Scaling It Up

With the final goal of the Minecrobe project large scale production, the process of scaling up manufacturing from the lab bench to the factory needs to be thoroughly examined and researched. For the final manufacturing procedure we need to consider the following aspects of the process.

· Culturing the Minecrobe

· Inducing Sporulation

· Separating Spores from Live Cells

· Producing Granule-form Product

· Packaging and Shipping

- Field Use

Lab Scale

On the scale of the lab, streaks of live bacteria were taken from lawn plates and inoculated in 10 mL of Luria broth. The liquid cultures were left to grow overnight to O.D. of 1.96 – 2.00, .5mL of the culture was pipetted directly onto the seeds which were then planted in sterile soil. For our lab scale tests live cells were used instead of spores due to time and financial restrictions.

Culturing and Sporulation

For large scale production the Minecrobe cells will be cultured and induced to sporulation at the same time. This protocol is from a 1963 Donnellan, Nags, and Levinson paper in which they outline a method and broth that induces sporulation while allowing B. subtilis to divide. Following their protocol and recipe, a 12L flask containing sporulation medium will be inoculated with spores or live bacteria. The cultures will then be incubated at 29 C on a shaker (95 strokes/min) for 4 days. It is hypothesized that inoculating with live cells instead of spores would decrease the incubation time as there would be no need for the spores to reactivate.

For inoculation of the culture cells or spores may be used, spores can be taken from the separation step and kept until needed for inoculation. If cells are desired they can be frozen in glycerol stock until they are needed. Cultures with an O.D. of 2.00 are desire but any high concentration will be effective. To create frozen stock add equal parts culture and 50% glycerol into a cold resistant tube and store at -80 C. To inoculate using frozen culture thaw until liquid and pipette directly into/onto desired medium.

Separating Spores from Live Cells

According to the Donnellan et. al. article after 4 days 60-80% of the cells completed sporulation. To purify the spores from the live cells we will follow the Donnellan et. al. protocol. We will split the culture into 12 1L flasks which will be centrifuged at 4 C then washed with 500 mL of 50 mM phosphate buffer (pH 5). The pellet will be resuspended in 250 mL water and 200 mL 1:1 polyethylene glycol-600 and 400 mL 3M phosphate buffer (pH 7.1). Rest the spores in an ice bath for 3 minutes and remove the top phase by suction. Add and mix in 500 mL 2M phosphate buffer (pH 7.1), remove the top phase and wash 7 times with 250 mL water by centrifuging at 4 C. The resulting pellet will be suspended in 15-25 mL water, frozen and vacuum dried for 2 days.

If the final cost of separating the spores from the live cells prove to be too expensive due to the labor and reagent costs this step should be considered for exclusion. A similar product named Milky Spore which contains Bacillus popilliae, a species inhabiting the soil which infects and kills grubs. Due to the similarities between Milky Spore and the Minecrobe many analogies can be made especially in the manufacturing and packaging processes. Many brands of Milky Spore are produced from live culture, undergo sporulation, and the whole culture including the live cells are packaged and shipped. The material they are packaged in provides few nutrients so many live cells die or undergo sporulation. This can then be seen as a way to further reduce costs, making our product more available to those it would benefit the most.

Granulation

Donnellan et. al. reported that from a 1 liter culture they were able to recover 250 mg of B. subtilis. Given the 12 L culture we will be able to purify 3 grams of spores which will be mixed with 13.6 Kg of calcium lignosulphonate giving a final concentration of .02% spores. Calcium lignosulphonate is a common binding agent which will dissolve in water, releasing the Minecrobe spores which will then activate in the soil, begin to reproduce, and produce siderophores. The spore-calcium lignosulphonate mixture will be compressed and extruded into granules using a roll compactor. The resulting granules will then be divided into three 10 lb. bags for shipping. Dry granulation was chosen for several distinct benefits that it offered to packaging and transporting the Minecrobe. By mixing the spores with a binding agent we overcame two distinct problems with transportation. Firstly by packaging the spores in a water sensitive binding agent they are prevented from activating prematurely. Any water that is able to penetrate the shipping bag will be absorbed by the binding agent and prevented from activating the spores. Only a catastrophic failure of the shipping bag would allow the spores to become active, and that would render the product useless in this form as with any other. The second problem that is avoided by using a binding agent is the accidental inhalation of spores. The original design was to package and ship the spores without any additives, the problem being that inhalation of B. subtilis spores may resulting possible lung irritation and may require hospitalization for the severely immunocompromised.

Packaging and Shipping

For shipping the granulated spores, 5 L of space are needed to fit 10 lbs. of granules. The density of calcium lignosulphonate is 800-965 kg/m^3, which was rounded to 1000 kg/m^ 3 for the purposes of calculation given the decreased packing efficiency of granules compared to powder. 10 lb (4.5 Kg) of granules require .0045 m^3 of volume, or 4.5 L. Rounding up to a more common 5 liter bag size gives room for variances in the density of the granules and a more easily obtainable bag size. Shipping three 5-L bags per box necessitates a box of 15 liters minimum volume. This is 915.356 cubic inches which was rounded to 1000 cubic inches. Because a 10in x 10in x 10in box can be easily shipped anywhere in the world, this size was chosen to contain the three 5-L bags.

Field Use

The final Minecrobe product will be shipped out in a box containing three 10 lb bags. Since a 2.5 lb. bag of Milky Spore can treat a 10,000 sq. ft. area. Therefore, each 10 lb. bag of Minecrobe should be able to treat an estimated 40,000 sq. ft. or 0.913 acres. However, because B. subtilis is more active in the soil than Milky Spore, a 10 lb bag of Minecrobe likely will be able to treat a full acre. Thus, to disperse Minecrobe, a 10 lb. bag should be used per acre and spread as evenly as methods will allow. This is best done before rain or watering to dissolve the calcium lignosulfonate. Multiple applications may be considered to increase Minecrobe concentration, but the first application will be effective.