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.
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.
- High efficient
- Non-thermal, ideal for temperature sensitive substances
- Reliable, repeatable results
- Precise processing control
- Linear scalable to larger throughputs
- Available for industrial production capacities
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:
|Batch Volume||Flow Rate||Recommended Devices|
|1 to 500mL||10 to 200mL/min||UP100H|
|10 to 2000mL||20 to 400mL/min||UP200Ht, UP400St|
|0.1 to 20L||0.2 to 4L/min||UIP2000hdT|
|10 to 100L||2 to 10L/min||UIP4000hdT|
|n.a.||10 to 100L/min||UIP16000|
|n.a.||larger||cluster of UIP16000|
Contact Us! / Ask Us!
Literature / References
- Sun, W., Jiao, C., Xiao, Y., Wang, L., Yu, C., Liu, J., Yu, Y., Wang, L. (2016):Salt-Dependent Aggregation and Assembly of E Coli-Expressed Ferritin. Dose-Response, March 2016.
- Rodrigues, M.Q.; Alves, P.M.; Roldão, A. (2021): Functionalizing Ferritin Nanoparticles for Vaccine Development. Pharmaceutics 2021, 13, 1621.
- Farid Chemat, Natacha Rombaut, Anne-Gaëlle Sicaire, Alice Meullemiestre, Anne-Sylvie Fabiano-Tixier, Maryline Abert-Vian (2017): Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. Ultrasonics Sonochemistry, Volume 34, 2017. 540-560.
- Villaverde, Antonio (2010): Nanotechnology, bionanotechnology and microbial cell factories. Microbial Cell Factories 2010 9:53.
Facts Worth Knowing
Ultrasound is used on the one hand to disrupt cells in order to release intracellular compounds, but applied with milder amplitudes and / or as pulsating ultrasound bursts, sonication can greatly enhance metabolic productivity of microbial, plant and animal cells in bioreactors thereby boosting biotechnology processes. Ultrasonic probes can be simply integrated in bioreactors (so-called sono-bioreactors) in order to intensify the efficiency of live biocatalysts. Hielscher ultrasonicators allow for precisely controlled ultrasound conditions, which can be optimally fine-tuned to high catalytic conversion of living cells. Learn more about Hielscher ultrasonic probes for sonobioreactors and the effects of ultrasonically-enhanced biocatalysis!
Cell Factories and the Synthesis of Metabolites
Different microorganisms can synthesize similar metabolites, for instance, for the production of amino acids Corynebacterium, Brevibacterium, and Escherichia coli have been successfully used; vitamins hae been synthesized using Propionibacterium and Pseudomonas; organic acids are derived from Aspergillus, Lactobacillus, Rhizopus; whilst enzymes can be made by Aspergillus and Bacillus; antibiotics can be produced by Streptomyces and Penicillium; whilst for the production of biosurfactants commonly formed Pseudomonas, Bacillus, and Lactobacillus are used as cell factories.
E. Coli as Microbial Cell Factories
The bacteria E. coli and its numerous strains are widely used molecular biology ans has become as one of the first efficient cell models used asmicrobial cell factories for the production of recombinant proteins, biofuels, and various other chemicals. E. coli features a natural ability to produce several compounds, which has been improved by bio-engineering and genetic modifications. For instance, by transferring heterologous enzymes, the capability of E.coli to produce numerous products has been modified in order to develop new biosynthetic pathways.
(Antonio Valle, Jorge Bolívar: Chapter 8 – Escherichia coli, the workhorse cell factory for the production of chemicals. In: Editor(s): Vijai Singh, Microbial Cell Factories Engineering for Production of Biomolecules, Academic Press, 2021. 115-137.)
Streptomyces as Microbial Cell Factories
Streptomyces is the largest group of actinomycetes; Streptomyces species are widespread in aquatic and terrestrial ecosystems. Members of Streptomyces genus are of commercial interest because of their capacity to produce a tremendous number of biomolecules and bioactive secondary metabolites. It produces clinically useful antibiotics such as tetracyclines, aminoglycosides, macrolides, chloramphenicol, and rifamycins. In addition to antibiotics, Streptomyces also produce other highly valuable pharmaceutical products including anticancer, immunostimulatory, immunosuppressive, antioxidative agents, insecticides, and antiparasitic drugs, which have broad medical and agricultural applications.
Streptomyces species produce a range of enzymes that is medically important, including L-asparaginase, uricase, and cholesterol oxidase. Many actinomycetes can produce industrially important enzymes as cellulases, chitinases, chitosanases, α-amylase, proteases, and lipases. Many actinomycetes can produce different pigments that are potentially good alternative of synthetic colors. Streptomyces species have great capacity to produce active surface biomolecules including bioemulsifiers and biosurfactants. Antidiabetic acarbose was produced by strains of Streptomyces via microbial fermentation. Species of Streptomyces have shown the ability to synthesize cholesterol synthesis inhibitors, like pravastatin. Recently, Streptomyces species can be used as environmentally friendly “nanofactories” for nanoparticles synthesis. Some Streptomyces species are a promising for vitamin B12 production.
(Noura El-Ahmady El-Naggar: Chapter 11 – Streptomyces-based cell factories for production of biomolecules and bioactive metabolites, In: Editor(s): Vijai Singh, Microbial Cell Factories Engineering for Production of Biomolecules, Academic Press, 2021. 183-234.)