Solvent tolerance in bacteria

Shreemoyee Mitra, Amity University Kolkata

Role of Solvent tolerance in biofuel production and its trend in the species-

Microorganisms have been considered as a boon that shapes all biological sciences. Microorganisms form an integral part of our everyday lives as they are not only present around us but also within us, some also involved in the functioning of various bodily functions.  They are extensively used for various purposes across multiple industries ranging from food to detergent to pharmaceutical and many more. The idea that not every microbe lives to cause disease was established after years of research and better understanding the story of the otherwise invisible microbial kingdom. Only then were we able to identify their kinds and distinguish ones that were harmful from others that were harmless and useful. These useful microbes are often the ones that are used for a variety of the aforementioned purposes. 

One such use that has revolutionized the fuel industry was brought about by the production of biofuels using microbes. In today’s world, where the use of fuel-energy has taken the eco-friendlier route, the creation of more efficient, sustainable, clean energy fuels has become necessary. This is where biofuels come into play. Biofuels are a substitute for fossil fuels, which are produced using industrial biotechnological techniques. However, commercial production of biofuels is still in its growing stage due to the occurrence of various obstacles. One of the most concerning of which is poor tolerance in microorganisms for their solvents (i.e., alcohols, like isobutanol, which can be used as a biofuel). This leads to adverse outcomes like insufficient yield. Scientists, by engineering solvent tolerance, have found that this property is strain-specific and every microorganism has a tolerance level of its own, which is determined genetically or is influenced by its environment.

Enhancing solvent tolerance in microorganisms-

Omics studies have revealed lists of genes, proteins, and intracellular compounds that are involved in the onset of responses to solvent stresses. To understand how these compounds contribute to solvent stress responses, scientists grew cells in the presence and absence of stressors (conditions that induce stress) thereby helping in the determination of the role of specific genes in solvent tolerance in terms of specific alcohols (solvent). The genes involved in reducing alcohol toxicity once detected and determined through wet lab experiments can be used to design typical solvent-tolerant bacterial strains. Amino acids like isoleucine, valine, glycine, glutamate, and trehalose were some of the essential metabolites that were identified to have caused protection against solvent stress in E. coli against the application of ethanol, n-butanol, and isobutanol as the solvent stressors.

Use of Lactococcus lactis strain for increasing isobutanol tolerance-

Isobutanol is a biofuel that inherently depicts better efficacy in comparison to ethanol due to several advantageous properties that is unique to it. Therefore, to increase its production, scientists had to ensure that the solvent tolerance for isobutanol in microorganisms like bacteria was desirably high. The authors selected the lactic acid bacteria (LAB)–  L. lactis NZ9000 strain for this purpose. It like other LABs also finds its use in the commercial production of important products for human use. One of the main reasons for choosing this strain was due to the availability of both the independently functioning catabolic and anabolic pathways, thereby unrelating product formation from the bacterias’ growth. This allows the bacteria to continue the product formation even when there is a complete lack of suitable growth conditions. Moreover, it depicts appreciable levels of tolerance to multiple stressful conditions. 

They conducted ALE (Adaptive Laboratory Evolution), which is a process that helps achieve directed evolution. E. coli cells with enhanced succinate production were obtained efficiently by allowing the evolution to take place in a continuous stirred tank reactor (CSTR) which is desirable here as it allows the cells to grow for a large number of generations while maintaining a specific growth rate.

The results achieved can be listed below as follows:-

1. When the bacteria depicted tolerance up to a particular concentration of isobutanol in the feed with a stable biomass quantity, the isobutanol concentration was thereafter increased in minuscule amounts to induce improved resistance. After reaching a particular limit, the biomass declined steeply with complete inhibition of growth. The feed was stopped to allow potential subcultures of tolerant phenotypes to multiply and grow, thereby attaining bacteria that were completely resistant to solvent stress. This entire procedure was repeated cumulative to attain a hyper-tolerant phenotype.
2. A 163-bp product, specific to L. lactis subsp. cremoris was identified to be present in both the native and evolved NZ9000 strains, confirming that the strain growing in CSTR was indeed L.lactis.
3. Cells show higher alcohol tolerance when supplemented with amino acids.
4. Surface changes in the cell wall or membrane structure takes place while combating solvent tolerance.
5. An evident increase in cell size was determined through SEM (Scanning Electron Microscopy).
6. Transcriptomics revealed a total of 578 genes to have differentially expressed in comparison to the native strain, even though mutations were lesser in number. Further analysis established the fact that multiple genes were responsible for tolerance.
7. The solvent tolerance was not gene(s) specific but was owing to the whole reprogramming of the cell.

However, just like everything has pros and cons, similarly scientists put forth some of the potential problems that might arise from developing such high tolerant strains, which can be stated as follows:

1. It is difficult for antibiotics to show their effects on these hyper-tolerant strains, as they can tolerate very high levels of toxicity, whether it be antibiotics or solvents like isobutanol.
2. The aforementioned point further makes genetic manipulation more challenging and complex.
3. Accumulation of mutations that are not related to stress tolerance might adversely affect the bacteria in other aspects. 
4. There is a wide range of changes in the transcriptomic landscape of the bacteria which might affect it adversely.

Thus, this study explores the solvent stress property of bacteria, and how it can be an important aspect for selecting candidates in biofuel production. Thus, we can say that microorganisms act as vehicles for biofuel production besides other major uses. 

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Reference:

Gupta J. A., Thapa S., Verma M., Som R., & Mukherjee K. J. (2020). Genomics and transcriptomics analysis reveals the mechanism of isobutanol tolerance of a laboratory evolved Lactococcus lactis strain. Scientific Reports, 10(1), 10850. https://doi.org/10.1038/s41598-020-67635-w 

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