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Bacillus Cultivation

Bacillus Cultivation

Bacillus is a gram-positive bacteria. Bacteria of this genus can produce spores, are aerobic or facultative anaerobic bacteria, most strains are motile, have no capsule, most are hemolytic, and are usually catalase positive. The G+C molar content in DNA is 32-62%. An important characteristic of bacteria of this genus is the ability to produce spores that are exceptionally resistant to adverse conditions.

Bacillus species are found in various habitats, and a few species are pathogenic to vertebrates and invertebrates. The genus includes strains such as Bacillus anthracis, which is pathogenic to humans and animals, Bacillus cereus, which can cause food poisoning, and non-pathogenic Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus thuringiensis.

Bacillus Culture Service

Creative Biogene offers customizable Bacillus strain culture services. This service can help you obtain Bacillus cultures for subsequent scientific research. This service allows you to skip the complicated and tedious groping of culture conditions, which helps to speed up research on this strain.

Bacillus licheniformis

Figure 1. Bacillus licheniformis. (From wikipedia.org)Figure 1. Bacillus licheniformis. (From wikipedia.org)

Bacillus licheniformis is a facultative anaerobic gram-positive bacterium commonly found in soil. The bacterial cells are rod-shaped and the colonies are solitary. The spores are oval, and the sporangiums are slightly enlarged. They are non-pathogenic bacteria. The optimum temperature for bacterial growth is about 50 °C, and the optimum temperature for enzyme secretion is 37 °C. Bacillus licheniformis is widely used in industrial production due to its excellent characteristics of heat resistance, high enzyme content and high enzyme production. Bacillus licheniformis can exist in the form of endospores in unfavorable external environments such as high temperature and acidity.

Bacillus subtilis

Bacillus subtilis has a cell size of 0.7-0.8 x 2-3μm and is uniformly colored. The bacteria are non-capsular, pericytoflagellated, and able to move. Bacillus subtilis is a gram-positive bacteria. The spores are elliptical to columnar, with a size of 0.6-0.9×1.0-1.5 microns, located in the center of the cell or slightly offset, and the cell does not expand after the spore is formed. The surface of the bacterial colony is rough and opaque, and it is dirty white or yellowish. Wrinkles are often formed when grown in liquid media.

Figure 2. Bacillus subtilis.(From wikipedia.org)Figure 2. Bacillus subtilis. (From wikipedia.org)

Bacillus subtilis is an aerobic bacteria. It can use protein, various sugars and starch to decompose tryptophan to form indole. The bacterium is widely distributed in soil and decaying organic matter, and it is easy to multiply in subtilis extract, hence the name.

Bacillus amyloliquefaciens

Gram stain for Bacillus amyloliquefaciens was positive. The bacterial cells are rod-shaped and can form endospores. The spores are oval in shape with obtuse ends. The sporangia do not expand, show mesophyte to subterminal, and have athletic ability.

Figure 3. Gram staining of Bacillus amyloliquefaciens.(AA Prihanto, et al.;2020)Figure 3. Gram staining of Bacillus amyloliquefaciens. (AA Prihanto, et al.;2020)

Bacillus amyloliquefaciens is a facultative anaerobic bacterium. The colonies on LB medium and beef extract peptone medium are light yellow opaque colonies, with rough surface, raised, irregular edges, and no pigment is produced on various mediums. Biofilms formed when the liquid culture was stationary.

Bacillus amyloliquefaciens can hydrolyze starch and gelatin, and the acetyl methyl carbinol (V-P) test, nitrate reduction test, phenylalanine deaminase test, indole test, methyl red (MR) test and hydrogen sulfide test were all negative. Studies have shown that different strains of Bacillus amyloliquefaciens have different requirements for medium components and culture conditions, but they are generally maintained within a certain range and can grow well in basic medium. The culture temperature is generally 31 ~ 37℃. The pH of the culture medium is neutral, 180-200 r/min and the appropriate incubation time is 16-24 h.

Bacillus thuringiensis

Figure 4. Gram stain of Bacillus thuringiensis. (From wikipedia.org)Figure 4. Gram stain of Bacillus thuringiensis. (From wikipedia.org)

The bacteria of Bacillus thuringiensis are purple oval rod-shaped under the microscope, the size is 1.2-1.8×3.0-5.0μm. The bacterial cells are arranged in short or long chains. The spores of the bacteria are oval, growing near the middle, and the sporangia are slightly enlarged. The colony of Bacillus thuringiensis on NA medium (Nutrient Agar) is round or oval, pale yellow, with irregular edges, opaque and slightly raised and waxy.

Bacillus anthracis

Bacillus anthracis is the largest bacterium among the pathogenic bacteria due to its thick body, truncated or sunken at both ends. Its cells are arranged like bamboo knots, without flagella, without motility, and are Gram-positive. The bacteria are easy to form spores under the conditions of sufficient oxygen and suitable temperature (25-30℃).

Bacillus anthracis is obligate aerobic, easy to cultivate in common medium and easy to multiply. During the cultivation process, the optimum temperature was 25-30°C; the optimum pH was 7.2-7.4. After culturing on agar plates for 24 hours, they grow into rough colonies with a diameter of 2-4 mm. The colony is ground glass-like, with irregular edges, curly hair-like shape, and one or several small tail protrusions, which are caused by the outward extension and reproduction of this bacteria. On the 5-10% sheep blood agar plate, there is no obvious hemolytic ring around the colony, but mild hemolysis may appear after a long period of culture. The optimal time for colony characteristics to appear was 12-15 hours.

Cultured in ordinary broth for 18 to 24 hours, flocculent precipitates grow at the bottom of the tube, a sterile membrane, and the bacterial liquid is clear. The virulent strains were cultured on sodium bicarbonate plates under 20% CO2 to form mucoid colonies (with capsules), while the avirulent strains were rough.

Bacillus coagulans

Bacillus coagulans was originally isolated from rancid canned milk by Hammer and described as a new species. These bacteria were later isolated from rancid preserved foods, and they produced high concentrations of L-lactic acid, which caused the rancidity of carbohydrate-containing canned foods, thereby attracting attention. The cells of Bacillus coagulans are rod-shaped, Gram-positive bacteria. The spores are born at both ends and have no flagella. The bacteria can decompose sugars to generate L-lactic acid, which is a homotype lactic acid fermentation bacteria. The optimum growth temperature is 45-50℃, and the optimum pH is 6.6-7.0.

Bacillus stearothermophilus

The cells of Bacillus stearothermophilus are rod-shaped, generally 0.6-1.0 μm×2.0-3.5μm. Spores elliptic, subterminal or terminal, variable in size. The Gram staining reaction is variable and the cells can move. Most strains grow actively under anaerobic conditions in glucose medium and are not active until the pH is lowered to 5.3-4.8. A few strains do not grow under anaerobic conditions. The product of anaerobic fermentation is mainly L(+) lactic acid, with a small amount of formic acid, acetic acid and ethanol in a ratio of 2:1:1. Nitrate promotes anaerobic growth and gas production in some strains. Some strains do not have this effect or only reduce to nitrite. The lowest nutritional requirements vary widely among strains. The spores are more resistant to heat and other hostile environments than any other mesophilic species of the genus Bacillus. Vegetative cells are obviously sensitive to adverse conditions, such as cooling to room temperature and their viability may be immediately lost. The minimum growth temperature is 30-45°C. The bacterium can be found in habitats such as soil, desert sand, hot springs, marine sediments, compost, and food. Its vegetative cells can germinate and grow rapidly in many foods, such as pH>5.0 and suitable temperature.

Bacillus cereus

Bacillus cereus is a gram-positive aerobic bacillus, a facultative aerobic bacterium that can grow under anaerobic conditions, and is a relatively common bacterium that causes bacterial food poisoning. The optimum temperature for the growth and reproduction of the bacteria is 28-37°C, and no reproduction is performed below 10°C. Its propagules are more heat-resistant and can be killed only after 20 minutes at 100 °C, while spores can withstand 100 °C for 30 minutes, or dry heat at 120 °C for 60 minutes. In the pH range of 6-11, the bacteria can grow; if the pH is below 5, its growth and development are significantly inhibited. Bacillus cereus is commonly found in plants and many raw and cooked foods.

Microbial GMP Production

Creative Biogene's fermentation platform has Good Manufacturing Practices (GMP) and can provide customers with a wide range of high-quality microbial fermentation products such as active pharmaceutical ingredients, enzymes and various fine chemicals. In addition, our microbiology experts have completed the transformation and innovation of traditional processes through continuous breakthroughs in key technologies of microbial fermentation processes, and fully contributed to the smooth delivery of the project.

Production Capacity

Creative Biogene builds a world-class microbial fermentation technology platform, providing a variety of services from strain screening and optimization to fermentation production and product purification. We have many years of rich experience and provide good technical support for microbial GMP production.

Facility Display

As a leader in microbial production, Creative Biogene has comprehensive production process technology and high-volume manufacturing capabilities. Our goal is to help our customers develop streamlined and controlled manufacturing processes and to support customers throughout the entire product development process, from the R&D stage to market launch.

Device Example:

  • Fermentation, centrifugation and filtration upstream process equipment;
  • Fully automatic fermenters ranging in volume from 4,000L to 12,000L with a total capacity of over 100,000 liters;
  • From industrial-scale chromatography systems, membrane systems to larger-scale continuous centrifuges;
  • Recycling and Downstream Equipment;
  • Waste treatment equipment.

Why Choose Us?

The culture of Bacillus requires specific formulations of growth media for use in cloning, plasmid DNA preparation, and protein expression. Creative Biogene offers a selection of bacterial growth media and custom services for your specific application. If you are interested in our microbial anaerobic and aerobic culture platform, please contact us for more details.

References

  1. Turnbull PC (1996). "Bacillus". In Baron S, et al. (eds.). Barron's Medical Microbiology (4th ed.). Univ of Texas Medical Branch. ISBN 978-0-9631172-1-2.
  2. Christie G, et al.; Bacillus spore germination: Knowns, unknowns and what we need to learn. Cellular Signalling. 2020, 74: 109729.
  3. Beladjal L, et al.; Life from the ashes: survival of dry bacterial spores after very high temperature exposure. Extremophiles: Life Under Extreme Conditions. 2018, 22 (5): 751–759.
  4. Nigris S, et al.; Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera. BMC Microbiology. 2018, 18 (1): 133.
  5. Brenner DJ (1984). "Family I. Enterobacteriaceae Rahn 1937, Nom. fam. cons. Opin. 15, Jud. Com. 1958, 73; Ewing, Farmer, and Brenner 1980, 674; Judicial Commission 1981, 104.". In Krieg NR, Holt JG (eds.). Bergey's Manual of Systematic Bacteriology. Vol. 1 (first ed.). Baltimore: The Williams & Wilkins Co. pp. 408–420.
  6. Alcaraz LD, et al.; Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics. BMC Genomics. 2010,11: 332.
  7. AA Prihanto, et al.; Identification of Protease-Producing Halophilic Bacteria Isolated from Salt-Pond Soil. Jurnal Ilmiah Perikanan dan Kelautan. 2020, 12(1):181.
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