Ultrasonic Lysis of Bioengineered Cells in Industrial Production

Bioengineered bacteria species such E. coli as well as genetically modified mammalian and plant cell types are used widely in biotech to express molecules. In order to release these synthesized bio-molecules, a reliable cell disruption technique is required. High-performance ultrasonication is a proven method for efficient and reliable cell lysis – easily scalable to large throughputs. Hielscher Ultrasonics offers you high-performance ultrasonic equipment for efficacious cell lysis in order to produce large volumes of high-quality bio-molecules.

Extraction of Molecules from Cell Factories

For the production of a wide range of biomolecules, various engineered microbes and plant cells can be used as microbial cell factories, including Escherichia coli, Bacillus subtilis, Pseudomonas putida, Streptomyces, Corynebacterium glutamicum, Lactococcus lacti, Cyanobacteria, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Nicotiana benthamiana and algae, among many others. These cell factories can produce proteins, lipids, biochemicals, polymers, biofuels and oleochemicals, which are used as food or raw material for industrial applications. Cells used as cell factories are cultured in closed bioreactors, where they can achieve high efficiency, specificity and low energy requirements.
To isolate the target molecules from the bioengineered cell cultures, the cells must be disrupted so that the intracellular material is released. Ultrasonic cell disrupters are well established as highly reliable and efficient technique for cell disintegration and compound release.

Microbial cell factories are metabolically engineered cells used for the synthesis of various valuable compounds. Ultrasonic cell disruption is an efficient and reliable method to release the valuable compounds from the cell interior.
study and graphic: ©Villaverde, 2010.

Advantages of Ultrasonic Cell Disruptors

As a non-thermal, mild, yet highly efficient technology, ultrasonic disruptors are used in lab and industry to lyse cells and to produce high-quality extracts, e.g. used for the isolation of molecules from cell factories.

Power-Ultrasound for Efficient Disruption of Microbial Cell Factories

Mechanism and Effects of Ultrasonic Cell Disruptors:
Ultrasonic cell disruption used the power of ultrasound waves. The ultrasonic homogenizer / cell disruptor is equipped with a probe (aka sonotrode) made from titanium alloy that oscillates at a high frequency of approx. 20 kHz. This means the ultrasonic probe couples 20,000 vibrations per second into the sonicated liquid. The ultrasound waves coupled into the liquid are characterized by alternating high-pressure / low-pressure cycles. During a low pressure cycle, the liquid expands and minute vacuum bubbles arise. These very small bubbles grow over several alternating pressure cycles until they cannot absorb any further energy. At this point, the cavitation bubbles implode violently and create locally an extraordinary energy-dense environment. This phenomenon is known as acoustic cavitation and is characterized by locally very high temperatures, very high pressures and shear forces. These shear stresses break efficiently cell walls and increase mass transfer between the cell interior and the surrounding solvent. As a purely mechanical technique, ultrasonically generated shear forces are widely used and the recommended procedure for bacterial cell disruption, as well as for protein isolation. As a simple and rapid cell disruption method, sonication is ideal for the isolation of small, medium and large sized volumes. Hielscher’s digital ultrasonicators are equipped with a clear menu of settings for precise sonication control. All sonication data are automatically stored on a built-in SD-card and are simply accessible. Sophisticated options of heat dissipation such as external cooling, sonication in puls mode etc. during the ultrasonic disintegration process ensure the maintenance of the ideal process temperature and thereby the intactness of extracted heat-sensitive compounds.

Research Underlines the Strengths of Ultrasonic Cell Disruption and Extraction

Prof. Chemat et al. (2017) resumes in their study that “ultrasound-assisted extraction is a green and economically viable alternative to conventional techniques for food and natural products. The main benefits are decrease of extraction and processing time, the amount of energy and solvents used, unit operations, and CO₂ emissions.”
Gabig-Ciminska et al. (2014) used a high-pressure homogenizer and an ultrasonic cell dsintegrator in their study for the lysis of spores in order to release DNA. Comparing both cell disruption methods, the research team concludes that regarding the cell lysis for spore DNA, “analysis has been done by employing cell lysates from the high pressure homogenization. Afterwards, we realized that an ultrasonic cell disruption has outstanding advantages for this purpose. It is rather fast and can be processed for small sample volumes.” (Gabig-Ciminska et al., 2014)

Biomolecules from Cell Factories for Food Production

Microbial cell factories are a viable and efficient production methodology using microbial organisms to produce high yields of native and non-native metabolites by metabolic bio-engineering of microbial microorganisms such as bacteria, yeasts, fungi etc. Bulk enzymes are for instance produced using microorganisms auch as Aspergillus oryzae, fungi, and bacteria. Those bulk enzymes are used for food and beverage production, as well as in agriculture, bioenergy and household care.
Certain bacteria such as Acetobacter xylinum and Gluconacetobacter xylinus produce cellulose during fermentation process, where nanofibers are synthesized in a bottom-up process. Bacterial cellulose (also known as microbial cellulose) is chemically equivalent to plant cellulose, but it has high degree of crystallinity and high purity (free of lignin, hemicellulose, pectin, and other biogenic components) as well as a unique structure of cellulose nanofiber-weaved three-dimensional (3D) reticulated network. (cf. Zhong, 2020) In comparison to plant-derived cellulose, bacterial cellulose is more sustainable and the cellulose produced is pure not requiring complex purification steps. Ultrasonication and solvent extraction using NaOH or SDS (sodium dodecyl sulfate) are very effective for the isolation of bacterial cellulose from the bacterial cells.

Biomolecules from Cell Factories for Pharmaceutical and Vaccine Production

One of the most prominent pharmaceutical products derived from cell factories is human insulin. For bioengineered insulin production, predominantly E. coli and Saccharomyces cerevisiae are used. Since bio-synthesized nano-sized molecules offer a high biocompatibility, biological nanoparticles such as ferritin are advantageous for numerous biomanufacturing applications. Additionally, the production in metabolically engineered microbes is often significantly more efficacious in the obtained yields. For instance, the production of artemisinic acid, resveratrol and lycopene has increased tenfold to several hundredfold, and is already established or is in development to industrial scale production. (cf. Liu et al.; Microb. Cell Fact. 2017)
For instance, protein-based nano-sized biomolecules with self-assembling properties such as ferritin and virus-like particles are especially interesting for vaccine development as they mimic both the size and structure of pathogens and are amenable to surface conjugation of antigens to promote the interaction with immune cells. Such molecules are expressed in so-called cell factories (e.g., engineered E. coli strains), which produce a certain target molecule.

Protocol for Ultrasonic Lysis and of E. coli BL21 for Ferritin Release

Ferritin is a protein, which primary function is the storage of iron. Ferritin shows promising capabilities as self-assembling nanoparticles in vaccines, where it is used as vaccine delivery vehicle (e.g. SARS-Cov-2 spike proteins). The scientific research of Sun et. al. (2016) shows that recombinant ferritin can be released as a soluble form from Escherichia coli at low NaCl concentrations (≤50 mmol/L). In order to express ferritin in E. coli BL21 and to release the ferrtin, the following protocol was successfully applied. The recombinant pET-28a/ferritin plasmid was transformed into the E coli BL21 (DE3) strain. The ferritin E coli BL21 (DE3) cells were cultured in LB growth media with 0.5% kanamycin at 37°C and induced at an OD600 of 0.6 with 0.4% isopropyl-β-D-thiogalactopyranoside for 3 hours at 37°C. The final culture was then harvested by centrifugation at 8000g for 10 minutes at 4°C, and the pellet was collected. Then, the pellet was resuspended in LB medium (1% NaCl, 1% Typone, 0.5% yeast extract)/lysis buffer (20 mmol/L Tris, 50 mmol/L NaCl, 1 mmol/L EDTA, pH 7.6) and different concentrations of NaCl solution (0, 50, 100, 170, and 300 mmol/L), respectively. For bacterial cell lysis, sonication was applied in pulse mode: e.g, using the ultrasonicator UP400St at 100% amplitude with a duty cycle of 5 seconds ON, 10 seconds OFF, for 40 cycles) and then centrifuged at 10 000g for 15 minutes at 4°C. The supernatant and precipitate were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All sodium dodecyl sulfate-stained gels were scanned with high-resolution scanner. Gel images were analyzed using Magic Chemi 1D software. For optimal clarity, protein bands were detected by adjusting parameters. The data for the bands were generated from technical triplicates. (cf. Sun et al., 2016)

Ultrasonic Cell Disruptors for Industrial Lysis of Cell Factories

Ultrasonic lysis and extraction is a reliable and comfortable method to release metabolites from cell factories thereby assisting an efficacious production of target molecules. Ultrasonic cell disruptors are available from lab to industrial size and processes can be scaled completely linear.
Hielscher Ultrasonics is your competent partner for high-performance ultrasonic disruptors and has long-time experience in the field of implanting ultrasonic systems in bench-top and industrial settings.
When it comes to sophisticated hardware and software, Hielscher Ultrasonics cell disruption systems fulfils all requirements for optimum process control, easy operation and user-friendliness. Customers and user of Hielscher ultrasonicators value the benefit that Hielscher ultrasonic cell disruptors and extractors allow for the precise process monitoring and control – via digital touch-display and browser remote control. All important sonication data (e.g. net energy, total energy, amplitude, duration, temperature, pressure) are automatically stored as CSV file on an integrated SD-card. This helps to obtain reproducible and repeatable results and facilitates process standardization as well the fulfilment of Good Manufacturing Practices (cGMP).
Of course, Hielscher ultrasonic processors are built for 24/7 operation under full load and can be therefore reliably operated in industrial production settings. Due to high robustness and low maintenance, the downtime of the ultrasonic equipment is really low. CIP (clean-in-place) and SIP (sterilize-in-place) features minimize laborious cleaning, especially since all wet-parts are smooth metal surfaces (no hidden orifices or nozzles).

The table below gives you an indication of the approximate processing capacity of our ultrasonicators: