1. General Concept of Quality Control

Quality assessment of inoculants has been a matter of interest for years. While examining peat-based rhizobial inoculants for moisture, viable counts, contaminants, and effectiveness using plate counts and MPN it was found that rhizobial counts were variable but that contaminants were present in most of the inoculants, even exceeding the number of rhizobia and affecting inoculation effectiveness. Similar results were obtained with a wide range of inoculants produced and used in different parts of the world. It was stated that inoculants prepared with nonsterile peat contained 100-fold fewer rhizobia than those made with sterilized peat. In an information bulletin on production and quality control of legume inoculants, it was indicated that most of the products tested in India contained suboptimal level of rhizobia (<108 rhizobia/g of inoculant) together with a large quantity of nonrhizobial organisms. Other autors found that rhizobia counts were inversely related to contaminants level. After analysis of 40 rhizobial inoculants produced in North America it was reported a constantly high level of contaminants (108 to 1010 cells/g of product), outnumbering the rhizobia in all the products but one. In some products, rhizobia could not even be detected. These results were confirmed with another study with 60 more samples among which the majority of the products contained more contaminants than they did rhizobia. Similar tests were run on commercial soybean inoculants from Argentina and showed that out of 18 products, 17 were highly contaminated, with rhizobia being outnumbered by contaminants in 14 of them. More recent studies report comparably alarming results on rhizobial inoculants but also on products containing PSB or free N₂-fixing bacteria. Moreover, among the isolated contaminants, several strains were found to be opportunistic pathogens for human, plant, or insects. Evaluation of the quality of AMF inoculants showed that they generally contain a very low quantity of viable propagules and a reduced (or an absence of) host infection and colonization potentials, resulting in highly inconsistent performance under field conditions.

There are a number of factors influencing the quality and the efficacy of an inoculant during production and after inoculation into soil. The main are presented in Fig. 1.

Figure 1. Main factors affecting the quality of the inoculants from production to inoculation

In this respect many technical difficulties related to a large-scale production of inoculant must be overwhelmed. For example, media and growth conditions (temperature, pH, time) for bacteria must be optimal in order to ensure that the cells are in good physiological conditions. For AMF, hosts might be chosen on the basis of strain–host specificity providing possibilities of AMF strain(s) to multiply. The type of cultivation and the corresponding required space are the major disadvantages for large-scale production of AMF. In all cases, the provision of competent and well trained operators is of critical importance, thus assuring implementation of the right methodologies. Other important factors are minimization of the production cost and maintenance of the pure microbial culture throughout the process. In this way better quality of the product is ensured.

Other important step in quality provision of biofertilizers is the formulation. New carriers are needed to overcome the limitations of peat (availability, environmental impact, toxicity) and provide a more suitable environment for the microorganisms. They should maintain microbial viability and fitness during storage, as well as on seed and in soil after inoculation.

One of the critical stages in biofertilizers production is the inoculation of the carrier. It has been broadly perceived that the utilization of a sterile carrier offers a few favourable fetures over nonsterile ones. These are higher populations of the target strain(s) and a longer shelf life. Moreover, contaminating microorganisms are generally able to grow faster than the target ones (especially in the case of rhizobia), thus easily replacing them in a short period of time. They compete for space and nutrients and may also produce toxic compounds reducing the growth of other cells, or be pathogenic for plants, humans, or environment. Sterility is generally obtained by using a steam (autoclaving) or gamma irradiation. The last one is considered as slightly better to steam sterilization but is more expensive and slow, requiring specific costly and not easily available equipment. Other technologies such as electron acceleration have also been developed, but they are economically unjustified as well.

Other major aspect important for quality assurance is the maintenance of cell viability during transport and storage. It is affected by many factors. The moisture is of primary importance for peat-based products and generally reaches 45 to 60 % on a wet weight basis. For the granular inoculants, the speed of drying was shown to be of great importance. Slow drying affects less severely the cells than fast drying. Addition of substances providing higher desiccation tolerance (such as osmoprotectants) could permit the production of biofertilizers more resistant to severe storage conditions. Low temperatures (4 °C) are generally recommended as the best storage conditions. However, it was shown that temperatures during both storage and transport can be above 26 °C and sometimes even 40 °C. These conditions are detrimental for rhizobial strains. It is very important to note that effects of water content, temperature, and time are not mutually exclusive Several studies have reported that over time microbial populations in inoculants decline, leading to a lower inoculation efficiency and increased contaminant strains. This is especially true for products that have not been stored under optimal conditions. Generally, inoculant expiry date is about 12 months after production, but some products are likely to be older when used.

Another detected problem is that most of the literature reports evaluating the quality of biofertilizers (or strain selection) are made under controlled conditions but not under field ones. Available studies generally reported variable performances (even of very promising products under controlled conditions) due to interactions between the target plant, microorganisms, soil and environmental conditions. Other factors such as the mode of application (seed coating, on-site seed application, or soil inoculation) may also affect inoculation efficiency depending on the kind of crop (size and fragility of the seeds) and anterior seed treatments. The type and the density of the native populations in the soil can be major barriers for successful inoculation. This is due to the fact that recently introduced cells must not only survive in the new potentially harmful conditions, but compete for protective niche and nutrients, dominating over the indigenous, better-adapted populations. In this aspect, the success of the inoculation is related to the persistence of the introduced strain, i.e., its ability to establish high population levels despite of the unfriendly environment and to live as a continuing member of the soil microflora even in the absence of its host plant.

A better understanding of these complex interactions is highly required since it significantly influences the effectiveness of the inoculants and their perseverance in soil. Up to now, the variability and the unpredictability of the results from crop to crop, place to place, and from season to season have restricted a wider use of inoculants.

Successful commercialization of new inoculants principally depends on the on the cooperation between the research (to formulate the best inoculant, using the right strain for the right crop in the right conditions), the private sector (to scale up the production, establish an economically viable and sustainable market chain), and the acceptance by farmers. The need for farmers' education is great. If the end users are convinced of the efficacy of the biofertilizers on their crops, they will be more willing to buy and use them instead of expensive and harmful chemical fertilizers. To accomplish that, the improvement of the biofertilizer quality is a critical issue. Demonstration trials with high-quality products and regular training of the farmers for the use of inoculants would lead to a greater confidence from the farmers and a significant increase in the use of biofertilizers.

2. Quality control parameters

2.1. Microbial Functions

Numerous soil bacteria which reside in the plant rhizosphere and which may grow in, on, or around plant tissues, stimulate plant growth. These bacteria are known as plant growth promoting rhizobacteria (PGPR).

Some of PGPR can promote growth by acting as both biofertilizer and biopesticides.

Figure 2: Integrated microbial actions in soils.

The screening for PGPR and investigation of their activities are expanding at a fast pace as endeavors are made to exploit them commercially as biofertilizers.

The most valuable activities of PGPR include fixing N₂, increasing the availability of nutrients in the rhizosphere, positively influencing root growth and morphology, and promoting other beneficial plant-microbe symbiosis. The blend of these modes of actions in PGPR is also addressed, as well as the difficulties facing the more broad usage of PGPR as biofertilizers.

Two types of materials are used in agriculture, fertilizer or pesticide. It can be assumed that fertilizer is required for nourishment, and pesticide for medication of plants in conventional agriculture. On the other hand, biofertilizer and/or biopesticide represent respectively both materials in sustainable or environmentally friendly system (Figure 2).

The main sources for biofertilizer are nitrogen fixing becteria, phosphate solubilizer, and mycorrhizae. Similar to the functional foods, like restoratives and/or adjuvant, who are required for human health care; plant growth promoting rhizobacteria may be one of the compatible substances for better crops yield.

However, several limitations exist in the use of biofertilizer for agricultural system. Primarily, the efficacy for most biofertilizer is not reliable. This is due on the scarce data available about the mechanism of action of different biofertilizer in promoting plant growth. However, research into biofertilizer is increasing, trying to manage these issues.

Moreover, different parameters should be also assessed, such as: soil type, managements practices, and weather effect on biofertilizer efficacy. Furthermore, there is a block in biofertilizer development. It is difficult to test inoculant in field as routine experiments, as shown in Figure 3.

Figure 3: Experimental process for biofertilizer testing.

2.2. Properties of Microbial Products

The microorganisms used for development of biofertilizers are bacteria of genera Bacillus, Pseudomonas, Lactobacillus, photosynthetic bacteria, nitrogen fixing bacteria, fungi of Trichoderma and yeast. Among the microbes, the most employed microorganism is Gram (+) endospore-forming bacteria from genus Bacillus. Usually, several species of microbes are used in microbial products with an available period of by- products of about 1~2 and/or 2~3 years.

Biofertilizers can be solid or liquid. Carriers used in solid type biofertilizers are generally clay mineral, diatomaceous soil, and white carbon as mineral. Other materials used are rice, wheat bran, and discarded feed as organic matter. However, the effects of carriers and/or supplements on microbial growth are of great importance and should be seriously consider in the control of microbial products. In fact, often farmers misunderstood this carrier effect as microbial action.

As displayed by producers, microbial products stimulate plant growth, decrease pest occurrence, stimulate composting and ameliorate the soil. However, the main effect generally is the plant growth stimulation. Nevertheless, in 40 % of the commercial biofertilizers manufacturers declare presence of multiple effects.

In this respect controlling the quality of biofertilizer is one of the most important factors. Thus their success or failure and acceptance or rejection by end-user, the farmers will be assured. Principally, quality represents the number of selected microorganism in the active form per gram or milliliter biofertilizer. Up to now quality standards are developed only for Rhizobium. Moreover, specifications of biofertilizer differ from country to country and maybe comprise parameters like: microbial density at the time of manufacture, microbial density at the time of expiry, the expiry period, the permissible contamination, the pH, the moisture, the microbial strain, and the carrier. Quality has to be monitored at different production stages (during pre-culture stage, carrier selection and preparing, broth formulation, mixing of broth and culture, packaging and storage). Main quality parameters to be respected during biofertilizer production are summarized in Table 1.

Table 1: Key quality parameters of biofertilizers

Forms	Liquid	Powder	Granular
Appearance of living target bacteria	Without strange smell	Brown or black	Brown
Fast-growing Rhizobium	>0.5x109/ml	>0.1x109/g	>0.1x109/g
Slow-growing Rhizobium	>1.0x109/ml	>0.2x109/g	>0.1x109/g
N fixation bacteria	>0.5x109/ml	>0.1x109/g	>0.1x109/g
Si bacteria P bacteria	>1.0x109/ml	>0.2x109/g	>0.1x109/g
Organic P	>0.5x109/ml	>0.1x109/g	>0.1x109/g
Inorganic P	>1.5x109/ml	>0.3x109/g	>0.2x109/g
Multi-strain biofertilizer	>1.0x109/ml	>0.2x109/g	>0.1x109/g
pH	5.5-7.0	6.0-7.5	6.0-7.5
Water content (%)		20-35	10
Non-target bacteria Contamination (%)	<5	<15	<20

3. Quality Management

Quality management is very important process, and must be performed repeatedly to monitor the microbial products in favor of the customers.

The current guidelines used for evaluating quality of biofertilizers are restricted to controlling the: density of the available microorganisms, their viability and preservation. However, it is also important to set control points that do not contain available microorganisms, but are focused on the consistency of the other compositions in the final microbial products. Also it is highly desirable that the biofertilizer demonstrates the major effects for quality management of the final biofertilizer products. It is a crucial requirement to discriminate between the role of the available microorganisms and the supplementary compositions on the effects of the biofertilizer guaranteed by the suppliers. If the final results of the two experimental schemes (microorganisms / supplements) are the same or cannot be confirmed statistically, then the product is only an organic matter. This means that the effects of microbial products should resulted from the activity of the guaranteed microorganisms, and the target of the substances should be presented in details as a prescription. It is important to assess precisely the functions under the given usage manifested by the end-user (Figure 4).

Figure 4: Procedure of biofertilizer quality control.

Biofertilizers, being microbial products, supply soil with nutrients, diminish the agricultural burden and conserve the environment. Good soil condition is imperative to improve crop yields, as well as to assure human and/or animal health welfare. That’s why, the materials, as biofertilizers, used to sustain good soil condition, are treated as environmental matters. However, as mentioned earlier, there are still some problems to be met on the use of microbial products. More accurate quality control must be performed in favor of the customers. With this in mind, the need to develop better production techniques and to improve the management system for microbial products is defined.

Although the effects of biofertilizers vary in different geographical regions due to the peculiarities in climate and soil conditions, the importance of biofertilizer on environmental preservation in the 21^st century must not be ignored. In the same time, development of various biotechnological approaches should be considered in order to increase the biofertilizer effects with concern for the environment.

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.

5. Quality standards for Rhizobium and Azotobacter

Several quality standards have been formulated for Rhizobium and Azotobacter inoculants. These specifications are shown in Table 2.

Table 2: general standards specified for Rhizobium and Azotobacter biofertilizers

Parameters	Rhizobium Biofertilizer	Azotobacter Biofertilizer
Cell no. at the time of manufacture	108/g carrier within 15 days of manufacture	107/g carrier within 15 days of manufacture
Cell no. at the time of expiry date	107/g carrier within 15 days before expiry date	106/g carrier within 15 days before expiry date
Expiry date	6 months from the date of manufacture	6 months from the date of manufacture
Permissible contamination level	No contamination at 108 dilution	No contamination at 107 dilution
pH	6.0–7.5	6.5–7.5
Strain	Should be checked serologically	Nothing specific. But A. chroococcum species is mentioned
Carrier	Should pass through 150–212 microns IS sieve	Should pass through 160 microns IS sieve
Nodulation test	Should be positive	––
Nitrogen fixation	Above 20 mg/g of glucose	Not less than 10 mg/g of sucrose

The variability in quality standards specified for Rhizobium in various countries are as follows (Table 3).

Table 3: Quality standards of commercial Rhizobium culture in different countries

Country	Cells/gm of culture (total viable count on Congo-red agar)
	Very satisfactory	Satisfactory	Doubtful
U. S. A.	109	–	106 – 107
Australia	–	2 × 108	106 – 107
Russia	109	–	–
India	More than 109	107–109	Less than 107

Although quality control standards for biofertilizer Azospirillum and PSM has not been in force, the proposed standard specification of PSM and Azospirillum are given in Table 4.

Table 4: Proposed standard specifications of PSM and Azospirillum

No.	Parameter	PSM	Azospirillum
1.	Base	Carrier (Lignite/ Charcoal)	Carrier (Lignite/ Charcoal)
2.	Carrier	>100 micron	>100 micron
3.	pH	6.5–7.5	7.0–8.0
4.	Moisture	35–40%	35–40%
5.	Viable count at manufacture	107/g carrier	107/g carrier
6.	Viable count at expiry	107/g carrier	107/g carrier
7.	Level of contaminant	No at 104 dilution	No at 104 dilution
8.	Growth in Pikovskaya medium	+ve	–
9.	Growth in S. S. Malate medium	–	+ve
10.	P Solubilization zone	1mm	–
11.	Pellicle formation	–	+ve
12.	Shelf life	6 months	6 months
13.	P Solubilization	30–50%	–
14.	N-fixation	–	15 mg/g of malic acid

6. Quality control measures

The biofertilizer should be evaluated for the following quality standards:

1. Inoculant should be carrier based or liquid based.
2. The inoculant should contain minimum of 10⁸ viable cells of bioinoculant/g of carrier on dry weight basis when it is stored at 25–30°C.
3. The inoculant should have at least 6 months shelf life from the manufacturing date in case of carrier based and 9 months in case of liquid based.
4. The pH of inoculant should be between 6.0 and 7.5.
5. Inoculant should show effective nodulation/nitrogen fixed on particular crop before expiry date.
6. The carrier material should be in the form of powder, i.e. peat, lignite, peat soil, and humus, etc.
7. Inoculant should be packed in 50–75 microns low-density polythene bags.
8. Each package should be marked legibly to give the information about name of the product, name of microbial inoculants, activity of bioinoculant, intended crop, name and address of manufacturer, type of carriers, batch and code numbers, date of manufacture, date of expiry, net quantity meant for 0.4 hectare, and storage instructions.
9. It should be free from any contaminant/contamination with other microorganisms.

References:

1. Herrmann L & Lesueur D. Challenges of formulation and quality of biofertilizers for successful inoculation. Appl Microbiol Biotechnol, 2013, 97:8859–8873
2. Malus E, Sas-Paszt L, Ciesielska J. Technologies for Beneficial Microorganisms Inocula Used as Biofertilizers. The Scientific World Journal, 2012, Article ID 491206
3. Yadav AK, Chandra K. Mass Production and Quality Control of Microbial Inoculants. Proc Indian Natn Sci Acad, 2014,80 (2): 483-489
4. Organic Farming :: Organic Inputs and Techniques: http://agritech.tnau.ac.in/org_farm/orgfarm_biofertilizertechnology.html
5. Biofertilizer Manual by FNCA Biofertilizer, 2006, Japan Atomic Industrial Forum (JAIF)
6. Borkar SG. Microbes as Biofertilizers and their Production Technology, 2015, Woodhead Publishing India Pvt. Ltd.