Background

Fermentation technology is widely used to produce a variety of economically important compounds that have been used in the energy production, pharmaceutical, chemical and food industries. Although fermentation processes are handed down from generation to generation, there is a challenging need for sustainable production of products that meet market demand in an economical and efficient manner. At present, there are some problems in the production process of fermentation industry, such as extensive production, poor product quality, high consumption of resources and energy, and serious environmental pollution.

Fig. 1. Workflow from proof-of-principle strain development to an optimized conversion system for industrial scale-up. (Wehrs, et al., 2019)

In view of this, the industry continues to improve the development and optimization of fermentation processes at many levels. For example:

• Develop and optimize medium components to support optimal growth and product synthesis of microorganisms or cells;
• Screening and improvement of high-yielding fermentation strains by mutagenesis, genetic engineering or other biotechnological means;
• Scale-up fermentation processes while maintaining or improving yield and efficiency;
• Design and optimize the configuration of bioreactors to meet different fermentation needs;
• Develop and implement process control system to realize real-time monitoring and automatic control of fermentation process;
• By modifying the metabolic pathway of microorganisms, the synthesis efficiency of target products is improved and the generation of by-products is reduced.
• Optimize product recovery and purification processes after fermentation to reduce costs and increase product purity;
• Ensure that fermentation products comply with relevant industry standards and regulations, and establish a strict quality control system.

Creative BioMart Microbe centered on the key technology of the fermentation process optimization and control, in order to obtain high yield, high substrate conversion rate and high production strength relatively unified as the goal, from optimization of microbial genes, regulating intracellular microenvironment and optimize the macro environment, the development of the comprehensive consideration biology, kinetics and physics phenomenon of the fermentation process optimization technology to meet the market demand and research needs.

Service Procedure

Platforms

Services Details

Creative BioMart Microbe's fermentation production laboratory mainly carry out metabolite production, fermentation, development and optimization services through the following points (including but not limited to other conventional techniques relating to fermentation):

• Identification/design of fermentation strains to match industrial fermentation strains or relevant fermentation strains for vaccine production
  Use genetic engineering, metabolic engineering or synthetic biology methods to modify existing industrial fermentation strains, or to screen microorganisms with production potential from nature through high-throughput screening techniques.
• Systematic method and optimization of fermentation medium design
  Methods such as Statistical Design of Experiments (DoE) are used to optimize the medium composition, including determining the nutrients required for microbial growth and product synthesis, such as carbon sources, nitrogen sources, vitamins, and trace elements, to maximize microbial growth rates and product yield.
• Analysis and optimization of fermentation conditions
  Explore the effects of various conditions such as pH, temperature, dissolved oxygen level, stirring speed, etc. on microbial growth and product synthesis during the fermentation process, adjust these conditions to optimize the fermentation process.
• Nutritional control of metabolites
  The metabolic pathway of microorganisms is controlled by regulating the supply of nutrients, thereby guiding the microorganisms to synthesize more of the required metabolites. In some cases, the addition of key precursor substances of metabolic pathways can increase the production of specific metabolites.
• Optimization of fermentation kinetics
  Study the dynamic models of microbial growth and product synthesis, and use these models to optimize the fermentation process, such as adjusting the substrate flow strategy or changing the batch, continuous or semi-continuous mode of fermentation, so as to improve the efficiency of microbial conversion of substrates, shorten the production cycle, and increase the concentration and yield of products.
• Expansion of culture stage
  This usually involves the adjustment of bioreactor design and the development of scaling up strategies. Maintaining consistency between microbial growth and product synthesis during scale-up is a challenge, requiring consideration of factors such as oxygen transfer, heat removal, and mixing efficiency.
• High-throughput experimental designs
  With the integration of the high-throughput bioreactor system, we can help researchers to swiftly carry out intricate experiments for the purpose of discovering novel protocols or refining current methods. The system features 12 individual tanks, each furnished with a suite of sensors including pH, dissolved oxygen, biomass, and offgas sensors, which are essential for meticulously capturing and managing all facets of the fermentation process.

Our Advantages

• Full Staffing

• Have researchers from the field of fermentation engineering and microbiology, who can develop and optimize the whole fermentation process
• Have operators engaged in fermentation industry for many years, familiar with the operation of fermentation tank and various analytical equipment
• Have a professional QC team, able to complete APQP/PPAP and other documents and technical documentation

• High Quality Fermentation Equipment

• Large scale fermenter, pilot scale fermenter and experimental fermenters
• Hollow fiber filtration system
• Frozen centrifuge

• A Variety of Expression Systems

We have a variety of expression systems, including E. coli expression system, Bacillus subtilis expression system, Pichia pastoris expression system, Saccharomyces cerevisiae expression system, etc.

Case Study

Case Study 1: The yield of the bacterial non-ribosomal peptide indigoidine hinges on the respiratory metabolic status within S. cerevisiae.

Non-ribosomal peptide synthetases (NRPS) constitute a significant group of enzymes that facilitate the synthesis of diverse secondary metabolites. Through genetic modification, Saccharomyces cerevisiae was tailored to produce a bacterial NRPS, which converts glutamine into indigoidine—a notable non-ribosomal peptide (NRP) esteemed for its intrinsic value as a sustainable pigment. Indigoidine synthesis exclusively occurs during the respiratory phase of cell expansion. Researchers have successfully scaled up production to a 2-liter bioreactor by fostering respiratory conditions through meticulous nutrient administration, achieving a peak concentration of 980 mg/L.

Fig. 2. Regulated environment in 2 L bioreactor enables control over metabolic state. (Wehrs, et al., 2018)

Case Study 2: The potential to link physiological alterations observed under regulated cultivation environments with substantial shifts in the genetic makeup of yeast barcoded variants is explored.

In this research, specific deletion mutants were consistently identified as enriched across all tested cultivation processes, irrespective of the conditions applied. Moreover, pronounced fluctuations in genetic diversity during fed-batch processes indicate the experience of considerable stress. For the yeast deletion strain collection, the choice of feeding strategy, which influences the buildup of the fermentation byproduct ethanol, has a more pronounced effect on the diversity of the mutant strains than the pH level of the culture medium. The loss of certain mutants during periods of extreme population selection implies that particular biological mechanisms may be necessary to endure such specific stressors.

Fig. 3. Beta-diversity of mutant populations in different generalized feeding regimes over time. (Wehrs, et al., 2020)

Case Study 3: Genome-scale metabolic rewiring improves titers, rates and yields of the non-native product indigoidine at scale.

High titer, rate, yield (TRY), and scalability are challenging metrics. To achieve these metrics, researchers take the minimal cut set (MCS) approach. They compute MCS solution-sets for a non-native product indigoidine. From the 63 solution-sets, the omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. Researchers implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. The system achieves 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield. These phenotypes are maintained from batch to fed-batch mode, and across scales.

Fig. 4. Analysis of indigoidine yield across cultivation formats for both glucose-fed and galactose-fed strains. (Banerjee, et al., 2020)

FAQs

References:

1. Wehrs M.; et al. Engineering Robust Production Microbes for Large-Scale Cultivation. Trends Microbiol. 2019;27(6):524-537.
2. Wehrs M.; et al. Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb Cell Fact. 2018;17(1):193.
3. Wehrs M.; et al. Investigation of Bar-seq as a method to study population dynamics of Saccharomyces cerevisiae deletion library during bioreactor cultivation. Microb Cell Fact. 2020;19(1):167.
4. Banerjee D.; et al. Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale. Nat Commun. 2020;11(1):5385.