Bacteriophages as antimicrobial agents against bacterial contaminants in yeast fermentation processes

Abstract

Background: The emergence of biofuels produced through yeast fermentation represents an important avenue for sustainable energy production. Despite all its advantages, this process is vulnerable to contamination by other organisms - most commonly lactic acid bacteria - that are present in raw feedstocks and/or in production lines. These contaminants compete with the yeast for nutrients, reducing the overall biofuel yield, and release substances that inhibit yeast growth. Here, we investigated the application of bacteriophages as potential antibacterial agents in yeast fermentation.

Results: Experiments conducted to understand the impact of pH on yeast, bacterial, and phage development showed that the yeast Saccharomyces cerevisiae Superstart™ grew in a similar fashion at pH levels ranging from 3 to 6. Growth of Lactobacillus plantarum ATCC® 8014™ was inhibited by pH levels of less than 4, and phages ATCC® 8014-B1™ (phage B1) and ATCC® 8014-B2™ (phage B2) displayed different infectivities within the pH range tested (pH 3.5 to 7). Phage B1 showed the best infectivity at pH 6, while phage B2 was most virulent at pH levels ranging from 4 to 5, and the cocktail of these phages showed highest infectivity in the range from pH 4 to 6. Population dynamics studies in MRS medium at pH 6 showed that, in the presence of bacteria inoculated at 10(7) cells/ml, yeast cultures were impeded under aerobic and anaerobic conditions, showing major decreases in both cell yield and ethanol production. The addition of the phage cocktail at a low initial multiplicity of infection was sufficient to reduce contamination by over 99%, and to allow yeast and ethanol yields to reach values equivalent to those of axenic cultures.

Conclusions: From the results observed, phages are good candidates as antimicrobial agents, to be used in place of or in conjunction with antibiotics, in yeast fermentation processes. Their implementation with other common contamination abatement/prevention methods could further increase their efficacy.

Keywords: Antibacterial; Bacteriophage; Biofuel; Contamination abatement; Ethanol; Lactic acid bacteria; Yeast.

Figure 1

Growth of saccharomyces cerevisiae under aerobic conditions. Experiments conducted in M9 medium at initial pH levels ranging from pH 3 to pH 6. Values are averages of duplicate experiments.

Figure 2

Growth of lactobacillus plantarum under aerobic conditions. Experiments conducted in M9 medium at initial pH levels ranging from pH 3.5 to pH 7. Error bars indicate the standard deviation of three experiments.

Figure 3

Growth of lactobacillus plantarum infected by phages under aerobic conditions. (a) Phage B1, (b) phage B2 and (c) cocktail of phages B1 and B2. Experiments were conducted at an initial multiplicity of infection (MOI) of 0.1 in M9 medium at initial pH levels ranging from pH 3.5 to pH 7.

Figure 4

Quantification of phage infectivity. Infectivity was determined for M9 medium at initial pH levels ranging from pH 3.5 to pH 7. (a) Phage B1, (b) phage B2, and (c) cocktail of phages B1 and B2. Infectivity was calculated as the difference in the integrals of the curves in Figures 3 and 2.

Figure 5

Growth of yeast, bacteria, phage B1, and phage B2 under aerobic conditions. Experiments conducted in MRS medium at pH 6. Counts of (a) yeast cells, (b) yeast buds, and (c) bacterial cells, measured using microscopy and a counting chamber, are presented. Data are reported for cases where yeasts were grown alone (Δ); in the presence of bacteria (O); and in the presence of bacteria and phages B1 and B2 (X). Values are averages of duplicates.

Figure 6

Final cell counts for cultures of yeast, bacteria, phage B1, and phage B2 growing under aerobic conditions. Experiments conducted in MRS medium at pH 6. Counts of (a) yeast and (b) bacterial cells, measured using microscopy and a counting chamber, are presented. Data are reported for cases where yeasts were grown alone (I); in the presence of bacteria at low inoculation level (II); in the presence of bacteria at high inoculation level (III); in the presence of bacteria at low inoculation level and phages B1 and B2 (IV); and in the presence of bacteria at high inoculation level and phages B1 and B2 (V). Measurements were taken at 20 hours of fermentation. Error bars indicate the standard deviation of three experiments.

Figure 7

Sample pictures for the population dynamics experiment presented in Figure 6 . Data are reported for cases where yeasts were grown alone (Δ); in the presence of bacteria (O); and in the presence of bacteria and phages B1 and B2 (×). Examples of yeast cells, yeast buds, and bacterial cells are indicated in the pictures. All samples were diluted fivefold.

Figure 8

Final cell counts for cultures of yeast, bacteria, phage B1, and phage B2 growing under anaerobic conditions. Experiments conducted in MRS medium at pH 6. Counts of (a) yeast and (b) bacterial cells, measured using microscopy and a counting chamber, are presented. Data are reported for cases where yeasts were grown alone (I); in the presence of bacteria at high inoculation level (II); and in the presence of bacteria at high inoculation level and phages B1 and B2 (III). Measurements were taken after 65 hours of fermentation. Error bars indicate the standard deviation of three experiments.

Figure 9

Ethanol production. Experiments conducted in MRS medium at pH 6 under (a) aerobic and (b) anaerobic conditions. Data are reported for cases where yeasts were grown alone (I); in the presence of bacteria at high inoculation level (II); and in the presence of bacteria at high inoculation level and phages B1 and B2 (III). Measurements for aerobic conditions were taken at 24 hours and for anaerobic conditions at 65 hours of fermentation. *Value was below the detection limit of 0.1% ethanol. Error bars indicate the standard deviation of three experiments.