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Lactiplantibacillus pentosus CECT4023T |
20 g L-1 xylose
32 °C
pH 7.0 |
Batch mode |
Shaking:
150 rpm
Cycles: NS
Time between passages: 24 h
Generations: 850 |
Lactic fermentation tests in bioreactor
LA yield
Xylose consumption |
The evolved strain showed 2-fold more production of LA and 1.5-fold more consumption of xylose compared with the original strain. |
Cubas-Cano et al., 2019Cubas-Cano, E., González-Fernández, C., & Tomás-Pejó, E. (2019). Evolutionary engineering of Lactobacillus pentosus improves lactic acid productivity from xylose-rich media at low pH. Bioresource Technology, 288, 121540. PMid:31174085. http://dx.doi.org/10.1016/j.biortech.2019.121540
http://dx.doi.org/10.1016/j.biortech.201...
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Lacticaseibacillus paracasei
4564 |
100 g L-1 sugar beet molasses
pH 6.5
41 °C |
Batch mode (first phase)
Fed-batch mode (second phase)
and pulsed fed-batch mode (third phase) |
Shaking: 100 rpm
Cycles: NS
Time between passages: 20-24 h
Generations: NS |
Antioxidant activity
DPPH Free Radical-Scavenging Activity
LA-production capability |
The evolved strain showed: one-fold higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging rates compared with the original strain.
The LA production
(89.4 g L-1) of the evolved strain was superior to the original.
The evolved strain showed a 59% higher capability to produce LA. |
Mladenović et al., 2019Mladenović, D., Pejin, J., Kocić-Tanackov, S., Djukić-Vuković, A., & Mojović, L. (2019). Enhanced lactic acid production by adaptive evolution of Lactobacillus paracasei on agro-industrial substrate. Applied Biochemistry and Biotechnology, 187(3), 753-769. PMid:30058018. http://dx.doi.org/10.1007/s12010-018-2852-x
http://dx.doi.org/10.1007/s12010-018-285...
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|
Lacticaseibacillus casei
|
37 °C
pH 5.5, 5.0, 4.6, and 4.3
75 days |
Batch mode |
Shaking: NS
Cycles: NS
Time between passages: NS
Generations: NS |
Cell counts
Intracelular pH
Intracelular amino acid determination
Lactate and acetate analyses
Membrane permeability |
The evolved strain showed a 318-fold higher survival rate than the original strain and exhibited a slightly higher production of lactate (13.6%) and 65.6% higher amount of acetate.
The intracellular pH of the evolved strain was higher with lower inner membrane permeability.
The survival rate of the evolved strain was increase in 1.36-, 2.10-, or 3.42-fold by the addition of 50 mM aspartate, arginine, and both, respectively. |
Zhang et al., 2012Zhang, J., Wu, C., Du, G., & Chen, J. (2012). Enhanced acid tolerance in Lactobacillus casei by adaptive evolution and compared stress response during acid stress. Biotechnology and Bioprocess Engineering, 17(2), 283-289. http://dx.doi.org/10.1007/s12257-011-0346-6
http://dx.doi.org/10.1007/s12257-011-034...
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Lacticaseibacillus casei
Zhang |
Amoxicillin (0.5 μg/mL) and Gentamicin
(1 μg/mL) |
- |
Shaking: NS
Cycles: NS
Time between passages: 24 h
Generations: 1200 |
Evaluation of fitness as represented by minimum inhibitory concentrations (MICs)
Identification of new mutations |
The MIC for amoxicillin increased to a maximum level (8 μg/mL) for the evolved strains.
There was a 4-fold increase in mutation accumulation (including SNVs, short insertions and deletions) in the evolved strains comparing to the control. |
Wang et al., 2017Wang, J., Dong, X., Shao, Y., Guo, H., Pan, L., Hui, W., Kwok, L. Y., Zhang, H., & Zhang, W. (2017). Genome adaptive evolution of Lactobacillus casei under long-term antibiotic selection pressures. BMC Genomics, 18(1), 320. PMid:28438179. http://dx.doi.org/10.1186/s12864-017-3710-x
http://dx.doi.org/10.1186/s12864-017-371...
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|
Lacticaseibacillus casei
|
37 °C
pH 5.5, 5.0, 4.6 and 4.3
70 days |
Batch mode |
Shaking: NS
Cycles: NS
Time between passages: 12 h
Generations: 50 |
Cell counts
Bile tolerance
Intracelular pH |
ALE resulted in 1.6-fold higher biomass in evolved strain and higher resistance to bile salt.
The evolved strain showed the capability of maintaining a higher pH compared to that of the original strain. |
Ming et al., 2016Ming, H., Xu, D., Guo, Z., & Liu, Y. (2016). Adaptive evolution of Lactobacillus casei under acidic conditions enhances multiple-stress tolerance. Food Science and Technology Research, 22(3), 331-336. http://dx.doi.org/10.3136/fstr.22.331
http://dx.doi.org/10.3136/fstr.22.331...
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Lactobacillus delbrueickii
Uc-3 (NCIM 5219) |
pH 4.5 |
Fed-batch mode |
Shaking: NS
Cycles: 40
Time between passages: 48 h
Generations: NS |
LA concentration, productivity, and yield
H+-ATPase activity determination
Analysis of H+-ATPase gene expression by one-step quantitative real-time PCR |
The evolved strain demonstrated a 1.80-fold increase in LA production, but its viability was reduced due to the acidic pH and/or end-product inhibition
There were higher H+- ATPase activity and a 415-fold increase in H+-ATPase gene expression compared to the original strain. |
Singhvi et al., 2018Singhvi, M., Zendo, T., & Sonomoto, K. (2018). Free lactic acid production under acidic conditions by lactic acid bacteria strains: Challenges and future prospects. Applied Microbiology and Biotechnology, 102(14), 5911-5924. PMid:29804138. http://dx.doi.org/10.1007/s00253-018-9092-4
http://dx.doi.org/10.1007/s00253-018-909...
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Lacticaseibacillus rhamnosus
|
−30 °C
13 °C
37 °C |
Batch mode |
Shaking: NS
Cycles: 150
Time between passages: 16 h
Generations: NS |
Viability
Growth Dynamics
Whole Genome Resequencing |
The evolved strain viability increased up to 93.65%.
The exponential growth rates were faster, with a 13-min shorter mean doubling time than the original strain.
Six gene regions and an intergenic space, related with encoding exopolysaccharide biosynthesis protein that could be responsible for the strain adaptation were discovered. |
Kwon et al., 2018Kwon, Y. W., Bae, J. H., Kim, S. A., & Han, N. S. (2018). Development of freeze-thaw tolerant Lactobacillus rhamnosus GG by Adaptive Laboratory Evolution. Frontiers in Microbiology, 9, 2781. PMid:30524399. http://dx.doi.org/10.3389/fmicb.2018.02781
http://dx.doi.org/10.3389/fmicb.2018.027...
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Lactobacillus delbrueckii subsp. bulgaricus ET45
|
37, 40 and
45 °C |
Batch mode |
Shaking: NS
Cycles: NS
Time between passages: NS
Generations: NS |
Enzymatic hydrolysis
Production of D-LA by SHF |
The enzymatic hydrolysis of the evolved strain showed 8% higher cellulose to D-LA conversion.
There was an increase in the LA production by the evolved strain (108 g/L D-LA) and 60% conversion of cellulose to D-LA. |
Prasad et al., 2020Prasad, J. V., Sahoo, T. K., Naveen, S., & Jayaraman, G. (2020). Evolutionary engineering of Lactobacillus bulgaricus reduces enzyme usage and enhances conversion of lignocellulosics to D-lactic acid by simultaneous saccharification and fermentation. Biotechnology for Biofuels, 13(1), 171. PMid:33088341. http://dx.doi.org/10.1186/s13068-020-01812-x
http://dx.doi.org/10.1186/s13068-020-018...
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|
Lactobacillus delbrueckii subsp. bulgaricus
|
52 °C |
Batch mode |
Shaking: NS
Cycles: NS
Time between passages: 48 h
Generations: NS |
Morphology
Lactic acid production
pH and viscosity
Amino acids
Volatile compounds |
Changes were observed in the morphology of the evolved strain after ALE (changed from rod to filament (52 °C) to cocci after frozen storage).
The evolved strain did not show LA production. The pH remains 6.84 ± 0.13.
There was a 115- and 275-fold higher production of Arginine and Methionine (237.24 ± 5.94 and 98.83 ± 1.78 µg/100 g, respectively) by the evolved strain.
The evolved strain showed an increase in diacetyl formation in milk. |
Liang et al., 2021Liang, J., Yoo, M. J. Y., Seale, B., & Grazioli, G. (2021). Nutritional and volatile characterisation of milk inoculated with thermo-tolerant Lactobacillus bulgaricus through Adaptive Laboratory Evolution. Foods, 10(12), 2944. PMid:34945497. http://dx.doi.org/10.3390/foods10122944
http://dx.doi.org/10.3390/foods10122944...
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Lactococcus lactis TM29 |
38, 39 and
40 °C |
Batch mode |
Shaking: NS
Cycles: NS
Time between passages: NS
Generations: 860 |
Growth rate
Physiological and transcriptomic characterization
Next-Generation Sequencing |
The evolved strain showed a 33% faster growth rate compared to the original strain and a 12% higher specific lactate production.
Changes in the expression of single proteins (chaperone; riboflavin transporter) were observed. Two had pleiotropic effects (RNA polymerase) in the evolved strain after ALE.
Deletion in 10 genes was also found to affect thermal tolerance significantly. |
Chen et al., 2015Chen, J., Shen, J., Hellgren, L. I., Jensen, P. R., & Solem, C. (2015). Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate. Scientific Reports, 5(1), 14199. PMid:26388459. http://dx.doi.org/10.1038/srep14199
http://dx.doi.org/10.1038/srep14199...
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|
Lactococcus lactis
|
10 AU/mL of Lcn972
30 °C for 8 transfers |
- |
Shaking: NS
Cycles: NS
Time between passages: 16 h Generations: 850 |
Milk Acidification and Production of Lactic Acid
Minimal Inhibitory Concentration
Nisin Production
Resistance to Antimicrobials |
The evolved strain was capable to acidify the milk (pH below 5.3).
ALE increased the MIC of Lcn972, between 4- and
32-fold.
Nisin production was not compromised by ALE.
Genes were involved in stress response, detoxification modules, cell envelope biogenesis and/or nucleotide metabolism. |
López-González et al., 2018bLópez-González, M. J., Escobedo, S., Rodríguez, A., Neves, A. R., Janzen, T., & Martínez, B. (2018b). Adaptive evolution of industrial Lactococcus lactis under cell envelope stress provides phenotypic diversity. Frontiers in Microbiology, 9, 2654. PMid:30455679. http://dx.doi.org/10.3389/fmicb.2018.02654
http://dx.doi.org/10.3389/fmicb.2018.026...
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Lc. lactic ssp. cremoris MG1363 |
5 mM hydrogen peroxide
30 °C |
Batch mode |
Shaking: 200 rpm
Cycles: NS
Time between passages: 72 h
Generations: NS |
Determination of Bacterial Growth and Survival
Acidification Test
Vitamin K2 Extraction and Analysis
Genome Sequencing
Proteomics Analysis |
The evolved strain showed a higher survival (dropped to 107 CFU/mL at 48 h from the initial 109 CFU/mL but further decreased to 104 CFU/mL at 72 h).
ALE improved the production of vitamin K2 (50-110% increase) by the evolved strain.
Common mutations in ldh and gapb genes were found in the evolved strain.
Proteomics analysis showed that 16 proteins were differently expressed by the evolved strain: glyceraldehyde 3-phosphate dehydrogenase (A2RIN9), universal stress protein A2 (A2RK64) and form amidopyrimidine-DNA glycosylase. |
Liu et al., 2021Liu, Y., de Groot, A., Boeren, S., Abee, T., & Smid, E. J. (2021). Lactococcus lactis mutants obtained from Laboratory Evolution showed elevated vitamin K2 content and enhanced resistance to oxidative stress. Frontiers in Microbiology, 12, 746770. PMid:34721346. http://dx.doi.org/10.3389/fmicb.2021.746770
http://dx.doi.org/10.3389/fmicb.2021.746...
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