Effect of benzoic acid concentration on the growth of strain GJ2008
To determine the effect of benzoic acid on ethanol fermentation by S. cerevisiae, we first analyzed its effect on the growth of strain GJ2008. The growth curves of S. cerevisiae GJ2008 with the addition of different concentrations of benzoic acid are presented in Fig. 1. With increasing benzoic acid concentration (0–1.8 g/L), the degree of cell growth inhibition gradually increased.
Growth curves and growth rate of S. cerevisiae GJ2008 under different concentrations of benzoic acid.
As shown in Fig. 1A, the control group (without benzoic acid) entered the rapid growth phase at 2 h and reached the stable phase at 8 h. The time required to enter the rapid growth phase and stable phase markedly increased as the benzoic acid concentration increased to the range of 0.5–1.4 g/L. When the concentration of benzoic acid was greater than 1.6 g/L, the growth of GJ2008 was almost completely inhibited. According to Fig. 1B, the OD560 in the control group increased at a faster rate than that in the benzoic acid treatment group in the first 6 h, and the increase rate was close to zero in the control group after 8 h, indicating that the growth of the yeast cells had reached a stable stage. With increasing benzoic acid concentration, the maximum increase rate of OD560 was delayed. According to the results, 1.2 g/L benzoic acid was selected for further study of the effects of benzoic acid on the ethanol fermentation process, physicochemical parameters and transcriptome analysis of S. cerevisiae GJ2008.
Effect of benzoic acid on the ethanol fermentation process of strain GJ2008
To explore the effect of benzoic acid on ethanol fermentation by S. cerevisiae GJ2008, 250 g/L sucrose was used to simulate the fermentable sugar in sugarcane molasses for ethanol fermentation experiments.
In the process of ethanol fermentation by S. cerevisiae using sucrose, the intracellular sucrose hydrolase of yeast is secreted to the extracellular space, and sucrose is hydrolyzed to produce glucose and fructose, which are used by yeast cells to grow and produce ethanol. The changes in sucrose, fructose and glucose concentrations and the associated rates during ethanol fermentation using 250 g/L sucrose under 1.2 g/L benzoic acid stress are shown in Fig. 2.
Changes in sucrose, fructose and glucose concentrations (A) and the associated rates (B) during ethanol fermentation using 250 g/L sucrose under benzoic acid stress. CK, control group; BA, benzoic acid treatment group.
The results shows that the trends of sucrose hydrolysis, glucose consumption and fructose consumption were similar between the treatment group and the control group (Fig. 2). In the control group, sucrose hydrolysis and glucose consumption were basically complete at 21 h of fermentation, indicating that by this stage, a large amount of sucrose hydrolase had been released, and the cells could grow rapidly using glucose and fructose. In the benzoic acid treatment group, sucrose was completely hydrolyzed at 36 h of fermentation, and the utilization of glucose and fructose was lower than that in the control group. It is speculated that benzoic acid may affect the sucrose hydrolase activity and sugar utilization ability of yeast. In addition, the consumption of glucose is greater than that of fructose, which may be due to the different affinities of the hexose transmembrane transporter for glucose and fructose17. The above results indicate that the sucrose hydrolysis rate and sugar utilization rate were low under the stress of benzoic acid, which was likely due to incomplete ethanol fermentation using 250 g/L sucrose.
As shown in Fig. 3, in the control group, there was no obvious lag phase in cell growth, and the cells could rapidly use sugar to grow and reproduce and generate a large amount of ethanol (122.69 g/L). However, in the benzoic acid treatment group, the residual sugar concentration was still as high as 97.39 g/L, and the alcohol concentration was only 75.35 g/L at 36 h of fermentation. The residual sugar content increased by 91.24%, the ethanol content decreased by 38.59%, and the OD value (5.77) was much lower than that of the control group. In general, benzoic acid inhibited the growth of yeast cells and reduced the ability of cells to hydrolyze sucrose, utilize sugars and produce ethanol.
Biomass, total residual sugar and ethanol production curves for ethanol fermentation using 250 g/L sucrose under benzoic acid stress.
The total sugar fermentation efficiency is an important index for evaluating the effect of ethanol fermentation. When ethanol fermentation was carried out at the same sugar concentration (25%), a lower residual total sugar concentration and a higher ethanol concentration indicated a better fermentation effect. As shown in Table 1, compared with those of the control group, the final ethanol concentration, glucose utilization rate, fructose utilization rate, total sugar utilization rate, total sugar fermentation efficiency and sugar consumption fermentation efficiency of the benzoic acid treatment group decreased by 35.58%, 24.76%, 44.76%, 34.37%, 37.61% and 4.93%, respectively. The final ethanol concentration (75.35 g/L) and total sugar fermentation efficiency (55.15%) of the treatment group were lower, which was related to the high residual total sugar content (97.39 g/L) and low utilization rates for glucose and fructose (75.24% and 51.74%, respectively).
In summary, the exogenous addition of 1.2 g/L benzoic acid significantly inhibited the growth of yeast cells, reduced cell viability, and caused yeast cells to metabolize sugar to produce ethanol, resulting in a high residual sugar content, low ethanol production and low fermentation efficiency.
Effect of benzoic acid on cell morphology
Under high permeability stress, strong acid or alkali stress, and inhibitor stress, the cell membrane is destroyed first24. Therefore, the effect of benzoic acid on the morphology of S. cerevisiae cells was observed by SEM. Figure 4 shows that the cells in the control group were oval or round in shape and were full, smooth, flat, and compact, with many bud marks and no ruptures or pores. The shape of the yeast cells in the treatment group was irregular, the surface was uneven, and the number of buds was small. A large number of voids were observed, along with intercellular adhesion. In summary, on the one hand, benzoic acid destroyed the integrity of the cell membrane and cell wall of S. cerevisiae, causing intracellular leakage and intercellular adhesion; on the other hand, it affected the budding of yeast cells, thereby affecting the normal growth and reproduction of cells.
Scanning electron microscopy images of S. cerevisiae GJ2008 in the control group (CK) and benzoic acid treatment group(BA).Rred circles indicating the damage on the surface of the strain.
Effect of benzoic acid on membrane permeability
Nucleic acids and proteins are important biological macromolecules in cells that do not leak out of cells when the cell membrane permeability is normal25. The conjugated double bonds of purine and pyrimidine bases in nucleic acids result an absorbance peak at 260 nm. The tryptophan and phenylalanine residues in proteins contain benzene rings, which result in an absorbance peak at 280 nm. Therefore, the OD260 and OD280values of the fermentation supernatant determined by spectrophotometry can not only reflect the leakage of nucleic acids and proteins but also indirectly reflect the degree of cell membrane damage26,27. Figure 5 shows that after treatment with benzoic acid, the protein and nucleic acid release from yeast cells increased significantly (p < 0.05), and the longer the treatment time was, the greater the release. Compared with those in the control group, the amount of nucleic acid and protein released increased 1.80 and 2.06 times, respectively, after 3 h of benzoic acid treatment. After 6 h of treatment, the nucleic acid and protein contents increased 2.67- and 2.31-fold, respectively. The above results indicated that under benzoic acid stress, intracellular nucleic acids and proteins leaked out of the cell, and the permeability of the yeast cell membrane increased as a result of severe damage.
Changes in the membrane permeability of S. cerevisiae GJ2008 under benzoic acid stress. ns indicates that there was no significant difference between the benzoic acid treatment group and the control group; * and ** indicate p < 0.05 and p < 0.01, respectively.
Effect of benzoic acid on the MDA content in yeast cells
MDA is produced by polyunsaturated fatty acid peroxidation catalyzed by active oxygen28,29. The change in MDA content can indicate the degree of membrane lipid peroxidation. In addition, MDA can destroy protein structure and increase the toxic effect of reactive oxygen species, further changing the fatty acid composition of the cell membrane, reducing the fluidity of the cell membrane, increasing cell membrane permeability, and damaging cell membrane integrity30. MDA and TBA can produce reddish-brown trimethylamine under acidic heating conditions, and trimethylamine has an absorption peak at 532 nm. The MDA content in S. cerevisiae cells before and after benzoic acid treatment was measured and is shown in Fig. 6. The intracellular MDA content in yeast cells increased significantly after benzoic acid treatment (p < 0.05) and was positively correlated to the treatment time. The MDA content of the yeast cells increased 4.25- and 4.51-fold after benzoic acid stress for 3 h and 6 h, respectively, compared to that in the control group. The MDA content increased significantly at 3 h, indicating that benzoic acid is highly toxic to yeast cells and can cause severe oxidative damage in a short time. The above results showed that S. cerevisiae caused lipid peroxidation of the cell membrane under benzoic acid stress, and the MDA content increased significantly. This finding is consistent with the above results of SEM showing intracellular nucleic acid and protein leakage and changes in cell morphology.
Changes in the intracellular MDA content in S. cerevisiae GJ2008 under benzoic acid stress. ns indicates that there was no significant difference between the benzoic acid treatment group and the control group; *** indicates p < 0.001.
Effect of benzoic acid on the intracellular glycerol content
The intracellular glycerol content is an important indicator of yeast tolerance. Many studies have shown that yeast cells increase intracellular glycerol production to balance osmotic pressure and reduce cell damage under unfavorable growth conditions31,32. As shown in Fig. 7, there was no significant difference in the glycerol content in the control group within 12 h of benzoic acid treatment. In the treatment group, the intracellular glycerol content increased significantly with increasing treatment time (p < 0.05). At 6 h and 12 h, the intracellular glycerol content increased 1.48- and 1.50-fold, respectively, compared with that in the control group. These results indicated that benzoic acid stress induced glycerol synthesis and accumulation in cells to protect cells from damage.
Changes in the intracellular glycerol content in S. cerevisiae GJ2008 under benzoic acid stress. * indicate that there was a significant difference between the benzoic acid treatment group and the control group; * and ** indicate p < 0.05 and p < 0.01, respectively.
FTIR spectroscopy
FTIR spectroscopy was used to characterize the changes in the cell structure components of S. cerevisiae GJ2008 after 1.2 g/L benzoic acid treatment, and the results are shown in Fig. 8. The characteristic FTIR bands shown are basically consistent with previous research reports33,34,35. There was a strong and wide absorption peak between 3500 and 3100 cm−1, which are attributed to the deformation and vibration of O-H and N-H bonds in proteins, fatty acids, polysaccharides, chitin and other substances36. The absorption peaks at 3100 –2800 cm−1 represent lipid functional groups and are attributed to the antisymmetric stretching vibration of the -CH group of fatty acids in triacylglycerol37. The amide I, II and III bands near 1650 cm−1, 1542 cm−1 and 1242 cm−1 are the most important bands for protein characterization. The absorption peak of the amide I band is attributed to the stretching vibration of C = O and the bending vibration of N-H, which indicates that the protein structure in yeast cells consists mainly of α-helices. The absorption peak in the amide II zone is attributed to N-H bending vibrations and C-N stretching vibrations. The absorption peak at the amide III band is attributed to the bending vibration of the C-H bond, the stretching vibration of C-O and the deformation vibration of P = O in the carboxyl group, which indicates that it is related to the phospholipid bilayer and represents the asymmetric stretched phosphodiester bond. The characteristic peaks around 1454 cm−1 are attributed to the shear vibration of CH2, the asymmetric bending vibration of CH3 and the antisymmetry of the carboxylate group RCOO-. The characteristic peak at 1080 cm−1 is attributed to the vibration of the polysaccharide hydroxyl skeleton in the nucleic acid or cell wall. The absorption peak below 1000 cm−1 is the characteristic absorption peak of yeast36.
Changes in the Fourier transform infrared spectra of Saccharomyces cerevisiae GJ2008 under benzoic acid stress.
Changes in the structure or configuration of the compound leads to an energy level difference between the ground state and the excited state and to changes in the first excited state and the ground state, resulting in changes in the energy of photon absorption or emission, leading to a redshift or blueshift in the spectrum. As shown in Fig. 8, the absorption peaks of protein amide II, amide III, and polysaccharide hydroxyl groups at 1542, 1242, and 1080 cm−1 shifted to 1537, 1238, and 1070 cm−1 under benzoic acid stress, respectively, indicating that the vibration spectrum moved toward a low wavenumber and that a redshift occurred, the energy required for vibration decreased, and the groups became more unstable. The characteristic peak at 3355 cm−1 shifted to 3424.5 cm−1, and a blueshift occurred, indicating that benzoic acid affected the lipid composition of the yeast cell membrane. In summary, benzoic acid affects the structure of phospholipid fatty acids, proteins and the cell wall polysaccharide chitin on the cell membrane, increases the permeability of the cell membrane, causes the leakage of intracellular nucleic acids and proteins, and inhibits the growth and metabolism of cells. This result is consistent with the results of physical and chemical parameter measurements and similar to the conclusion that formic acid destroys proteins, lipids, and polysaccharides in yeast cells, as reported by Zeng et al.23 .
Transcriptome analysis
Quality assessment of RNA-seq and genome comparison results
Filtered data were obtained after a series of raw data processing steps. The sequence comparison of the filtered high-quality reads with the reference genome showed that the number of clean reads of each sample was above 46.79 M. After data quality control, the mean Q20 values of the control group and the benzoic acid treatment group were 98.44% and 98.40%, respectively, and the mean Q30 values were 95.00% and 94.91%, respectively, which are in line with the requirement that the Q20 and Q30 generally be greater than 85% and 80%, respectively. Assuming that there was no contamination in the experiment and that the reference genome annotation was complete, the total mapped area was generally greater than 65%. For the clean reads produced by Illumina sequencing, the total number of mapped reads was greater than 65% in both the control group and the benzoic acid-treated group. On average, 95.55% and 94.73% coverage of the reference genome was obtained in the control and treatment groups, respectively. The average proportions of uniquely aligned reads were 90.96% and 89.78%, and the average proportions of reads with multiple alignment positions were 4.60% and 4.95%. The sequencing data showed that the quality control data were well compared with the reference genome sequence; that is, the original sequencing data were reliable and could be used for subsequent analysis.
Differentially expressed gene screening
Differentially expressed genes (DEGs) were screened using |log2FC|≥1 and p-adjusted < 0.05 as the screening criteria, and the results are shown in Fig. S1. As shown in Fig. S1, the volcano plot directly shows the overall distribution of DEGs, and the number of significantly downregulated DEGs was greater than that of significantly upregulated DEGs. A total of 1670 DEGs were screened in the benzoic acid treatment group, of which 770 were upregulated and 900 were downregulated.
GO enrichment analysis
After the DEGs were identified, GO enrichment analysis was performed, as shown in Fig. S2. The GO database divides gene ontologies into three categories: biological process (BP), cell component (CC), and molecular function (MF). As shown in Fig. S2, the DEGs under benzoic acid stress were mainly enriched in the metabolism of purine nucleoside phosphate and purine ribonucleoside monophosphate. This was followed by β-fructofuranase activity, sucrose α-glucosidase activity, cytoplasmic large ribosome subunit, small ribosome subunit, cytoplasmic small ribosome subunit, rRNA ribosome binding, 90 S precursor, ribosome large subunit protein, fructose and mannose transmembrane transport activity, pyruvate decarboxylase activity, oxidoreductase, ribosome, riboprotein complex, ribose body biosynthesis, etc. The results showed that benzoic acid mainly affected nucleotide, ribosome and glucose metabolism in yeast.
KEGG pathway enrichment analysis
KEGG metabolic pathway enrichment analysis of the screened DEGs was helpful for understanding the stress metabolism pathways activated by cells under benzoic acid stress. Figure 9 and Fig. S3 show the KEGG pathway classification statistics and metabolic pathway enrichment maps under benzoic acid stress.
KEGG pathway classification of differentially expressed genes under benzoic acid stress.
As shown in Fig. 9, the metabolic processes of the DEGs under benzoic acid stress that were predominant in the KEGG classification included mainly carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, lipid metabolism, metabolism of other amino acids, energy metabolism, glycan biosynthesis and metabolism, and biosynthesis of other secondary metabolites. Second, in the processing of genetic information, on the main pathways were translation, transcription, folding, classification and degradation, replication and repair. In environmental information processing, membrane transport and signal transduction were the main pathways. In the cell process category, cell growth and death, transport and catabolism were the main pathways involved. In biological systems, cell aging was the main pathway.
In Fig. S3, the most frequently annotated DEGs in the KEGG enrichment pathway under benzoic acid stress were associated with ribosomes, followed by starch and sucrose metabolism; purine metabolism; RNA polymerase; galactose metabolism; neomycin, kanamycin and gentamicin biosynthesis; ribosomal biogenesis; arginine and proline metabolism; a carbon pool composed of folic acid; homologous recombination; pyrimidine metabolism; sugar metabolism; thiamine metabolism; valine, leucine and isoleucine biosynthesis; ABC transport; fructose and mannan metabolism; β-alanine metabolism; the Hippo signaling pathway; the biosynthesis of unsaturated fatty acids; and ascorbic acid and aldose metabolism.
Real-time fluorescence quantitative PCR validation
To verify the reliability of the transcriptomic results, the expression levels of 9 genes (3 upregulated and 6 downregulated genes) in the benzoic acid-treated group and the control group were measured by RT‒qPCR, as shown in Fig. 10. The results showed that the expression levels of HXT2 (involved in glycolytic metabolism), PDR15 (involved in plasma membrane ATP-binding transporters), HXK1 (encoding hexokinase), PDC6 (encoding pyruvate decarboxylase subtype), GAL2 (encoding galactose permease) and PDR18 (encoding putative transporters of the ATP-binding box (ABC) family) were downregulated. The expression levels of MNN11 (involved in cell wall synthesis regulation), OPT2 (encoding an oligopeptide transporter) and ERG11 (encoding sterol synthesis) were upregulated. The RT‒qPCR expression data were highly consistent with the transcriptome sequencing data, indicating that the transcriptome sequencing data were reliable.
RT‒qPCR results for some differentially expressed genes under benzoic acid stress.
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- Source: https://www.nature.com/articles/s41598-024-80484-1