2. Production of inoculants

Development of an effective inoculant represents a multistep procedure comprising the attachment of one or more strains of microorganisms in a particular carrier together with sticking agents or other additives which assure the protection of the cells during storage and transportation. Since the inoculants are often stored under non-optimum conditions (e.g., high temperature, light exposure), they must have an extended shelf life, i.e., the microorganism should be either robust or to have greater capacity to survive in high numbers under harsh conditions. A good formulation will also provide effective introduction of microorganisms in the soil and will enhance their activity in order to obtain the maximal benefits after inoculation to the host plants. To be easily accepted by the farmers, an inoculant must be cost effective and simple to deal with and use, to guarantee that the microorganisms are delivered to the target plant in the most suitable way and form. Formulation is a crucial issue and limited investigations were performed in this subject. Available data showed that since the 1980s, most rhizobial research are concentrated on the bacterial genetics and physiology and less than 1 % - on formulation aspects of rhizobia inoculants. In any case, there is a real need for improved formulations of inoculants, to develop and commercialize new biofertilizers that will be more successful, more stable over time, of better quality, and addressing agricultural needs.

The ideal formulation does not exist and obviously every type has its own particular advantages and constrains. However, there are some critical steps which must be precisely considered during the biofertilizers production. The choices made at these steps can lead to the success or the failure of the inoculation. The decision of the microorganisms to be inoculated is of crucial importance. Some of the most important desirable characteristics of the inoculant strain (bacterial or fungal) include its genetic stability, its ability to be beneficial for the target crops, to be competitive to the indigenous populations, to migrate from inoculation site to the hosts, and to survive in hostile soil without the presence of the host. Other important features sought during production is the ability of the strain to grow in laboratory conditions (exception is made for AMF which cannot grow without a host plant), grow or survive in carriers (during curing or storage), on seeds and in soil and to be compatible with agrochemical products that might be applied on seeds. The live inoculant must also be able to overcome the various technological processes during production and maintain its functional properties. Bacterial inoculants are generally cultivated in liquid medium to reach high biomass yields. The composition of the media and growth conditions (temperature, pH, agitation, aeration, etc.) are directly related to the physiology-biochemical properties of the particular strain and the kind of inoculant that is to be produced. Obtained bacterial cultures are then used to inoculate the different carriers (encapsulation or impregnation of peat and granules), or after addition of various additives liquid formulations could be produced. The large-scale production of bacteria in pure cultures using bioreactors is wildly spread common practice (Fig. 2).

Fig.2. Mass-production of Azolla

In this way, once the specific strain/s for the inoculum has been chosen, an industrial standardized procedure of production can be defined. However, for biofertilizers, dissimilar to biopesticides, the cost of production is an important limitation. This is due to the fact that the price of the biofertilizer shall not exceed that of the conventional ones. Hence, several cheap raw materials (e.g., whey, water sludges, composts, etc.) have been utilized as growth media for PGPM. Another approach to diminish the production costs is by using agro-industrial residues enriched with rock phosphate. During composting or fermentation, free or immobilized microorganisms that produce organic acids are added to the matrix, enhancing the solubilisation of phosphate and thus making it more available to plants.

Recently, the use of biofilms has also been applied as possible means to produce effective plant inocula. A biofilm comprises of microbial cells embedded into a self-produced polymeric matrix (known as an extracellular polymeric substance—EPS) and adherent to an inert or living surface, which provides structure and protection to the microbial community. Three major types of biofilms are observed in the soil: bacterial (including Actinomycetes), fungal, and fungal-bacterial biofilms). Both bacterial and fungal biofilms are formed on abiotic surfaces, while fungi act as the biotic surface in formation of fungal-bacterial biofilms. The majority of plant-associated bacteria found on roots and in soil are forming biofilms. Therefore, applying PGPM strains that form biofilms could be a successful strategy in formulation and production of biofertilizers. While ectomycorrhizal fungi can be produced under fermentation conditions, the production of AMF inocula is much more difficult due to the need of a plant host for the multiplication of the mycorrhizal fungi. The first attempts in AMF production are based on pot cultures with soil mixtures, or aeroponics. However, the development of monoxenic cultures in the late 1980s has allowed the production of AMF under strictly controlled conditions. A method was developed for production of spores by using split-plate cultures and Ri T-DNA transformed roots of carrots. However, although the method allows production on average of 15.000 spores per Petri dish in 4-5 months after beginning the production cycle, it has been used mainly for physiological and laboratory studies. The improvement of this method was achieved through replacing the media in the distal compartment every 2 months with parallel replenishing the carbon source in the proximal compartment with glucose. Obtained results lead to the production of about 65.000 spores in 7 months. Yet, such methods are mainly used for experimental batch production of spores or for maintenance of gene banks. The reason is that the estimated annual cost for producing of one spore is up to 30–50 USD, depending on the method utilized. Recently, a large-scale in vitro production of mycorrhizal fungi, feasible for implementation on a commercial scale, has been proposed. It is based on several key points: selection of appropriate Ri T-DNA transformed host roots for different AMF species, selection and maintenance of optimal growth medium, and application of quality assurance procedures.

However, commercial inoculants containing AMF species are still produced mainly by growing host plants in controlled conditions, with the addition to the inoculant of various fungal structures (spores, mycelium hyphae) and containing mycorrhizal roots residues from the plants used as the propagating material (i.e., sorghum, maize, onion, or Plantago lanceolata) (Fig. 3). This could be considered a classical method where substrates of sand/soil and/or other materials (e.g., zeolite, perlite) are used to mass-produce AM fungal inoculum in pots, bags, or beds, for large-scale applications. Critical issues in this production strategy are:

1. usage of known AMF species,
2. selection of host species with a short life cycle, adequate development of the root system, a good colonization level by a large range of AM fungi, and tolerance to relatively low levels of phosphorus,
3. control of mineral nutrients level in soil,
4. suitable combination of AMF species and host plant.

With this technique, it is possible to achieve inoculum densities of 80–100 thousand propagules per litre. This implies the need of diluting the inoculum with a carrier for the preparation of a commercial product.

Fig.3. Plantago lanceolate root nodules

Considering that microbial associations between bacteria and mycorrhizal fungi occurring naturally in the soil can promote the mycorrhizal symbiosis, it could be suggested that formulations including two or more species of different PGPM would have enhanced beneficial effect on plants. Microbial consortia can stimulate plant growth through a range of mechanisms that improve nutrient uptake and supress fungal plant pathogens. The different approaches proposed to explain such growth stimulation are based on the increased rate of nutrients cycling. The last is due to the greater microbial content and biodiversity found in the soil where mycorrhizal plants are grown. Simultaneous inoculation with different PGPR and/or AMF often resulted in increased growth and yield, compared to single inoculation through improved nutrient uptake. Indeed, the interactions between bacteria and AM fungi have positive effect on nutrient uptake, particularly when PGPR and N₂-fixing bacteria are combined. Inoculation of maize and ryegrass with A. brasilense and AMF resulted in N and P contents comparable to plants grown with fertilizer. Co-inoculation with different AMF species is generally more effective due to the lack of AMF fungi colonization specificity for define plant species/cultivars. Synergistic interaction between AM fungi and several PGPR, including Azospirillum, Azotobacter, Bacillus, and Pseudomonas species, has also been reported as favourable for plant growth. Improved root colonization by AMF was observed when mycorrhizal fungi were co-inoculated with such PGPR. Four times higher nodule number was reported when plants were inoculated with a mixture containing Glomus deserticola and Rhizobium trifoli, in comparison to single R. trifoli, inoculation, and enhanced mycorrhization and nodulation was observed with co-encapsulated R. trifoli and Yarrowia lipolytica. Inoculation with nodule-inducing rhizobia and AM fungi resulted in increasing both P and N uptake efficiency. Application of PGPM as commercial biofertilizers containing consortia of different microorganisms often leads to diminishing the infection rate, better mineral nutrition, and increased plant growth. All these examples are are indicating the convenience and higher adequacy of biofertilizers composed by more species having different mechanisms of growth promotion. The possibility for testing of several strains of PGPR and AMF in different crops species and under different field conditions should allow the definition of consortia suitable for commercial uses.