Background

In most industrial fermentations, biological or eukaryotic cells are immersed in a liquid medium. Scaling up is an important factor in industrial fermentation, which is an important process for converting laboratory procedures to industry. Scale up process are studies carried out at the laboratory or even pilot plant scale fermenters to yield data that could be used to extrapolate and build the large scale industrial fermenters with sufficient confidence it will function properly with all its behaviors anticipated.

It is important to note that before scaling up, the feasibility of the production process needs to be demonstrated and the stability of the selected strains ensured, which is crucial for cell banks and large-scale fermentation. Other methods such as:

• Optimizing codon use of genes and screening high-expression clones by shaking bottle experiments;
• Systematic optimization of culture conditions, including medium, temperature, pH, dissolved oxygen level, etc., which affect microbial metabolic pathways and target protein expression;
• Select the right fermentation mode (feed batch or continuous culture) to achieve high cell density and high yield;
• Temperature has an important effect on protein solubility and productivity, while dissolved oxygen levels generally limit microbial growth at high growth rates;
• By reducing the scale to simulate the production conditions, gradually scale up to the industrial scale, while considering the mixing efficiency, mass transfer coefficient and shear force and other factors to determine the scale up strategy.
• Data-driven modeling based on machine learning (ML) helps to quickly select biological models and determine strains, extracellular conditions, and medium conditions.

Fig. 1. Schematic of constraint-based modeling (CBM) methods. (Du, et al., 2022)

Creative BioMart Microbe's fermentation scale-up laboratory and optimization laboratory make joint efforts to solve the key technology and problems 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 scale-up laboratory mainly carry out metabolite scale up studies services through optimization the following points:

• Inoculum development

The quality and density of inoculants directly affect the initiation of fermentation and the efficiency of the entire production process, and the number of inoculants needs to be expanded by laboratory-scale shaker culture to meet large-scale fermentation.

• Establishing the correct sterilization cycle at larger loads

When scaling up the load, the sterilization time, temperature and pressure need to be adjusted to ensure that the medium and equipment are completely sterilized without destroying the nutrient content.

• Optimization of environment parameters

Parameters including nutrient availability, pH, temperature, dissolved oxygen and dissolved carbon dioxide have a significant impact on the microbial metabolic activity and the synthesis of target products, and these parameters must be maintained within an optimal range through real-time monitoring and control systems.

• Simulation of the production environment

Use computer simulations and scale-down models to test different operating conditions and parameters.

• Evaluation of amplification effects

Amplification effects refer to the various effects that can occur in small scale to large scale production processes, such as changes in mixing, mass transfer and heat transfer. The effects of these effects on microbial growth and product synthesis need to be assessed and necessary adjustments made.

• Optimization of process parameters

Process parameters such as inoculation amount, culture time, ventilation and stirring speed need to be optimized according to production objectives.

• Selection and adjustment of equipment

Select suitable fermenters and related equipment such as stirrers, ventilation systems and temperature control systems.

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 abide by the FDA terms and conditions and other documents and technical documentation
• Have a timely technical support and customer service team, can real-time technical communication with customers, tracking services

• 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.

• Many Years of Experience in Scale-Up

Creative BioMart Microbe has many years of experience in fermentation process scale-up services and can meet the needs of different customers.

Case Study

Case Study 1: Selecting, identifying, and profiling molds for rice wine fermentation.

The superior strain for rice wine brewing was identified through the use of traditional Qu rice wine. Notably, strains YM-8, YM-10, and YM-16 stood out for their robust saccharification and fermentation capabilities, along with their contribution to an appealing flavor profile. Among them, YM-16 showed the highest enzymatic activities with α-amylase at 220.23±1.88 U/g, glucoamylase at 1,269.04±30.32 U/g, and protease at 175.16±1.81 U/g. When these three strains were fermented in a 20-L bioreactor, the resulting rice wine had a higher amino acid content than the control, except for arginine, which was notably lower. The total amino acid levels were in the order of YM-16 surpassing YM-8, YM-10, and the control group. Strain YM-16, with its potent enzymatic profile and enhancement of rice wine's taste, proved to be an ideal candidate for commercial rice wine production.

Fig. 2. Free amino acid content of rice wine. (Yuan, et al., 2024)

Case Study 2: Utilizing Corynebacterium glutamicum for the bioconversion of UF-SSL in a simplified medium.

This research devised a bioprocessing approach to harness ultra-filtered spent sulfite liquor (UF-SSL) as a carbon source in a minimal medium, bypassing initial detoxification. The strategy integrated a biomass quantification method tailored for matrices with significant water-insoluble solids, validated through elemental analysis. Kinetic insights were derived from a mechanistic model grounded in Monod kinetics to ascertain the process dynamics.

Fig. 3. Normalized concentration changes of biomass (top), glucose (middle) and mannose (bottom). (Waldschitz, et al., 2024)

Case Study 3: Large-scale fermentation of recombinant beta-mannanase by E. coli BL21 in a microaerobic condition.

The production of beta-mannanase was scaled up efficiently from shaker cultures to a 5-L fermenter. A cost-effective minimal medium (M9+e), devoid of vitamins, was identified as optimal for cell cultivation. This medium maintained stable pH levels during beta-mannanase production in both shake flasks and fermenters. Furthermore, E. coli cells produced comparable dry cell weight and quantities of recombinant beta-mannanase under both microaerobic and aerobic conditions. The medium demonstrated consistent pH stability throughout the protein production process. In a one-liter culture, 2.0314 g of E. coli dry cell weight resulted in 1.8 g of purified recombinant beta-mannanase.

Fig. 4. Optimization of different factors to get the maximum cell biomass of recombinant beta-mannanase producing BL21(DE3) cells in shake flask. (Purohit, et al., 2024)

FAQs

References:

1. Du YH.; et al. Optimization and Scale-Up of Fermentation Processes Driven by Models. Bioengineering (Basel). 2022;9(9):473.
2. Yuan H.; et al. Screening, identification, and characterization of molds for brewing rice wine: Scale-up production in a bioreactor. PLoS One. 2024;19(7):e0300213.
3. Waldschitz D.; et al. Robust, fully quantifiable and scalable bioprocess utilizing spent sulfite liquor with Corynebacterium glutamicum. Bioresour Technol. 2024;406:130967.
4. Purohit A.; et al. Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment. Carbohydr Res. 2024;541:109150.