Bacillus subtilis is the most widely studied gram-positive bacterium in the world. It is found in soil and it produces endospores, which are small dormant bacteria cells that can withstand stressful conditions like extreme heat or lack of nutrients. B. subtilis is rod-shaped, relatively safe and easy to work with, and reproduces quickly. This makes it a wonderful model organism for microbiologists to study.
Figure 1 Rod-shaped Bacillus subtilis. Source: Science Photo Library
So, how can a common soil bacterium like B. subtilis be a potential solution for climate change like the title of this post suggests? Climate change is defined by persistent changes to weather patterns, primarily caused by human activity like the burning of fossil fuels. Bacillus subtilis is a particularly special bacteria because it has the ability to produce biofilms. Biofilms are colonies of bacteria held together by extracellular polysaccharide (EPS). These slimy conglomerates of microbes may soon become a game changer in creating renewable energy sources that can help us move away from relying on fossil fuels. They have three-dimensional structures and a variety of functions ranging from defense against antibiotics, cell-cell communication, and genetic transfer. B. subtilis bacteria can produce biofilms in a variety of different environments. This includes at the liquid air interface, called pellicle formation, and completely submerged under water.
Figure 2 Biofilm formation. Source: AHV USA
Bacillus subtilis biofilms, also, have the unique ability to degrade organic matter like sugars. This capacity of biofilm is the most promising when it comes to environmental sustainability. One particular study by Deng and Wang in 2022 looked at the ability of Bacillus subtilis biofilm to degrade cellulose, a sugar that is the main component in the cell walls of plants. This study was carried out by genetically altering B. subtilis biofilm and removing two of its major components: extracellular polysaccharide (EPS) and TasA protein. Extracellular polysaccharides, as previously stated, are sugar polymers secreted by bacteria. They hold together microbes during biofilm formation. TasA is a protein that resides in the extracellular matrix of biofilm. It is the primary protein that forms amyloid fibers, which hold cells together in biofilm. TasA protein is also needed for flagellar genes to be expressed. Therefore, TasA protein not only assists in the formation of biofilm by forming amyloid fibers, but it also allows biofilm to become mobile by expressing genes that encode flagella.
By deleting the genes and/or gene operons that encode EPS and TasA protein, two vital elements of B. subtilis biofilm, biofilm formation can no longer occur. It was hypothesized that if either or both EPS and TasA genes were deleted, then biofilm would not form and, ultimately, less cellulose would be degraded.
Figure 3 Bacillus subtilis biofilm. Source: Harvard Gazette
First, Deng and Wang looked at the amount of biofilm formation when the genes encoding TasA protein and EPS were removed from B. subtilis. As expected, the wildtype, unaltered B. subtilis produced significantly more biofilm than the double EPS and TasA gene knockout. Unaltered B. subtilis also produced more biofilm than single knockout EPS and single knockout TasA strains, but results were less significant. After confirming that TasA and EPS are indeed essential in biofilm formation for B. subtilis bacteria, the next step was to examine whether or not biofilm could soak up and hold onto cellulase. Cellulase is the enzyme that degrades cellulose. If biofilms are efficient in soaking up and holding onto cellulase, then they will be better at degrading cellulose.
Results showed that the wildtype strain of biofilm soaked up the more exogenous cellulase and retained more self-made cellulase than the double EPS and TasA knockout biofilms could. These results are important because they suggest that more biofilm formation leads to more retention of the sugar degrading enzyme cellulase. This assumption is made because the wildtype B. subtilis strain produces the most biofilm and captures and retains the most cellulase. Whereas, the double knockout strain produces the least biofilm and captures and retains the least cellulase.
The final, and perhaps most important, experiment that Deng and Wang did was examining how efficient the various alterations of B. subtilis biofilm were at being able to degrade the sugar cellulose. The results are shown in Figure 4. On the X axis of this image you can see the four B. subtilis biofilm strains labeled accordingly. The first bar is labeled Wt(pBC), which stands for wild type strain of B. subtilis biofilm that carries a plasmid called pBC. A plasmid is a piece of DNA that is small and round. The pBC plasmid, in particular, is known to over-express the enzyme cellulase. Researchers inserted the pBC plasmid into the wildtype B. subtilis biofilm and all subsequent variants after determining the wildtype strains they were studying did not self-produce very much cellulase. The next two bars on the figure are labeled ΔtasA(pBC) and Δeps(pBC), this represents the biofilm with the TasA protein and EPS removed respectively. Both, again, carry the pBC plasmid that over expresses cellulase. The final bar in Figure 4 is labeled Double(pBC), this is the strain of biofilm with both TasA and EPS removed. In image A of Fig. 4, the Y axis is labeled cellulose degraded (mg/disc), whereas in image B the Y axis is labeled cellulose degraded (mg/million cells), this distinction just signifies that image B has been normalized for cell density.
Figure 4 Effect of missing biofilm components on cellulose degradation
The takeaways from this figure and the findings from Deng and Wang are that the Wt(pBC) strain does indeed degrade the most cellulose. In both images A and B, the wild type B. subtilis degrades significantly more than the double knockout and TasA knockout strain. These findings also further solidify the importance of cellulases in biofilm as a part of the degradation of cellulose.
Deng and Wang concluded from their experiments that B. subtilis biofilm forms compounds with cellulase and cellulose called bacteria-cellulose-cellulase complexes that increase the efficiency of cellulose degradation. The findings from this paper further confirm that EPS and TasA protein are important for biofilm formation, cellulase capture, and cellulose degradation. This allows for future research to be done that genetically engineers biofilm to become even more efficient at degrading cellulose. For example, inserting the cellulase-making plasmid pBC or overexpressing genes that code for TasA and EPS.
Now, we must circle back to the title of this blog post for a final time. What does this all have to do with environmental sustainability? Cellulose is a major energy source that has been notoriously hard for scientists to break down. Break down is complicated due to the sugar’s crystalline structure. Cellulose degradation is also costly and time consuming. However, when successful, breaking down cellulose is a way to create biofuels, which is energy produced by renewable biological sources. Biofuels are a more sustainable alternative to fossil fuels like petroleum and coal. If scientists like Deng and Wang can genetically engineer Bacillus subtilis biofilm to efficiently degrade cellulose in a cost effective manner, then this bacteria could revolutionize the way we use renewable energy.
Figure 5 Renewable energy and biofuel production cycle
Cellulose biofuels have a number of environmental benefits. Cellulose is one of the most abundant sources of biomass on the planet and it produces less greenhouse gas emission compared to traditional fossil fuels. Biofuels made from cellulose also reduce land competition because they do not tend to interfere with farming of crops for food, medicinal purposes etc. This is because there are crops that produce cellulose that can grow on land typically unsuitable for vegetation.
There is a considerable way to go before scientists, engineers, and policymakers make significant progress when it comes to cellulose breakdown and biofuel production. However, the bacteria B. subtilis and its ability to produce cellulose degrading biofilm provides a new and exciting avenue to explore.
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