3. Carriers

The carrier is the delivery material of live microorganisms from the processing plant to the field. It represents the major element (by volume or weight) of the inoculant and has a crucial significance in the delivery of the correct number of viable cells in good physiological condition. It provides a momentarily protective niche to microbial inoculants in soil: physically by provision of a protective surface of pore space (creating protective microhabitats) and nutritionally by provision of a particular substrate. Ideally, a good carrier possesses the following features:

• Provision of appropriate microenvironment to the target microorganism(s).
• Possession of appropriate physical and chemical properties: moisture absorption capacity (high water holding capacity), pH buffering capacity, and easy adjustable pH.
• Stability during the process: the carrier should be chemically and physically stable. It should be sterile or easy to sterilize (autoclaving or other methods), be free of protuberance materials, easily grinding and mixing with other substances (nutrients, adjuvants) using standard machinery equipment. It should also be applicable for as many bacterial or fungal species and strains as possible and simple to deal with and handle.
• Easy storage and inoculation: a good carrier should guarantee an adequate time span of usability (at least 2–3 months at room temperature), adhere well to and survive on seeds, and permit quick and controlled release of the microorganisms into the soil near the roots of the host.
• Economically and environmentally sustainable: that suggests a low cost and and reliable accessibility and quality. The carrier should be free of toxic materials, biodegradable, and non-polluting and minimize environmental risks (dispersal of cells to the atmosphere or ground water).

Selection of a carrier defines the physical form of the inoculant and clearly there can't be a perfect and widespread carrier for all microorganisms (Table 1). The carriers can be of various origins (organic, inorganic, or synthetic) and can be classified into four main categories:

• Soils: peat, coal, clays, lignite, inorganic soil
• Plant waste materials: charcoal, composts, farmyard manure, cellulose, soybean meal, soybean and peanut oil, wheat bran, press mud, corn cobs
• Inert materials: vermiculite, perlite, ground rock phosphate, bentonite, calcium sulfate, polyacrylamide gels, alginate beads
• Plain lyophilized microbial cultures and old dried bacteria: can be later incorporated into a solid carrier or used as they are

It is also possible to obtain carriers made of a combination of the above: mixture of soil and compost, of soil, peat, bark, and husks among others. Four dispersal forms are generally used: dry inoculant (powders), slurries (powder-type inoculants suspended in liquid), granules, and liquids. Peat is the most commonly used carrier, especially for bacterial inoculants. However, it is not easily accessible worldwide and its use has a undesirable impact on the environment and ecosystem from which it is extracted. This highlights the need of development of new formulations using alternative materials to compete with the existing inoculants.

3.1. Dry inoculants (powders)

Dry inoculants are delivered using soil, organic, or inert carrier. In many parts of the world, inoculants are formulated using peat (soil carrier). Peat is made of partially decomposed flora accumulated over the years. It provides a nutritive and defensive growth environment of an extensive variety of microorganisms which can develop and form microcolonies both on the surface of the particles and in fissures. To be appropriate for inoculant use, peat must be nontoxic (for microorganisms, plant, animals, and human), highly adsorptive and easily sterilized, have a high organic matter content and water-holding capacity, and be available locally at a reasonable cost. Peat has been principally utilized because it is widely available. However, its processing is expensive as it requires several steps before it can be used as carrier for inoculant. Harvested peat must be drained and sieved to remove coarse material before it is slowly dried to around 5 % moisture. This drying step is of crucial significance since it can prompt to the formation of toxic compounds. The drying should be carried out at the lowest possible temperatures and certainly never surpass 100 °C. Air drying is the preferable method instead of oven drying. The type of peat and the particle size desired defines the extent of drying. However, the moisture content must be decreased adequately to guarantee that the subsequent addition of liquid culture brings the final moisture content of the inoculant to the sought level. Once dried, peat is ground, commonly to pass through at least a 250-μm sieve. Generally the peat deposits have a low pH, which must be corrected to pH 6.5–7.0. The peat is then sterilized and an adequate amount of liquid inoculum is added to it.

In the case of bacterial inoculant, a final moisture content of 40–55 % is generally acceptable. Inoculated peat is incubated for a certain period of time to allow bacteria multiplication in the carrier. This step, also called maturing or curing is of major importance since it improves the bacteria survival rate during storage and on seeds. Peat can also be used for AMF and ectomycorrhizal inoculants though the latter are not broadly utilized, except for forest regeneration. Ectomycorrhiza generally are grown in glucose containing medium and produced spores are used for inoculation. Pure mycelia cultures are preferred as they supress growth of pathogens and contaminants. Ectomycorrhizal inoculants may be formulated using a carrier made of vermiculite and 5–10 % peat moisturized with salts and glucose nutrient medium. This formulation provides a strong buffering capacity (keeping pH below 6) and enhances the production of fulvic acid that stimulates growth.

Table 1. Advantages and limitations of the most common carriers

Carrier	Benefits	Restrictions
Peat	Suitable for a wide range of microorganisms: bacteria, AMF, ectomycorrhizal	Not readily available
Liquid	Easy to handle and apply	Lack carrier protection: low viability during storage and on seeds
Granules	Easy to store, handle, and apply	Bulky: high transport and storage costs
Lyophilized encapsulated cells	Suitable for all types of cells (all sizes)	High production cost

• Protective nutritive environment
• Moisture content can be adjusted to improve growth and survival of bacteria during curing, storage, and inoculation
• Strong buffering capacity
• Strong negative impact on the environment and the ecosystems
• Costly investment for extraction
• Toxic compounds released during drying and sterilization
• Highly variable in composition and quality depending on the origin
• Seed application: contact with other chemical compounds (toxicity)
• Easy addition of additives to improve growth or survival of the cells
• Composition easily defined and controlled
• High cells concentration → low application rates
• Cool temperatures for storage (4 °C)
• Limited shelf life
• More sensitive to stressful conditions
• Less dusty than peat
• Application rate easily assessed
• Soil application: no direct contact with the other chemical compounds (no toxicity)
• Especially efficient under stressful environmental conditions
• Higher application rates
• Often nonsterile carriers
• Cells protected in a nutritive shell against mechanical and environmental stresses and against predators
• Slow and controlled release of the microorganisms when the shell is degraded
• Wide variety of polymers: nontoxic, biodegradable
• High concentration of cells/shell → limited space for storage
• Storage at room temperature (dried capsules)
• More handling work at the industry level
• Specific equipment required
• Physiological, morphological, and metabolic changes occurring in the shell
• Several applications needed if strains cannot establish in soil
• No commercial product available

Inoculated peat is typically applied on-site on the seeds just before sowing. The required amount of product is relatively small. However, the quantity of microorganisms used per seed is not well controlled as they are in direct contact with the other chemicals which may have been covered on the seeds. The seed coating can be done by machines (large dough, cement mixers, and mechanical tumbling machines). This procedure allows the inoculation of a large number of seeds. The significant disadvantage of peat originates from the variability in its quality and composition, which are source-dependent. Peat is an undefined and complex material and different sources will vary in their ability to support cell growth and survival. Toxic compounds might also be released during sterilization, negatively influencing the growth and survival rate of desired microorganisms. This may bring about challenges to guarantee reliable quality and results in the field, as well as to identify the optimal storage conditions, or usage instructions. Regardless of these restrictions, peat remains the standard by which every other material is judged.

Coal, clays, and inorganic soils (i.e., lapillus, volcanic pumice or diatomite earths) are available in different areas and could be utilized as carriers. Their microbial load depends on the deriving place (about 10²-10³ CFU g−¹), but it is generally lower than in organic carriers. Vermiculite, perlite, and bentonite are also available in different countries, but their application in general is restricted due to the difficulties in preparing an effective formulation. In reality, the impact of these carriers on bacteria viability and growth is dependent on the pH, ion strength, and the electrolyte in solution. Expanded clay has been tested as a carrier for AMF and mycorrhized roots mixed with soil are also used for AMF inocula. Among other inorganic compounds, glass beads have also been proposed for AMF inocula. A mixture of organic and inorganic materials has been demonstrated successful in increasing activity and shelf life of Burkholderia sp. The majority of the previously mentioned carriers depend on the absorption of the microorganisms by the substance/matrix of the carrier. This strategy for incorporation has some disadvantages, especially in relation to the survival of the microorganisms and their protection during transport, storage, and handling. Nevertheless, some procedures with different carriers using such approach have been patented:

1. the Belgian patent no. 521.850 for use of diatomaceous earth and colloidal silica for Rhizobium,
2. the British patent no. 1.777.077 for the use of bentonite for Rhizobium,
3. French Patent no. 1.180.000 using a must juice, to which substances with an adsorbing action are added, such as cellulose, bone meal, kaolin, or silica gel, in the manufacture of preparations rich in bacteria of the Azotobacter group,
4. United States Patent no. 4956295 for the stabilization of dried bacteria extended in particulate carriers, where dried viable bacteria are mixed in a particulate carrier composed primarily of an inorganic salt of low moisture absorbing capacity together with a minor proportion of a silica gel absorbent. The inorganic salts may be sodium or calcium carbonates, bicarbonates, sulfates, or phosphates.

3.2. Granules

To overcome the disadvantages in application of peat, the interest in other types of formulations and especially in granular inoculants is increasing. Granules are made of peat prill or small marble, calcite, or silica grains that are wetted with an adhesive material and then mixed with a powder-type inoculum. Thus the granules are coated or impregnated with the target microorganism(s). The size of the granules varies, however the relation between initial microbial population density and finished product quality is direct: the better the initial microbial population, the better the product. Granules have many advantages over peat. They are less dusty and easier to handle, store, and apply. The placement and the application can be easily controlled and the limitations of seed applications are overcome: the inoculant is placed in a furrow near to the seed to facilitate lateral–root interactions but is not in direct contact with the chemicals or pesticides potentially toxic for the microorganisms. Limits in granules applications are related to the fact that they are bulkier and the transport and storage costs are therefore higher.

The prevalence of rhizobial granular inoculants over peat and liquid inoculants has been evaluated in several studies and obtained results are variable. A few reviews demonstrated that granular application of rhizobia did not display predominant nodulation or biological N₂ fixation compared with the other formulations (peat and seed coating), while other studies on inoculation of legumes showed that granular formulations are superior to peat-based products and liquid inoculants in terms of number of nodule formation and weight, N accumulation, N₂ fixation (% Ndfa), and total biomass generation. The benefits of using granular inoculants are particularly advantageous under soil stress conditions like high acidity, moisture stress, or cool, wet soils.

3.3. Liquid inoculants

Liquid inoculants are based on aqueous (broth cultures), mineral or organic oils, oil-in-water or polymer-based suspensions. Liquid products have been elevated as being simpler to handle and apply either on seeds or in soil. So, their ubiquity has expanded in the most recent decade. They are currently popular and have been applied for legume inoculation (in the USA and Canada for instance) due to their high cell concentrations. This characteristic allows the application of a lower quantity of inoculant for a similar efficiency. However, a number of limits blocked their utilization: inoculants based on liquid cultures lack carrier protection and quickly lose viability on the seed. They require more particular storage conditions (cool temperatures) and generally have a limited shelf life. It was additionally revealed that liquid inoculants were more sensitive to environmental stresses and poorly survived in the carrier. Application of some other components (sucrose, glycerol, gum arabic, PVP) may improve survival of microorganisms in liquid inoculants.

3.4. Polymer-based carriers (cell immobilization)

The advance made in formulation improvement has led to new types of microorganism entrapment and immobilization processes that seem particularly promising. Immobilization encompasses the different forms of cell attachment or entrapment into a matrix. These include flocculation, adsorption on surfaces, covalent binding to carriers, cross-linking of cells, and encapsulation in a polymer gel. Encapsulation has proven to be the most promising technique for development of microbial carriers. Once encapsulated, the living cells are protected in a nutritive shell (or capsule) against mechanical and environmental stresses (such as pH, temperature, organic solvent, or poison) and predators. When placed into the soil, soil microorganisms slowly degrade the capsules and the target cells are gradually released in large quantities. Usually this happens during the time of seed germination or seedling emergence. Different kinds of cells could be encapsulated, including bacteria, fungal spores, or small hyphal segments. In this way the encapsulation procedure represents a promising technology for development of single and multiple strain products, such as PSB–AMF or rhizobia–AMF-based ones.

Different kinds of polymers may be used for encapsulation: natural (polysaccharides, protein material) or synthetic (polyacrylamide, polyurethane) and homo-, hetero-, or co-polymers. There are more than 1,350 possible combinations of polymers which can be applied for encapsulation. Selection generally is made on the basis of their chemical composition, molecular weight (too low or too high molecular weights being considered as a disadvantage), and their ability to interact with other components. Polyacrylamide and alginate are the most commonly used polymers for cell encapsulation. However, alginate is preferred since polyacrylamide requires more specific handling precautions due to its toxicity. Alginate is a natural, biodegradable and nontoxic substrate which forms a 3D porous gel when mixed with multivalent cations (Ca²⁺). To form beads, microorganism cells are dispersed into the polymer matrix and the mixed solution is simply dropped in the cationic solution. Nutrients and other supplements can be included to prolonged shelf life and inoculation efficacy. The beads are then dried for simplicity of packaging and handling. Different technologies are applied (including spray drying, extrusion, emulsion technique, coacervation, solvent extraction/evaporation, thermal gelation, pre-gel dissolving technique) to control the size, the shape, and the texture of the beads. Smaller beads of 10–100 μm (microencapsulation) are preferred since they offer direct contact with seeds, while macroencapsulation (larger size, extending from a few millimetres to centimetres) requires the released cells to move through the soil toward the plants.

Inclusion of bacteria in alginate beads has been used for various species, either spore forming or not. Different AMF have also been entrapped into alginate matrixes or in beads formed with different polymers. Spores of mycorrhizal fungi were entrapped in alginate film formed in a PVC coated fibreglass screen. Roots of leek seedlings inoculated with this alginate film containing G. mosseae spores were heavily colonized after few weeks of growth in greenhouse conditions. Similar results were obtained with spores obtained from monoxenic cultures embedded into beads. Inclusion of filamentous microorganisms such as Aspergillus and Actinomycetes has been also proved possible.

Several positive effects over free cells (conventional formulations) have been reported. Besides the cell protection provided by the shell, different studies under numerous conditions have revealed that encapsulation has numerous advantages during storage and field applications. This process is not stressful to cells, aseptic conditions minimize contamination, and the carriers are biodegradable and nontoxic. As the beads can be highly concentrated, their volume is very low, and thus, limited space for storage is required and transportation and handling are facilitated. They have an extended shelf life, can even be stored dried at room temperatures for relatively long periods, are easy to use, and are of consistent quality. When are microencapsulated the cells are distributed uniformly to the targeted site, even on small seeds, thus enhancing the application efficacy. As a result the cell movement through soil and the possibility of off-site drift during application are significantly reduced. It was also demonstrated that encapsulation of PSB microorganisms increased their P solubilisation capacity and their potential to promote plant growth compared to free cells. Limitations include a high production cost, more handling work at the industry level, and special equipment requirements. It was also mentioned that physiological, morphological, and metabolic changes may occur in encapsulated cells and that repeat applications of beads may be required since cells may not establish outside of beads.

Despite the fact that encapsulation seems to have a relative success, the vast majority of the research was performed in laboratory conditions and up to now no commercial bacterial product is available on the market. One of the explanations of the non-adoption of the technology by the inoculant industry might be the high production costs and technical handling. New technologies have to remain affordable and cost effective to be easily implemented by manufacturers and farmers.

Reducing the cost of the production process and improving the quality of the beads were acheived by encapsulation and air-drying of bacteria into a mixture made of alginate (3%), standard starch (44.6%), and modified starch (2.4%). This process permitts to obtain beads that after drying have a water content of 7%, size of 4 mm, and a mechanical resistance of about 105 Newton (features similar to that of grain seeds). Encapsulated bacteria can be stored at room temperature or at 4◦C without losing their viability - they are able to survive up to six months maintaining a final population size of about 10⁸ CFU g⁻¹ (corresponding to about 10⁵ CFU bead⁻¹). However, with this composition, some problems can arise when standardizing and automating the beads formation due to the viscosity of the mixture and the need of a continuous agitation of the stock medium. Recently, a new procedure was proposed, using starch industry wastewater as a carbon source for the production of Sinorhizobium meliloti with simultaneous addition of alginate and soy oil as emulsifier. Results obtained showed a cell viability of more than 10⁹ CFU mL⁻¹ after 9 weeks of storage. Addition of synthetic zeolite to the alginate mixture did not improve the survival of the embedded microbial cells, nor the physical structure of the beads.

Different other polymers have also been tested with AMF. Carragenan was used to encapsulate AMF communities while hydroxyethylcellulose was used as a gel carrier. Two patents have also been registered:

1. French Patent application no. 77.10254 (corresponding to U.S. Patent no. 4.155.737) which makes use of a polymer gel based on polyacrylamide gel or a silica gel for different microorganisms,
2. the US patent 5021350 on the process for inclusion of mycorrhizae and actinorhizae in a polymer gel matrix based on at least one polymer from the polysaccharide group, with at least partial crosslinking of the polymer.

3.5. Other carriers

An extensive variety of materials, both natural and artificial, have been tested and assessed as alternative carriers for diverse microorganisms. The principle drivers for the utilization of another carrier appear to be its supply and cost rather than a requirement for better quality and that works against their more widespread adoption.

Several cheap organic matrixes including water sludge, composts, sawdust, sugarcane bagasse, whey, or enriched agro-industrial residues have been proposed. Sludge wastewater might be an appropriate carrier but it contains heavy metals and this pose legal problem in respect to its utilization. Good alternative to peat is the compost from the cork industry. It is better in maintaining the survival of different rhizospheric bacteria during 6 months of storage as well as survival on seeds. However, organic composts may not be applicable for AMF formulations as they can decrease the mycorrhization rate.

Coal, clays, and inorganic soils (lapillus, volcanic pumice, or diatomite earths) can be used where available, though microbial concentration is lower than in organic carriers. In Madagascar, AMF production was done using Pouzzolane, a volcanic rock. Utilization of perlite as an inoculant gave variable outcomes. It is a suitable carrier but less efficacious than cork- and peat-based inoculants. Its effectiveness was increased when sucrose was employed as adhesive.

Gels of various chemical compositions (including magnesium silicate, fluidized bed or cellulose-based gel) is regarded as having a potential but none of them have been adopted on-farm till now.

3.6. Promising New Technologies for Carriers Development

Water-in-oil emulsions seem to be a good, yet underutilized, method for storing and delivering microorganisms through liquid formulations. The oil traps the water around the organism and, therefore, slows down water evaporation once applied. This is especially helpful when microorganisms sensitive to desiccation are used or in case of horticultural crops where irrigation systems are in place. Water-in-oil emulsions permit the addition of substances to the oil and/or aqueous phases. In this way both cell viability and release kinetics are improved. However, cell sedimentation during storage is a major issue to be considered. Several studies are carried out trying to solve this problem through application of nanomaterials. Thickening the oil phase using hydrophobic silica nanoparticles essentially diminished cell sedimentation and enhanced cell viability during storage.

Recently, a new procedure for encapsulation of virus formulations based on the application of supercritical fluid properties has been proposed. Same idea could also be applied to prepare bacterial inocula. The process, named PGSS (Particles from Gas Saturated Solutions), is carried out at low temperatures and uses carbon dioxide as a supercritical fluid. Main advantages of proposed technic would be lack of negative effects on the microorganisms’ viability, and the low cost of production. The final product of the process is almost spherical particles that form a free-flowing powder which can be suspended in water. The possibilities of the PGSS process have already successfully been demonstrated for several solids and liquids.

Another interesting innovation is the exploitation of the natural production of bacterial biofilms as a possible carrier. It could be applied not only for the production of the bacterial inoculum but also for fungi-bacteria consortia. Biofilms are already obtained for different industrial applications (e.g., wastewater treatment, production of chemical compounds). Two types of biofilms are considered: biofilms growing onto inert supports (charcoal, resin, concrete, clay brick, and sand particles) and biofilms that are formed as a result of aggregate formation. In the first case, microorganisms grow all around the particles, and the size of the biofilm grows with time usually to several mm in diameter. Biofilms formed by aggregation is called granular biofilms and their formation may take from several weeks to several months.

There are four phases in the development of a mature biofilm: i) initial attachment, ii) irreversible attachment, iii) early development, and iv) maturation. Particularly critical is the irreversible attachment when cells bind to the surface and extracellular polymeric substances (EPS) are generated. Thus microorganisms are protected from the surrounding environment. EPS generally are composed form polysaccharides, proteins, nucleic acids, or phospholipids. A typical EPS excreted by bacterial cells in biofilms is the exopolysaccharide alginate (Fig. 4 and 5).

The rate of biofilms formation and maturation is affected by surface, cellular, and environmental factors. Rough surfaces, porous, and less hydrophobic materials tend to improve the biofilm formation. Biofilms tend to form more readily in the presence of optimum nutrients availability, particularly of phosphorous which increases the adhesion ability of cells. Other factors positively influencing the biofilm formation are high temperature, EPS production, and surface adhesion. Biofilm reactors can be assembled in a number of configurations including batch, continuous stirred tank, packed bed, trickling bed, fluidized bed, airlift reactors, up flow anaerobic sludge blanket, and expanded bed reactors.

Fig. 4. Fungal –bacterial biofilm (FBB)

Fig. 5. A fungal–rhizobial biofilm (FRB) on a wheat root.

Recently, with good practical efficacy for nonlegume species biofilms were used that were developed in in vitro cultures containing both fungal and bacterial strains. Application of this biofilmed fungalrhizobia consortium led to significantly increased N₂ fixation in soybean compared to a traditional rhizobium inoculant. Wheat seedlings inoculated with biofilm-producing bacteria exhibited an increased yield in moderate saline soils. Moreover, experimental data showed that biofilms protect microorganisms and assure their survival even under stress conditions. The last issue is from key importance for the effectiveness of PGPM inoculation under agricultural conditions. It was reported that biofilmed inocula allow rhizobia strains to survive at high salinity (400mM NaCl) by 10⁵-fold compared to rhizobial monocultures. Interestingly, it was observed that beneficial endophytes in biofilms produce higher acidity and plant growth-promoting hormones than their mono- or mixed cultures.

Another new frontier in the development of carriers for PGPMs is production of hybrid materials for inoculating microorganisms. Silica has appeared as a promising host for encapsulation: technic is based on dispersing of bacterial population into a silica gel and its immobilization. Cell can be either entrapped into alginate microbeads coated with silica membranes or into macrocavities created inside the silica matrix. Such hybrid material improves the mechanical properties of the alginate bead, reduces cell leakage, and enhances cell viability.

The application of bionanotechnologies could also provide new directions in the development of carrier-based microbial inocula. Nanoparticles made of inorganic or organic materials are employed in dimensions 100 nm and less. The integration of whole cells within hybrid nanostructures have numerous applications in many fields including agriculture. Already macroscopic filters, made of radially aligned carbon nanotube walls, able to absorb Escherichia coli, were fabricated. This technology was applied to collect bacterial cells from fermentation processes and deliver them to the plant. The physical stability and the high surface area of nanotubes, together with the ease and cost-effective fabrication of these membranes, may also expand in the production of biofertilizer.

The use of nanoformulations may improve the stability of biofertilizers and biostimulators with respect to desiccation, heat, and UV inactivation. The addition of hydrophobic silica nanoparticles of 7–14 nm to the water-in-oil emulsion formulation of the biopesticide fungus Lagenidium giganteum reduces the desiccation of the mycelium. The physical features of the formulation are improved and the microorganism are still viable and active after 12 weeks of storage at room temperature.