The production of microbeads for encapsulated bacteria in an alginate matrix is accomplished with a device (see the technical diagram) made mostly of stainless steel to allow sterilization by autoclave. The tool is positioned at 45° inclination, allowing the production of a stream of droplets. All fittings and other details are built of commonly used materials that can stand autoclaving.
The basic design can be modified or modularly-enlarged to produce a larger amount of microbeads without compromising the basic features. This tool produces microbeads that range in size from 100 to 200 mm.
Ten ml of a bacterial suspension from cultivating bacteria in a growth media are mixed under aseptic conditions with
2% sodium alginate with slow stirring at ambient temperature for 1 h, when all ingredients will be thoroughly mixed.
The concentration of bacteria at this stage is 1010 colony-forming units (CFU)/ml solution. To produce beads
that are more biodegradable, skim milk without Ca is added to the alginate-bacterial suspension. These suspensions are
placed in Erlenmeyer flasks or a beaker that are attached to the microbead-producing device (described above) and the
device is pressurized at 10-15 psi with a commercial air compressor. The suspension sucked from the Erlenmeyer flask is
then forced to pass through a capillary tube (222-mm diameter), which creates a fine spray of
miniature droplets. The mist is sprayed into a 25x40-cm stainless steel pan containing 0.1 M CaCl2 that is slowly rotating at 40 rpm.
Microbeads form instantly upon contact of the droplets with the solidifying solution. The microbeads are allowed to cure
in the CaCl2 solution for 30 min. This procedure produces microbeads ranging from 100 to 200
mm. At this stage, the of bacteria is 2x1011 CFU/g microbeads on average
in fresh beads. After extracting the wet beads from the CaCl2 solution, they are rinsed in 500 ml saline solution
(0.85% (w/v) NaCl) four times under aseptic conditions and transferred into fresh, growth medium for the bacteria.
Bacteria multiplied in this mixture for an additional 12 h at 30 ± 2°C at 100 rpm. Then the microbeads are separated
from the suspension by filtration, using Whatman no. 4 filter paper, and rinsed three times with 500 ml saline solution.
The population in fresh beads, after this secondary multiplication, always reaches a concentration 1x1012
CFU/ g microbeads. Usually, wet preparations of microbeads at this stage are used for inoculation of plants almost
immediately or after overnight storage at 4 ± 1 °C. This does not affect the number of bacteria in the preparation.
Ten g of microbeads are placed in a thin layer on filter paper in a Petri dish and dried at 38 ± 1°C for 48 h if the bacterial species can tolerate this temperature. Then the dry microbeads are collected and stored in hermetically-sealed containers with silica gel until used. If the bacteria species is susceptible to this temperature, dry beads are prepared by standard lyophilization (Freeze-drying technology) or by using a drying tool (see technical diagram).
Seed coating with microbeads and microbead counts from seeds
Dry or wet microbeads are manually mixed with dry seeds. Alternatively, a very dilute solution of an adhesive (lecithin, solidified fatty acids derived from soybean, 0.1% and 0.5% dissolved in ethanol that evaporates immediately on exposure to ambient temperature and dried with an unheated hair dryer) or any other agricultural adhesive can be applied to the seeds. This solution creates a sticky surface, but does not allow the seeds to stick to one another. The microbeads are then manually mixed with the sticky seeds. Microbeads adhering to seeds can be counted under a stereoscopic microscope or by any counting equipment, such as an image analyzer.
Once the seeds are coated with the microbead-containing bacteria, they can be sown by any sowing machine and need no further treatment or special care.