4. Procedures for Quality Control of Biofertilizer

4.1. Rhizobium

Quality checks on Rhizobium biofertilizer can be divided into three parts:

• Pre - culture test
• Broth test
• Peat test

Before producing Rhizobium biofertilizer, the pre-culture should be checked on the following parameters:

• Growth
• Purity
• Gram stain
• Broth composition test
• pH

4.1.2. Cell morphology

Rhizobial are stained for observation of shape and size of the cells. Cells of rhizobia are rod-shaped, with one or two cells sticking together. Microscopical check for contaminates is performed.

4.1.3. Viable count

The number of living cells is counted by spread plate or drop plate methods in YMA + CR medium. Plates are incubated in incubator (28 - 30⁰ C) or at room temperature for 7 days.

4.1.4. Peat test

For the peat inoculant, the following quality parameters are checked:

• pH
• Moisture content
• Viable number
• Plant infection method (MPN)
• pH

The optimal pH for the inoculant is the neutral. Since peat is acidic the pH has to be adjusted with CaCO₃. The optimum moisture content of peat-inoculant is between 40 - 50 %. At low moisture rhizobia will die rapidly. If moisture is high, inoculant may stick to the plastic bag and, thus to compromise the rhizobial growth.

4.1.5. Plant Infection Analysis using Most Probable Number Method (MPN)

This is an indirect method of assessing plant infection on nodulation. It is widely used when peat is not sterile. It takes more time than spread plate method as to grow plants is required. This method is based on the assumptions that: if a viable rhizobia is inoculated on its specific host, nodules will develop on that roots. Nodulation on that inoculated plant is a proof of the presence of infective rhizobia.

4.2. Non-symbiotic N₂-fixer

In research aspect, microbial growth may be represented by the augmentation in cell mass, cell number or any cell constituent. Growth of the organism could be also assessed by the utilization of nutrients or accumulation of metabolic products. Growth, therefore, can be determined by various methods based on one of the following assays: (a) cell count, directly by microscopy or by an electronic particle counter, or indirectly by colony count, (b) cell mass, directly by weighing or measurement of cell nitrogen, or indirectly by turbidity; and (c) cell activity, indirectly by relating the degree of biochemical activity to the size of the population.

The growth rate of Azospirillum is expected to have reached its maximum at 3-5 days after inoculation. The recommended counting technique in this case uses the drop-plate method. Proper aseptic procedures should be observed, otherwise contaminants may be accidentally introduced during the injection of the broth culture and during serial dilution and plating. This contaminants are also detectable on the utilized indicator media and their number should be reported together with the number of viable cells as additional measure of the quality.

4.3. Mycorrhiza - the arbuscular mycorrhizal fungi, AMF

Quality control in the formulation of AMF inoculum is essential for product uniformity, reliability and reproducibility. This is applied to the laboratory, preparation room, growth room, storage room and the greenhouses, taking care into the design, to achieve the most efficient control in inoculum production.

4.3.1. Laboratory quality control

Laboratory quality control is applied in respect to the production of bacterial spores. They are extracted from monospecific spore cultures in the preparation room. The spores are transported in petri dishes to the laboratory and placed in a refrigerator before examination under stereoscopic microscopes. Spores from each petri dish is described and records are prepared.

4.3.2. Preparation room quality control

This room has to be isolated from the greenhouse and growth room, and unsterilized soil or media samples should not be stored in it. Materials (cultures; sterilized growth media) are clearly labeled and placed in specific containers. Floor should always be clean without presence of dust. All surfaces should be clean and disinfected. Containers are surface-sterilized with 10% sodium hypochlorite.

4.3.3. Growth room quality control

The growth room should be temperature controlled (22 °C). Air is exhausted to the outside and no recycling is applied. Surfaces should be painted with anti-microbial paint and sterilized periodically e.g. monthly. All samples are checked for contaminants and pathogens. Watering is done manually, avoiding cross-contamination.

4.3.4. Storage room quality control

All samples stored are placed in plastic bags, with proper labelling, and surface of bags should be cleaned before usage. Floors and surfaces are clened regularly, preventing generation of dust.

4.4. Phosphate Solubilizers

Phosphate solubilizers (PS) contain phosphate solubilizing bacteria or fungi. Commercially produced PS biofertilizers (PSB) are certified in respect to the guaranteed components such as type of strains, microbial density, and biological activity. If possible the rate of phosphorus absorption of target crops is also determined. The procedure shown in Figure 5 could be used for the quality control of PSB (Figure 5).

Figure 5: General procedure for quality control of PSB.

Phosphate solubilizing microorganisms play an important role in plant nutrition through increasing the available phosphate for plant. Accordingly, great attention should be paid to investigations and formulation of new combinations of phosphate solubilizing bacteria and other plant growth promoting rhizomicrobes for improved crop yields.