The rise of renewable energy has unsurprisingly coincided with a greater focus on renewable energy research within the scientific community. Among the many researched, the fuel ethanol industry has taken off in recent years, and is only going to increase. But an urgent problem facing the industry is meeting the increasing demands, while dealing with detrimental losses of ethanol due to contamination. This contamination is predominantly caused by lactic acid bacteria. The lactic acid bacteria contamination significantly reduces ethanol yields in the fermentation process. To remedy this problem, the industry typically uses antibiotic treatments, which do their job well enough, but are costly and aren’t a permanent solution. In the paper “Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations”, the authors hypothesized that lysins produced by bacteriophages have the ability to effectively degrade structural elements in lactic acid bacteria, causing exolysis in these cells. Lysins, also known as endolysins, are enzymes which play a critical role in the lysis portion of the lytic cycle. These enzymes degrade bonds in peptidoglycan, which makes up bacterial cell walls. The next step in the experimental process for the authors was to test their hypothesis. The lysins tested underwent a thorough process of expression and purification, zymography, and turbidity reduction assaying, all before being tested with a mock fermentation setup. Prior to the purification, the bacterial strains tested were grown in their respective optimal conditions to prepare them for use in this experiment. Next, the lysins went through Escherichia coli expression. This process synthesized the lysins used within the experiment. To purify the synthesized lysins, the authors used affinity chromatography, integrating the use of nickel-NTA Superflow resin. Affinity chromatography is a process used to separate biochemical substances by taking advantage of their specific biochemical properties. The nickel affinity chromatography used in the experiment is associated with histidine tags on proteins. After performing the affinity chromatography, gel electrophoresis was used to examine the purity of the samples. The next step taken was to perform gel zymography, which detects enzymes with their respective substrates. Following this, the authors created turbidity reductions assays and measured wavelength absorption to determine the amount of turbidity reduction. Finally, the authors were finally able to organize a mock fermentation environment to test their hypothesis. The authors acquired corn fiber and put the fiber in optimal conditions for fermentation, adjusting the pH to 5.5. They then inoculated the fermentation environment with strains of lactic acid bacteria that most commonly cause issues within the ethanol creation process. Seven bacteriophage strains were used to test against the lactic acid bacteria. Then, throughout the
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The turbidity reduction assay results showed that the phage LambdaSa2, derived from streptococcal bacteria, demonstrated effective lytic activity in 17 out of 22 of the bacteria strains tested. LambdaSa2 was able to maintain its effectiveness when tested under the typical conditions of fermentation (pH 5.5 and less than or equal to 5% ethanol concentration), however, LambdaSa2 when tested with Lactobacillus fermentum 0605-B44 and Lactobacillus fermentum BR0315-1 showed optimal specific activity at pHs 6.5 and 7.5, as shown in Figure 3. Along with this, LambdaSa2 when tested against Lactobacillus brevis 0605-48 had very little, and in some cases even none, from 2% concentration of ethanol and upwards. Other bacteriophages tested with turbidity reduction assays, including LysA, LysA2, and LysgaY, showed similar, yet not quite as successful results. Exolytic activity was observed in 60% of the lactic acid bacteria that were tested with the aforementioned bacteriophages. Yet, these bacteriophages all demonstrated optimal specific activity at pH 5.5. Also, LysA and LysgaY both demonstrated maximum activity at 5% ethanol concentration when tested against Lactobacillus brevis 0605-48, while demonstrating an inverse effect with the two L. fermentum strains in Figure 3. These bacteriophages also were able to prove successful when put under conditions similar to fermentation. LysA2, when tested on strains of bacteria that were not lactic acid bacteria, did not demonstrate any lytic activity. Along the same lines, no lysins had successful results when tested against Escherichia coli DH5alpha and Saccharomyces cervisiae Y-2034. The authors used pH and ethanol concentration as variables because within a real fermentation environment, all these factors are slightly variable. That necessitates testing the effectiveness of lysins under a variety of
The turbidity reduction assay results showed that the phage LambdaSa2, derived from streptococcal bacteria, demonstrated effective lytic activity in 17 out of 22 of the bacteria strains tested. LambdaSa2 was able to maintain its effectiveness when tested under the typical conditions of fermentation (pH 5.5 and less than or equal to 5% ethanol concentration), however, LambdaSa2 when tested with Lactobacillus fermentum 0605-B44 and Lactobacillus fermentum BR0315-1 showed optimal specific activity at pHs 6.5 and 7.5, as shown in Figure 3. Along with this, LambdaSa2 when tested against Lactobacillus brevis 0605-48 had very little, and in some cases even none, from 2% concentration of ethanol and upwards. Other bacteriophages tested with turbidity reduction assays, including LysA, LysA2, and LysgaY, showed similar, yet not quite as successful results. Exolytic activity was observed in 60% of the lactic acid bacteria that were tested with the aforementioned bacteriophages. Yet, these bacteriophages all demonstrated optimal specific activity at pH 5.5. Also, LysA and LysgaY both demonstrated maximum activity at 5% ethanol concentration when tested against Lactobacillus brevis 0605-48, while demonstrating an inverse effect with the two L. fermentum strains in Figure 3. These bacteriophages also were able to prove successful when put under conditions similar to fermentation. LysA2, when tested on strains of bacteria that were not lactic acid bacteria, did not demonstrate any lytic activity. Along the same lines, no lysins had successful results when tested against Escherichia coli DH5alpha and Saccharomyces cervisiae Y-2034. The authors used pH and ethanol concentration as variables because within a real fermentation environment, all these factors are slightly variable. That necessitates testing the effectiveness of lysins under a variety of