Development of Rhizobial Inoculant Formulations

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Ilungo J. Xavier, Greg Holloway, and Mary Leggett, Philom Bios Inc., 318-111 Research Drive, Saskatoon, Saskatchewan, Canada S7N 3R2

Abstract

A key constraint to successfully commercializing beneficial microorganisms is overcoming difficulties in formulating a viable, cost-effective, and user-friendly final product. The live nature of the active ingredient (i.e., the microbial agent) underscores the importance of formulation in maintaining the microbial cells in a metabolically and physiologically competent state in order to obtain the desired benefit when applied. The development of new microbial formulations is a challenging task and requires greater effort in terms of funding and research towards making significant advances in this field.

Introduction

Worldwide, legumes are grown on approximately 250 million hectares and fix about 90 Tg (or 9 × 10¹³ g) of dinitrogen (N₂) per year (12). Rhizobium spp. and Bradyrhizobium spp. are major contributors to overall N₂ fixation through the legume-rhizobium symbiosis and have been used for over a hundred years as beneficial microorganisms. For example, the improvement of crop production through mixing of soil containing naturally-occurring rhizobia with seeds became a recommended practice in the USA by the end of the 19th century (24). Since then considerable improvement in this inoculant technology has been made, resulting in the successful commercialization of rhizobial inoculants by several companies around the world.

Development of a successful inoculant involves several critical elements such as strain selection, selection of a carrier and mass multiplication, formulation of the inoculant, and packaging and marketing. Stringent quality assurance at various steps of production ensures the production of consistently high quality inoculants.

Formulation is a crucial aspect for producing inoculants containing an effective bacterial strain and can determine the success or failure of a biological agent (1). Formulation typically consists of establishing the active ingredient (i.e., microorganism) in a suitable carrier together with additives that aid in the stabilization and protection of the microbial cells during storage and transport, and at the target site. Whether a product is new or improved, it is imperative that the formulation be stable during production, distribution, storage, and transportation. The formulation should also be easy to handle and apply so that it is delivered to the target in the most appropriate manner and form, protects the agent from harmful environmental factors, and maintain or enhance activity of the organism in the field (11). Another important consideration is the cost-effectiveness of the formulation. Therefore, several critical factors including user preference have to be considered before delivery of the final product.

Current Inoculant Formulations

Commercial inoculant formulations are available as powder, granule, and liquid. Generally, peat has been the preferred carrier in powder form (3,29). The rhizobial cells in the inoculant are metabolically active and continue to grow and multiply as long as favorable nutrient and environmental conditions are maintained. Superior seed adhesion is achieved with a finer particle and therefore it generally is recommended the particle size should allow at least 50% of the peat particles to pass a 0.075-mm sieve (29).

Some factors affecting the titre of rhizobia and its long-term storage survival are the type of peat, the origin and batch, as well as sterility of the peat (26). Peat is acidic and is neutralized prior to use in formulations as rhizobial carrier. Sterility of the peat carrier prior to inoculation with the rhizobial broth culture is the choice of the inoculant manufacturer. For example, the use of non-sterile peat as an inoculant carrier is popular in the USA and different methods have been developed (16). However, there are clear advantages to using sterilized peat, such as the high sustaining levels of rhizobia and longer shelf life (7) and higher yield in the field (8). Furthermore, they offer the option of extending culture production by diluting the broth without sacrificing the final inoculant quality (25).

A major consideration in the use of sterile peat, however, is the higher production cost compared to non-sterile peat. Other drawbacks include the production of toxic byproducts following heat and gamma irradiation of peat, which are detrimental to the growth and survival of rhizobia (15,18).

In addition, peat powder can be blown away easily from seeds by air-seeders. This problem has been rectified by the addition of adhesives to peat formulations, which has also ensured enhanced seed coverage. Despite these drawbacks, peat continues to be the major carrier of Rhizobium and Bradyrhizobium inoculants. Carrier alternatives to peat are being investigated in many tropical and sub-tropical countries, as these countries do not have a reliable peat source. In North America, non-peat powders such as clay and vermiculite-based rhizobial formulations have been successfully commercialized. Examples of such products include Nitragin Gold Inoculant (Nitragin Inc., Milwaukee, WI) and Dormal Plus (Becker Underwood, Ames, IA).

Increasing interest in the granular form of inoculants in the North American market (22,23) may be because granules are easy to apply and less dusty than powders. The ability of the end-user to place these granules relative to the seed during seeding can be optimized to obtain enhanced nodule occupancy of the inoculant (14). For example, it has been shown that Bradyrhizobia are capable of competing with indigenous soil strains but have limited mobility (14). This precludes the inoculant strain from sustaining high numbers in the developing root system, resulting in low nodule occupancy by the inoculant strain in the root system. This limitation could be overcome by placing the inoculant granule in close proximity with the seed. Furthermore, the inoculant granules are not in direct contact with chemical pesticide-treated seeds, and therefore, their survival is enhanced (5). However, granular inoculants are bulkier and have higher storage and transportation costs. Granular inoculants must be free-flowing when applied through seeding equipment, and free of "sticky" or "tacky" granule aggregates. Granules containing rhizobia are generally available as peat prills and as hard mineral-based products. Examples of such products include Soil Implant (Nitragin, Milwaukee, WI) and TagTeam Granular (Philom Bios Inc., Saskatoon, Canada). Another granule inoculant use is to mix mineral granules with a classical peat inoculant at sowing (30).

Ease of application of a liquid inoculant either on the seed or in situ delivery has enhanced the popularity and use of liquid formulations in several countries in the last decade. Researchers have shown that the performance of liquid rhizobial formulations is comparable to that of peat-based products under field conditions (9,10). Liquid formulations typically are aqueous-, oil-, or polymer-based products. Polysaccharides such as gums, carboxymethylcellulose and polyalcohol derivatives are frequently used to alter the fluid properties of liquid formulations (19). Several liquid formulations available today sustain high viable rhizobial numbers for extended periods of time. However, physiological changes in aspects such as on-seed stability and their ability to form nodules have been shown in Rhizobium that have been stored in commercial liquid formulations for several years (27).

Challenges in Formulation

In spite of the central role of formulation in the successful commercialization of inoculant products, research in this pivotal area has been largely ignored. In addition to the limited availability of published scientific information with regard to inoculant formulation, the information available is at best fragmented. A survey of the bibliography databases such as Biological Abstracts, CAB Abstracts, and Agricola in the last 23 years show that a significant amount of research on Rhizobium spp. has focused on the genetics of rhizobia and is comparable to studies on inoculation of legumes with rhizobia. However, little attention has been paid to formulation aspects of inoculants (Table 1). In fact less than one-half of one percent of research articles on Rhizobium spp. have focused on rhizobial inoculant formulations. Even fewer reviews are available on legume inoculant formulation, production, and application (2,6,24,28). Although this gap could be partly attributed to issues on infringement of intellectual property rights, this demonstrates a lack of effort and resource allocation for formulation research and underscores the need for basic formulation research. Typically, such efforts are best directed through universities and/or government research institutions (GRI) since the inoculant industry has limited research and development resources to spend on basic research. This highlights the need for mutually benefiting research collaborations between universities, GRIs, and industry.

Table 1. Survey of research articles published on Rhizobium and Bradyrhizobium spp*.

Database (and review period)	Total no. of articles	Aspects of Rhizobium/Bradyrhizobium research (percentage of total)
Database (and review period)	Total no. of articles	Genetics	Inoculation	Formulation
Biological Abstracts (1990 to 2003)	12933	7383 (57.1)	3023 (23.3)	57 (0.44)
CAB Abstracts (1972 to 2003)	23392	10115 (43.2)	9198 (39.3)	136 (0.58)
Agricola (1970 to 2003)	10917	1553 (14.2)	2138 (19.5)	23 (0.21)

^* Number of research articles indexed in various databases retrieved using several variants of the descriptors indicated.

The primary challenge in formulating beneficial inoculants is in recognizing the inherent “live” nature of the active ingredient. This characteristic of the biological agent differs from that of chemical agroproduct formulations which set high standards for efficacy and long storage lives. The live inoculant must be able to overcome the various technological processes during production, be viable over long periods of storage, and maintain its functional properties. Improving shelf life of an inoculant with a concomitant retention of desired biological traits is a major challenge for the formulator. This can be further exacerbated by strain-to-strain variability, which necessitates discrete research protocols for individual strains that adds further to the research and development cost. Furthermore, often the same organism has to be formulated in different forms to accommodate different climates, soil-types, and user preferences.

The formulated material must remain at acceptable titres for long enough to ensure that at least the minimum number of rhizobia can be applied to seed at the time of sowing. In Canada, seed inoculants must deliver 10³, 10⁴, and 10⁵ rhizobia per seed for small, medium, and large seeds, respectively, but some Canadian scientists recommended raising these standards (17). Granular inoculants must deliver at least 2.5 × 10¹¹ rhizobia per acre. Long-term shelf life can be achieved by either of two methods. The number of viable cells in the product can be increased so that despite a decline in titer, enough cells remain alive at seeding time to meet inoculant standards. If the decline in cells is rapid (greater than one log drop in six months) this method can be expensive, as the inoculant must start with 10 times the required rhizobia. The alternative strategy is to decrease the rate of decline so that the initial numbers of rhizobia do not have to be as high. Establishing realistic goals for formulation development must take into account the minimum titre required at seeding, the maximum titre achievable during production, the cost of achieving this titre, the length of time the product will be stored, and the temperature of the storage area (generally, cooler conditions lead to slower decline).

Cost must be a major consideration when developing an inoculant formulation. A seed inoculant in Canada and the USA sells for between $1.00 and $2.50 (US) per acre, while granular inoculants range from $6.00 to $7.20 (US) per acre. The cost of formulation materials, along with the costs of production, packaging, marketing, and retailing, must be low enough to allow sufficient profit based on these selling prices.

Future Prospects

Formulation is a challenging and often success-limiting step in the successful commercialization of new microbial inoculants. Although formulation research is progressing slowly, several developments including liquid and granular formulations have contributed to the ease of use at the farm combined with the economic benefits of increased crop yield levels.

Rapid progress in the field of genetic engineering and the resultant molecular tools now available may allow scientists to develop Rhizobium strains with desirable genetic traits. However, at this time any improvement of rhizobial inoculants still relies exclusively on formulation improvement, mutation, and strain selection (20), which appears to be a likely development in the future. Improvement in formulation is key to the development of enhanced high-end inoculants as the identification of new isolates with specific beneficial activities is often not difficult.

Formulation research thus far has been focused on selection of alternative carriers to peat. Several solid materials generally grouped as soils, plant materials, and inert materials have been investigated as alternatives to peat (24). Encapsulation of microorganisms in a polymer matrix as immobilized microbial cells is being examined as they are easy to produce, store, and handle during production processes (4). However, the high cost of production will be a significant factor in the consideration of this technology by the inoculant industry.

Recent advances in the understanding of the Rhizobium-legume signal transduction pathway (31) have led to the identification of signaling molecules such as lipo-chitooligosaccharides, which can be included in formulations to ensure early and enhanced nodulation. Inclusion of a slow-release form of molybdenum in the inoculant formulation has been suggested as another possibility to enhance nodulation (21).

One of the factors that limit the potential of a powerful inoculant may be environmental stressors such as high temperature and acidity. The adaptation of inoculant strains to these stressors through simulations of appropriate positive pressure (i.e., appropriate growth conditions and formulations) is an area that needs to be examined. For example, would decreasing water potential in the growth medium enable rhizobia to increase tolerance to dessication? Other areas that warrant attention include the development of multi-strain inoculants. Although some inoculant companies offer multi-strain formulations for increased product efficacy, further research on the formulation requirements of such products is needed.

Co-cultures of Rhizobium and other beneficial microorganisms are a logical next subject for formulation researchers. This is because select microorganisms that interact intimately with the crop can influence the efficacy of Rhizobium. For example, Rice et al. (22,23) have successfully co-cultured and commercialized the phosphate solubilizing fungus Penicillium bilaii with Rhizobium as a legume inoculant. Similarly, soybean co-inoculated with bradyrhizobia and Bacillus megaterium was shown to have enhanced nodulation (13). The potential of improving rhizobial inoculants by co-culturing with disease control agents and plant growth promoting bacteria is immense. With recent advancements in the field of genetic engineering and the possibility of introduction of genetically modified inoculant strains, it is imperative that formulation technologies be tailored to the physiological requirements of the genetically modified inoculant strains.

Acknowledgments

The authors thank Dr. Lisette Xavier for her constructive suggestions in the preparation of this manuscript.

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