BETATECH INNOVATION CENTRE

Natural Hop Extract Boosts Performance

Compared to virginiamycin-based antibiotic in a recent study, the industry’s top natural antimicrobial alternative showed a clear overall benefit. By Michail Karavolos

The search for clean and sustainable energy sources has led to numerous industrial biotechnology developments, with ethanol production utilizing various feedstocks as the science has progressed. Three key factors are considered to determine the efficiency of ethanol production: enzymatic lysis into simple sugar molecules, alcohol fermentation rate, and ethanol tolerance of yeast cells.1

Regardless of the method or technology selected, a common problem is microbial contamination. The degree of contamination is a burden that can significantly affect ethanol production. Therefore, it is crucial to develop cost-effective processes with higher antimicrobial efficiency that meet both alcohol and coproduct standards.

For years, it has been common practice to use antibiotics to combat infections in biofuel production. Continued and repetitive use has also increased concerns about antibiotic-resistant strains. Alternative solutions, such as natural hop extracts, constitute a viable option to antibiotics enabling the industry to sell certified antibiotic-free DDGS to local and international markets where antibiotics are either banned or discouraged.

Screening for Variation

This study compared a natural hop extract to an industrial virginiamycin-based antibiotic from a different perspective: the role in yeast health and fitness, measured by the ability of yeast to grow faster (growth rate). During industrial fermentation, the yeast continuously interacts with its environment and senses and responds to various external stimuli. During ethanol fermentation, yeast fitness is affected by several factors that are crucial for its overall performance. Could industrial antimicrobials used during ethanol fermentation cause stress for the yeast? To investigate this, we examined the effect of a natural antimicrobial hop extract (150 ppm) in comparison to a leading brand of virginiamycin-based industrial antibiotic (2 ppm) on a large number of yeast strains (6,000 S. cerevisiae gene-disruption variants), covering a large landscape of potential variation in nature.

Each individual gene variant’s growth rate was scored in rich media containing specific antimicrobial compounds in a high-density array (ROTOR HDA Robot and the PhenoBooth, Singer Instruments Ltd.), using colony size as a proxy for fitness (Figure 1). The study and data analysis was conducted in collaboration with Professor Daniela Delneri at the Manchester Institute for Biotechnology, University of Manchester, UK. The data presented herein focuses on the effects of a virginiamycinbased industrial antibiotic compared to the natural hop extract on several gene categories contributing to yeast fitness.

FIGURE 1: 384-well plates combined with robotic colony printing and detection to assess the fitness of S. cerevisiae variants in the presence of either natural hop extracts or virginiamycin-based industrial antibiotic.

SOURCE: BETATEC INNOVATION CENTRE

Clear Benefits Observed

Our screening focused on the effect of either a natural hop extract or a virginiamycin-based industrial antibiotic on yeast gene variants involved in ethanol production and resistance (ETH), cell replication (REP), and metabolism (MET) pathways. Typically, an increase of more than 30% in colony size between the experiment and the untreated control is considered promising. The overall beneficial impact of hop extract over the antibiotics on yeast growth is evident, including the impact on specific ETH, REP, and MET categories (Figure 2).

Upon screening the fitness of 6,000 S. cerevisiae gene-disruption variants, we observed a clear overall benefit of 21% when using an antimicrobial based on

FIGURE 2: A total growth enhancement benefit of 21% is further examined per category as shown in green boxes. ETH (gene variants involved in ethanol production and resistance), REP (gene variants involved in yeast replication), MET (gene variants involved in general yeast metabolism).

SOURCE: BETATEC INNOVATION CENTRE

natural hop extracts versus a virginiamycin based industrial antibiotic (Figure 2). The majority of the benefit is evident in the ETH (8%) and REP (11%) categories (Figure 2). Our data indicated that natural hop extracts used in industrial yeast fermentation increases the opportunity for a favorable yeast growth environment and may result in faster-replicating yeast cells and improved ethanol yields.

Author: Michail Karavolos Laboratory and R&D Manager BetaTec Innovation Centre michail.karavolos@betatec.com

About: Karavolos manages the BetaTec Innovation Centre’s laboratory, overseeing research and development. He holds a Ph.D. in microbiology from the University of Leeds and has extensive academic and commercial experience in antimicrobial applications.

1. Vamvakas SS, Kapolos J. 2020. doi:10.1007/s11274-020-02881-8