisiae cells to acetic acid led to significant cell wall architecture alterations and increased cell wall stiffness, leading to higher acetic acid and lyticase activity resistance. Furthermore, yeast cell wall polysaccharide contents, specifically β-glucan and mannans, were also increased under acetic acid stress, making yeast cells more resistant to stress conditions. Regarding the morphological alteration of the P. kudriavzevii evolved strains, further studies should be performed, such as cell membrane and cell wall composition analysis.Figure 42. Therefore, to check whether the selected evolved strains acquire multistress tolerance ability, the growth performance of the P. kudriavzevii parental and selected evolved strains under different stress conditions was evaluated and compared. The effect of high-temperature stress, which has been shown to inhibit microbial cell growth and cause cell damage resulting in cell death, was first evaluated. As shown in Fig. P. kudriavzevii parental strain and all the evolved strains at 30 °C was similar. However, slightly higher growth of the evolved strains, particularly strains PkAC-8 and PkAC-9, occurred at 35 °C compared to the parental strain, which might be because these strains experienced a relatively high temperature of 35 °C during adaptation. The growth of the parental strain markedly decreased when the incubation temperature increased from 35 to 37 and 40 °C, in line with the results of Chamnipa et al.38 and Pilap et al.42, who reported a reduction in the cell viability of P. kudriavzevii under high-temperature conditions. Interestingly, all of the evolved strains exhibited significantly higher growth than the parental strain at 37 and 40 °C, suggesting that the evolved strains acquired thermotolerance ability during long-term adaptation in acetic acid. The acquisition of thermotolerance during long-term evolution adaptation to acetic acid or a mixture of lignocellulosic inhibitors, such as acetic acid, furfural, and vanillin, has also been reported in S. cerevisiae44 and Kluyveromyces marxianus53.As previously reported, the inhibitory effect of several stress conditions shares specific common characteristics, and resistance to one stress condition may lead to multiple stress tolerances29. Various concentrations of sorbitol as an osmotic stressor, including 0.6 and 1.2 M, were tested for their negative impact on yeast cell viability at 35 °C, and the results are illustrated in Fig. P. kudriavzevii parental strain can grow in medium containing up to 1.2 M sorbitol, its growth was significantly lower than in the control medium without sorbitol supplementation and in the medium with 0.6 M sorbitol. All evolved strains exhibited higher growth than the parental strain; specifically, strain PkAC-9 displayed the highest growth under all osmotic stress conditions. As previously reported, high osmotic pressure due to high concentrations of sorbitol had negative effects on yeast cell growth, cell viability, and metabolic activity55. Notably, the high growth performance of all the evolved strains under high sorbitol concentrations indicates that all these strains may have acquired osmotolerance ability toward osmotic stress. Similar work has also been reported in the thermotolerant yeast K. marxianus, in which a thermal-adapted strain exhibited significant osmotolerance toward a high glucose concentration of 16% (w/v) at 40 °C29. Another study by Gurdo et al.44 also showed that the acetic acid-adapted strain of S. cerevisiae exhibited osmotolerance toward osmotic shock due to a high sorbitol concentration.In addition to thermal stress, yeasts may also encounter other stress conditions during fermentation, particularly osmotic stress due to the high sugar concentration in the fermentation medium and ethanol stress due to ethanol accumulation during fermentation. These stresses have been shown to negatively affect microbial cell growth and viability, especially at relatively high temperatures56. Ultimately, ethanol stress also causes the modification of the plasma membrane and the denaturation of several macromolecules, such as DNA, RNA, and proteins, which can lead to cell death58. The inhibitory effect of ethanol stress on the growth of the P. kudriavzevii parental and evolved strains was monitored, and the results are presented in Fig. P. kudriavzevii evolved strains are more resistant to ethanol toxicity than the parental strain under all stress conditions, suggesting that these evolved strains acquired ethanol tolerance ability, similar to an acetic acid-adapted strain of S. cerevisiae44 and a thermal-adapted strain of K. marxianus29.Ethanol is one of the primary end products of sugar metabolism in ethanologenic microorganisms and can become a significant stressor if high ethanol concentrations accumulate in the culture broth during fermentation. High levels of ethanol inhibit cell growth, cell viability, and metabolic activity, resulting in lowered ethanol yield and productivity62. As reported in the literature, the acetic acid-adapted strain of S. cerevisiae and the thermal-adapted strain of K. marxianus exhibited multistress tolerance toward lignocellulosic inhibitors44. To check whether the evolved strains of P. kudriavzevii possess multistress tolerance properties against lignocellulosic inhibitors, the effect of individual and a mixture of lignocellulosic inhibitors, including acetic acid, formic acid, furfural, 5-HMF, and vanillin, on the growth of the P. kudriavzevii parental and evolved strains was evaluated. Regarding acetic acid, a byproduct from hemicellulose after releasing the acetate group, and formic acid, a product generated from the transformation of 5-HMF and furfural, low concentrations of these acids can suppress yeast cell growth. In addition, high concentrations of acetic and formic acids negatively inhibit transmembrane proton transport, interfere with pH homeostasis, and disturb cellular membrane structure and protein stability, leading to cell death64. The results in this study demonstrated that the growth of the P. kudriavzevii parental and evolved strains was similar when grown in medium without acetic acid supplementation. However, the growth of the evolved strains, particularly PkAC-8 and PkAC-9, was significantly higher than that of PkAC-7 and the parental strains when cultured at 35 °C in medium supplemented with 9 g/L acetic acid. Almost no growth of the parental strain was observed in medium containing 12 g/L acetic acid, similar to that reported in the wild-type strains of P. kudriavzevii 42 and S. cerevisiae (Y8)44. Interestingly, the evolved strain PkAC-9 can grow better than the other strains under this stress condition  in yeast cells, causing oxidative stress, and reducing the activity of several enzymes involved in glycolysis and ethanol production pathways, such as pyruvate dehydrogenase, alcohol dehydrogenase, and aldehyde dehydrogenase67. The effect of furfural and 5-HMF on the growth of the P. kudriavzevii parental and evolved strains was tested using YM medium containing 0, 1, and 5 g/L furfural and 0, 1, and 3 g/L 5-HMF. In the medium without furfural, the growth of the P. kudriavzevii parental and evolved strains was similar. A slight decrease in the growth of all yeast strains was observed when the medium was supplemented with 1 g/L furfural. However, a significant decrease in the growth of all yeast strains occurred when the furfural concentration in the medium increased to 5 g/L. The parental strain displayed the lowest growth, while PkAC-9 yielded the highest growth under all furfural stress treatments . Several macromolecules, such as DNA, RNA, proteins, chromatin, vacuoles, mitochondria, and cell membranes, can be damaged in the presence of these stress compoundsnts Fig. C. ConsidHMF Fig. D. These 68 and the fermentation efficiency of yeast cells. A recent study by Pattanakittivorakul et al.29 demonstrated that vanillin at 1 g/L inhibits the growth and ethanol production performance of the Kluyveromyces marxianus wild-type strain, a thermotolerant yeast for high-temperature ethanol production. The adverse effect of vanillin on the growth of the P. kudriavzevii parental and evolved strains was evaluated, and the results are summarized in Fig. K. marxianus53.In addition to weak acids and furan derivatives, phenolic compounds such as vanillin have also been shown to inhibit cell growth and metabolic activity. Vanillin, a degradation product of guaiacylpropane units of lignin, can inhibit yeast cell growth and fermentation activity at very low concentrations, making it one of the most effective inhibitors in lignocellulosic hydrolysates. It negatively impacts the structure and integrity of yeast cell membranesP. kudriavzevii during long-term adaptation to acetic acid. In particular, the evolved strain PkAC-9 displayed superior multistress tolerance toward heat, ethanol, osmotic, and lignocellulosic inhibitors, making it a robust yeast strain for second-generation bioethanol production.Based on the growth properties under different stress conditions, acquiring multistress tolerance may evolve in 69. Thus, the inhibitory effect of multiple inhibitors, including acetic acid, formic acid, furfural, 5-HMF, and vanillin, on the growth of the P. kudriavzevii parental and evolved strains was examined , the growth of the yeast cells was increased. The highest growth occurred in the evolved strain PkAC-9 under all inhibitor loading conditions, suggesting that this strain is more resistant to multiple stresses than the others, similar to the results observed in the experiments using a single stress condition.Considering that the lignocellulosic pretreatment process often generates a complex mixture of inhibitors, the synergistic effects of several inhibitors in lignocellulosic hydrolysate on the microbial growth, metabolic activity, and fermentation efficiency of yeast cells have also been reported70. This byproduct contains approximately (on a dry weight basis) 36–40% cellulose, 28–32% hemicellulose, and 12–14% lignin71, making it one of the highest-potential feedstocks for second-generation bioethanol production. The sugarcane bagasse used in the present study contained 49.09% cellulose, 29.30% hemicellulose, and 9.74% lignin on a dry weight basis, slightly different from values reported in the literature. For instance, 39.8% cellulose, 28.6% hemicellulose, and 22.5% lignin were reported by Oliveira et al.72, while 39.2% cellulose, 37.9% hemicellulose, and 11.8% lignin were reported by de Souza et al.73. Cellulose, hemicellulose, and lignin contents of 38.6%, 27.9%, and 17.8% on a dry basis, respectively, were also reported74, and most recently, Chamnipa et al.75 reported cellulose, hemicellulose, and lignin contents in sugarcane bagasse with values of 47.9%, 31.4%, and 10.3% on a dry basis, respectively. Since several factors, such as plant varieties, environmental growth conditions, cultivation areas, harvesting, and manufacturing processes, affect plant growth and physiological properties and the structure of plant biomass, the differences in the chemical compositions of sugarcane bagasse may be attributed to such factors76.Sugarcane bagasse, a byproduct of sugarcane manufacturing, is one of the least expensive and most significant readily available feedstocks in Thailand. In 2022, Thailand was the world’s fourth-largest sugarcane-producing country, producing approximately 10.3 million tonnes of sugar. Forty-seven sugar factories are in operation in Thailand, generating approximately 20 million metric tonnes of sugarcane bagasse annually80. In this study, dilute acid pretreatment of sugarcane bagasse using 3% (v/v) sulfuric acid at 121 °C and 15 psi for 60 min was performed, yielding a liquid fraction called acid hydrolysate, as mentioned in the Materials and Methods section. This acid hydrolysate contained 25.81 g/L of total sugar, in which xylose was the most predominant sugar, accounting for 20.32 g/L, followed by arabinose and glucose at concentrations of 2.90 and 2.59 g/L, respectively. Acetic acid was the most predominant lignocellulosic inhibitor in the acid hydrolysate, accounting for 3.77 g/L. Other inhibitors found in this hydrolysate included formic acid (0.26 g/L), furfural (0.08 g/L), 5-HMF (0.46 g/L), and vanillin (0.03 g/L).Although several lignocellulosic pretreatment processes have been reported, the chemical process using dilute acid is typically used since it has high efficiency in separating cell wall components, yielding high concentrations of fermentable sugars. It is also a simple process with a low-cost operation that can be adopted for wide-scale applications compared to physicochemical and biological processesThe solid fraction after acid pretreatment was hydrolyzed using cellulase, and the resulting hydrolysate (enzymatic hydrolysate) was collected and analyzed for its chemical composition. As a result, a total sugar content of 63.88 g/L was obtained, which was approximately 2.5-fold higher than that in the acid hydrolysate. Glucose, a major product of cellulose hydrolysis, was the most abundant sugar found in the hydrolysate, comprising 43.54 g/L. Xylose and arabinose were also detected at concentrations of 19.29 and 1.05 g/L, slightly lower than that of the acid hydrolysate. All of the inhibitors found in the acid hydrolysate, except vanillin, were also present in the enzymatic hydrolysate, i.e., acetic acid (0.89 g/L), formic acid (2.80 g/L), furfural (1.57 g/L), and 5-HMF (0.23 g/L).P. kudriavzevii parental and evolved strains using the acid hydrolysate as a primary feedstock was performed. Unfortunately, very low yeast cell growth and ethanol production were detected after 72 h of fermentation at 35 °C, probably due to the toxic effect of sulfuric acid and a low sugar level, mainly glucose, in the hydrolysate. Thus, a mixture of acid and enzymatic hydrolysates at a ratio of 1:1 was used as a feedstock instead of using acid hydrolysate alone. As a result, all yeast strains can grow and produce ethanol in different manners, possibly due to a reduction in sulfuric acid toxicity and an increase in the glucose concentration in the hydrolysate. Indeed, a mixture of acid and enzymatic hydrolysate contained 30.06 g/L of total sugar, in which glucose and xylose were the most predominant sugars, comprising 17.44 and 11.18 g/L, respectively. The concentrations of inhibitors in the acid hydrolysate were reduced from 3.77 to 1.90 g/L for acetic acid, 0.26–0.12 g/L for formic acid, 0.08–0.03 g/L for furfural, and 0.46–0.15 g/L for 5-HMF. Vanillin was not detected in the hydrolysate mixture.One of the aims of this study is to use undetoxified acid hydrolysate as feedstock for ethanol production. Therefore, ethanol production by the P. kudriavzevii parental and evolved strains was shown in Supplementary Fig. S2. The sugar utilization by the parental strain was lower than that of the evolved strains. PkAC-9 displayed the highest sugar utilization than the others. The maximum ethanol concentrations produced by different yeast strains were detected at 72 h after fermentation. The kinetic parameters of the ethanol production by P. kudriavzevii parental and evolved strains are summarized in Table P. kudriavzevii parental strain produced the lowest ethanol concentration of 6.60 g/L, with an ethanol yield of 0.33 g/g, while strain PkAC-9 produced the highest ethanol content of 11.02 g/L and ethanol yield of 0.50 g/g. Notably, all of the evolved strains consumed sugar in the SBH better than that of the parental strain, specifically strain PkAC-9 consumed approximately 16% higher sugar than the parental strain  or central composite design (CCD) based on the response surface methodology (RSM) should be performed to improve the fermentation efficiency.Regarding the biological fermentation process, not only the microbial species but also the environmental fermentation conditions significantly impact ethanol yield and productivity. Several fermentation parameters, such as microbial cell concentration87. For instance, the aquaglyceroporin Fps1, Jen1, and Ady2 transporters proteins and the ABC transporter Pdr18 have been reported to play a crucial role in cellular transportation of acetic acid as well as lipid composition and cell membrane permeability . The plasma membrane H+-ATPase (Pma1) and vacuolar ATPase assembly protein (VMA3) have been shown to contribute to acetic acid stress resistance, which is part of the pH homeostasis of yeast cells . Regarding the metabolism and stress-signaling pathways, several genes and proteins related to carbohydrate metabolism, protein folding, lipid metabolism, amino acid metabolism, cell wall function, and transport are involved in acetic acid tolerance . Of these, the transcription factors Haa1p and Hog1 are the significant players contributing to acetic acid resistance in yeast cells .Several cellular pathways are responsible for acetic acid stress in yeast cells. These pathways include cellular transport, pH homeostasis, metabolism, and stress-signaling pathways, in which many genes or proteins are involved in each pathwayICL1 (encoding isocitrate lyase), CIT3 (encoding citrate synthase 3), and ADY2 (encoding acetate transporter protein), are upregulated upon heat stress at 45 °C and are proposed to be involved in acetic acid tolerance in K. marxianus29. In S. cerevisiae, tolerance to acetic acid is also correlated with the expression of these three genes98 as well as other genes, including ASG1 , ADH3 , SKS1 (encoding protein kinase), and GIS4 (encoding a protein involved in ion homeostasis)48, while the ability to withstand oxidative stress is associated with the activity of antioxidative enzymes, specifically catalase (CAT) and glutathione S-transferase (GST)44. The synthesis of some metabolites in response to heat stress, such as trehalose and glycogen, also improved the thermotolerance and ethanol fermentability of S. cerevisiae37. The acquisition of thermotolerance and fermentation efficiency of S. cerevisiae are also associated with genes encoding the proteins involved in the ethanol production pathway, DNA repair, and oxidative stress85. Genes for ergosterol biosynthesis are required for tolerance to vanillin stress99, while several genes involved in ionic homeostasis, heat protection, trehalose synthesis, antioxidant defense, and ATP production have been shown to be involved in ethanol stress in S. cerevisiae100. A recent study by Rahman et al.101 demonstrated that the response of P. kudriavzevii toward acetic acid includes the activation of genes involved in the tricarboxylic acid cycle, ATP-binding cassette transporters, and protein folding, sorting and degradation. Thus, it could be concluded from this information that different organisms employ different mechanisms to deal with different stress conditions. Considering the molecular mechanism involved in multistress tolerance in P. kudriavzevii evolved strains, further studies such as transcriptomic, proteomic, and metabolomic analyses or whole-genome sequencing should be performed.Based on a literature review, the molecular mechanisms involved in multistress tolerance are complicated, involving several genes or proteins. For instance, three genes, P. kudriavzevii, namely, PkAC-7, PkAC-8, and PkAC-9, were obtained. Although their cell size and cell surface were slightly different, all of them exhibited multistress tolerance toward heat, ethanol, osmotic, and lignocellulosic inhibitors, including acetic acid, formic acid, furfural, 5-HMF, and vanillin, individually and in a mixture of inhibitor cocktails. Furthermore, the evolved strains, particularly strain PkAC-9, produced the highest ethanol concentration, productivity, and yield from undetoxified SBH. This study provides evidence that ALE using acetic acid as a selective pressure is an effective method to improve the multistress tolerance of P. kudriavzevii for industrial ethanol production using lignocellulosic biomass as a feedstock.Based on the repetitive long-term cultivation in acetic acid at 35 °C, three potential multiple-stress-tolerant strains of Supplementary Figures.


In this study, the importance of experimental parameters such as chiral selector type and concentration, sodium dodecyl sulfate (SDS) concentration, the ratio of methanol, and separation voltage in optimizing were investigated. The simultaneous enantioseparation of diniconazole, hexaconazole, and imazalil was successfully achieved in 30 mM borate buffer (pH 9.0) containing 10 mM HP-γ-CD and 20 mM SDS with methanol (8%) added as organic modifiers. The resolution of diniconazole, hexaconazole, and imazalil was 15.2, 2.12, and 2.78, respectively, and the peak efficiency (N) was over 566,825 plates/m. This study provides an alternative way to systematically separate chiral antifungal pesticides with high efficiency.Simultaneous enantioseparation of three commonly used chiral antifungal pesticides  was first studied based on micellar electrokinetic chromatography (MEKC) with hydroxypropyl- R-enantiomer of chiral pesticides showed great difference compared to S-enantiomers in biological activity, environmental behavior, and biological toxicity [S-enantiomer was higher than that of R-enantiomer for mosquito, armyworm, and mouse; however, the result was contrary to that of housefly [μgL−1 of hexaconazole, the (−)-enantiomer was found to accumulate more readily in zebrafish than the (+)-enantiomer, suggesting that hexaconazole has significant enantioselective bioaccumulation [Nowadays, developing green pesticides  has becotoxicity , for exahousefly . When exmulation . TherefoNumerous chromatographic methods have been used for enantiomeric separations of pesticides including high-performance liquid chromatography (HPLC) , 7, gas γ-CD and hydroxypropyl-β-cyclodextrin (HP-β-CD) [β-CD (S-β-CD). Besides, hexaconazole was separated using supercritical fluid chromatography (SFC) [Different kinds of chiral pesticides have been produced, including triazole fungicides, pyrethroid insecticides, and phenoxy herbicides. Diniconazole, hexaconazole, and imazalil are common fungicides used for crop sterilization . Many waHP-β-CD) , 16 and hy (SFC) . HoweverIn this study, we aimed to establish a simultaneous enantioseparation method of diniconazole, hexaconazole, and imazalil  by usingβ-CD, HP-β-CD, and 2-HP-γ-CD were purchased from Aladdin; C12H25SO4Na (SDS), Na2HPO4, Na2B4O7.10H2O, NaOH, H3PO4, and methanol were obtained from Sinopharm Chemical Reagent . All experimental water was purified by a Milli-Q apparatus . All samples and background solutions were filtered by a 0.22 μm nylon filter  and degassed by sonication.Diniconazole, hexaconazole, and imazalil were obtained from Aladdin ; 2B4O7.10H2O in ultrapure water to 100 mM. The running buffer was prepared by dissolving CD and SDS in the background buffer and adjusting the pH of butter with NaOH or H3PO4. All buffers were filtered through a 0.22 μm filter before use. Stocking solutions of signal chiral antifungal pesticides were prepared in methanol at a concentration of 2000 μg/mL and stored at 4°C. Working solutions were prepared by diluting the stocking solutions with a background buffer to a suitable concentration. All samples were filtered before sampling.Background electrolyte was prepared by dissolving Naμm microporous membrane, and 2 mL sterile syringe were used.Capillary electrophoresis (CE) with an ultraviolet detector (UV) , chromatography workstation , a fused-silica capillary column, ultrasonic cleaner, electronic scales, pH meter, 0.22 Preconditioning capillary: The capillary was flushed with 0.1 mol/L NaOH for 5 min followed by flushing with ultrapure water for 5 min and running buffer for 10 min before use. The capillary was flushed with 0.1 mol/L NaOH, ultrapure water, and running buffer for 3 min between injections to confirm the stability of the system and the reproducibility of results. At the end of the experiment, the capillary was rinsed for 3 min with ultrapure water to protect the column and UV detector.μm × 80 cm, 60 cm effective length; gravity injection 10 s at a height difference of 10 cm; voltage: +13 kV; buffer: 30 mM sodium borate, 20 mM SDS, 10 mM HP-γ-CD, and 8% methanol; wavelength: 220 nm; the analysis was performed at room temperature; sample concentrations: 0.1 mg/mL diniconazole, 0.05 mg/mL hexaconazole, and 0.05 mg/mL imazalil.The optimal analysis conditions were as follows: capillary: 50 α-CD, β-CD, and γ-CD. To expand the application range of CD, chemical modifications were introduced by using the derivatization of CD to enhance the enantioselectivity. Many modified CDs have been performed previously, for instance, hydroxypropyl-CD, hydroxymethyl-CD, and sulfobutyl-CD.CD, as an additive, is commonly used for chiral separation, especially for β-CD, HP-β-CD, and HP-γ-CD were studied in the 15 mM sodium borate containing 20 mM SDS with pH from 2 to 9, and all concentrations of CDs were 20 mM. Results showed that neither diniconazole nor hexaconazole could be enantiomerically separated from β-CD and HP-β-CD. In addition, β-CD was barely dissolved in phosphate buffer and turned into crystals after a night. The baseline was abnormal with high noise when HP-β-CD was used. We found that all samples were separated effectively by using HP-γ-CD, with constant baseline and low noise. Therefore, HP-γ-CD was used as a chiral additive for the following experiments.The separation abilities of γ-CD in the 15 mM sodium borate containing 20 mM SDS at pH 9. γ-CD concentration in the range from 5 mM to 10 mM and the chiral resolution was best at 10 mM HP-γ-CD, while the chiral resolution of samples was decreased with the HP-γ-CD concentration since the concentration was higher than 10 mM. The possible reason is that HP-γ-CD has no electric charges, so the moving speed is equal to μeo. The content of the sample in CD was increased with the increase of HP-γ-CD concentration; accordingly, the content of the sample in SDS was decreased. As a result, the sample's μapp was increased, so the retention time of the sample was shorter than before. The inclusion complexes formed by cyclodextrins and chiral substances were partitioned between the pseudostationary phase and aqueous solution. Eventually, the good enantioresolution of the three chiral antifungal pesticides was achieved at 10 mM HP-γ-CD, so 10 mM was the optimal concentration for HP-γ-CD.The concentration is an important parameter to control in enantiomeric separation. Migration times and resolution would be changed by the variation in the CD concentration, which impacts the enantiomeric separation. The chiral separation of all chiral antifungal pesticides was studied at 5, 10, 15, and 20 mM HP-γ-CD at pH 9 was also performed to obtain better enantiomeric separation of all chiral antifungal pesticides  to 50 μm ID with an effective length of 60 cm , and the separation voltage was decreased to 13 kV. The three pairs of chiral antifungal pesticides were ideally separated, and the resolution between two adjacent peaks was all more than 1.80. The chromatogram in optimal conditions can be seen in Not all enantiomers had been successfully separated yet after a series of optimization experiments, and the separation of hexaconazole II and diniconazole II remained the main problem. From the results of methanol optimization, the separation resolution increased with the reduction of methanol, so the ratio of methanol was reduced from 10% to 8%. Meanwhile, considering that the reduction of EOF might improve the resolution and a low capillary inside diameter (ID) could improve the peak profile and reduce the EOF and Joule heat, the capillary column was changed from 75 μm × 80 cm, 60 cm effective length; gravity injection: 10 s; voltage: +13 kV; buffer: 30 mM sodium borate, 20 mM SDS, 10 mM HP-γ-CD, 8% methanol; wavelength: 220 nm; at room temperature; sample concentration: 0.1 mg/mL diniconazole, 0.05 mg/mL hexaconazole, and 0.05 mg/mL imazalil. Meanwhile, the peak efficiency (N) and resolution peaks were analyzed based on the chromatogram. It can be seen from γ-CD-MEKC method for the simultaneous enantioseparation of diniconazole, hexaconazole, and imazalil had good efficiency (N ˃ 560000) and resolutions between two adjacent peaks. The resolutions of diniconazole, hexaconazole, and imazalil were 15.2, 2.12, and 2.78, respectively, which showed that the method met the requirement of enantiomeric separation for the three kinds of chiral antifungal pesticides.The influence of several experimental conditions including the type and concentration of chiral selector, the concentration of SDS, the ratio of methanol, the concentration of sodium borate, and the separation voltage was investigated. The optimal conditions were as follows: capillary column: 50 γ-CD as chiral selector, chiral separation for three kinds of chiral antifungal pesticides  was successfully achieved for the first time; the minimum chiral resolution for a pair of enantiomers was 2.12, and the maximum resolution was 15.2. This study provides an alternative way to systematically separate chiral antifungal pesticides with simplicity and practicability, high efficiency, and good resolution, and chiral columns and coat capillaries are not needed to be prepared. It can provide help for the study of enantiomers of diniconazole, hexaconazole, and imazalil in environmental behavior, biotoxicity, and so on. However, this method also has some shortcomings because the traditional capillary electrophoresis sample injection method has some disadvantages, which leads to the poor repeatability of the experimental method. It is expected to use quantitative capillary electrophoresis to improve repeatability. Meanwhile, the experimental method will be further verified to study the actual samples.Based on micellar electrokinetic chromatography (MEKC) including HP-