Bacteria for plants, plants for living

Written by: Martina Vavrusova Hedegaard, Ph.D. / Technologist / SD Freeze Drying

Plant production includes the food, feed, and fibre industries. Therefore, it represents a significant role in supplying goods for our daily lives. However, plant production is challenged by climate changes, environmental damages, continued population growth, and increasing demand for sustainable production.
With no time to waste, the industry needs to find smart, healthy, novel, and sustainable solutions (very fast) to meet the growing demands for plant food.
The use of microbial inoculants is not necessarily a new idea as it has been investigated since the ’80s. Microorganisms have been shown to be alternatives to chemical products in agriculture practices when applied as bio fertilisers or biopesticides.
Soil is full of microscopic life forms such as bacteria, actinomycetes, protozoa, fungi, and algae. Bacteria are by far the most common, often around 10⁸ to 10⁹cells per gram of soil.
The soil conditions like moisture, temperature, pH, presence of salts, minerals, and other compounds influence the number and the type of bacteria found in the soil.
Interestingly, bacteria are not evenly distributed in the soil. The most complex ecosystem is defined as the narrow layer of soil around the roots of plants and is known as the rhizosphere.
The concentration of bacteria around the roots of plants (rhizosphere) is much greater than in the rest of the soil due to the presence of nutrients. The abundance of root exudates such as amino acids, organic acids, fatty acids, phenolic compounds, plant growth regulators, sugars, sterols, vitamins, and other compounds is known to affect the microbial composition around the roots, the so-called rhizosphere effect.
There is more and more experimental evidence that underlines the importance of the root microbiome in plant health. The number of bacteria in the soil and the final effect may change as the conditions For example, the ability of bacteria to facilitate plant growth (by providing nitrogen or phosphorus) can be diminished when significant amounts of chemical fertilisers are added to the soil. Additionally, bacteria can stimulate the development of one plant but inhibit the development of another plant. However, as mentioned by Glick (2012), it is usually straightforward whether certain bacteria either promote or inhibit plant growth.

Plant growth-promoting bacteria in brief

In general, bacteria that promote plant growth and health are known as plant growth-promoting bacteria (PGPB).
PGPB are bacteria of different kinds like for example bacteria that form specific symbiotic relationships with plants, bacterial endophytes which colonise some or a portion of a plant´s interior tissue or cyanobacteria (formerly blue-green algae).
Despite the differences among the bacteria mentioned above, these bacteria utilise the same mechanisms, which promote plant growth directly or indirectly.
Direct mechanisms:
Facilitating resource/nutrients acquisition:
- Generally, bacteria provide plants with nutrients that they lack such as fixed nitrogen, iron, and phosphorus.

Modulating phytohormone levels:
- PGPB can alter the level of phytohormones and consequently influence the plant´s hormonal balance and its response to stress.

Indirect mechanisms:

Biocontrol bacteria can indirectly promote plant growth by decreasing the inhibitory effects of pathogenic agents. Therefore, understanding the mechanism used by biocontrol bacteria and using these bacteria instead of chemical pesticides are of considerable importance.

Importantly, the misconception about bacteria as agents causing sickness needs to be changed before beneficial bacteria are fully accepted by the public.
Therefore, a smart approach and good knowledge of the selection of microorganisms are required as well as the use of the best technologies for formulations and delivery approaches. The use of appropriate technologies will only ease an already bright future of PGPB in agricultural practice.

Formulation technology

Microbial products must exhibit a sufficiently long shelf life. This includes stability throughout the production, packaging, storage, and transport processes.
Subsequently, during application in the fields, the inoculant comes across factors that are detrimental to its viability. These factors include UV radiation and fluctuating soil properties such as temperature, pH, and texture.
Furthermore, actual biotic interactions with the native environment present a major challenge to any applied bacterial strains. It is not unusual, that the number of cells of introduced bacterial strains declines after the application to non-sterile soil because the bacteria are out-competed by indigenous microbes.
Obviously, already the production process causes pre-application stress factors which can lower the number of viable cells delivered to the field and therefore makes this problem even worse. The lower the number of viable cells delivered to the field, the less likely is a successful application at the target. Therefore, a mild formulation process is essential.
Microbial products are formulated as solids, liquids or slurries. Solid formulations may be divided into powders and granules depending on their particle sizes.
Standard carrier materials, apart from peat, for dry formulations are for example soil-derived carriers (e.g. clays and charcoal), organic carriers (e.g. sawdust, wheat/soy/oat bran, and cork compost), and inert material (e.g. perlite and kaolin).
In the case of liquid formulations, they are composed of oil- or water-based suspensions of cell concentrate, emulsions or slurries containing solid particles. Additives like nutritive substances, stabilisers, adhesives, and protectants are often added. The liquid formulations, like the solid ones, can be coated directly onto the seeds or may be delivered to the soil during sowing or at a later stage. However, liquid formulations may lack carrier protection.
In recent years, advanced formulation technologies like microencapsulation have also been developed and employed to produce diverse inoculants in morphology and composition.
As written in the review by Berninger (2018), the formulation technology depends on the application form, the available equipment, the farmer´s convenience, the plant inherent characteristics, the plant developmental stage, and the costs, etc.
In general, drying represents a challenge in formulation development. In solid formulations, drying occurs under controlled conditions during technological processing. Liquid formulations, when applied to seeds, face semi-controlled drying and after field application the conditions of drying are non-controlled. Furthermore, repeated drying-rewetting cycles concern both solid and liquid inoculants after field application.
Non-sporulating bacteria are sensitive to drying, as they do not form spores, because they are more susceptible to harmful factors occurring during processing and field application.
Therefore, drying is one of the main reasons for the loss of the viability of the bacteria prior to and after field application.
Importantly, there are many critical factors prior to and after drying, which need to be considered carefully. They include the composition of the growth and the rehydration media, the type of cryoprotectant, the cell concentration, the water content, the temperature, and the rate of cooling and thawing etc. These factors together with suitable packaging and storage conditions ensure the viability and longevity of microorganisms.

Freeze drying and life without water

Drying, in general, has been recognised as an efficient way of long-term preserving.
Life without water – anhydrobiosis – means the remarkable ability of some organisms to survive the loss of almost all The organism’s metabolism in such a state comes reversibly to a standstill (state of dormancy). Upon rehydration, the organism resumes its vital functions.
Dry products have a long shelf life, reduced costs during storage and distribution, and are less prone to contamination.
However, despite the benefits of drying bacterial cells, the drying process itself often leads to a pronounced initial drop in bacterial viability. Therefore, the bacterial survival rate represents the main quality parameter to be considered when screening for adequate drying techniques.
Among the other drying techniques, freeze-drying is one of the most frequently applied methods in the formulation and conservation of different bacterial strains.
Freeze drying is the preferred method of drying in the microbiological industry. A typical freeze-drying cycle consists of three stages: freezing, primary drying, and secondary drying.

Freezing is the process, in which the samples are exposed to a sufficiently low temperature to become a solid. A sample can be frozen before drying either in the freeze dryer chamber (using cooled shelves) or in a separate freezing unit. The way and the rate of freezing affect the crystal structure of the final product. Since the viability of the cells after drying is very important, a faster rate of freezing is preferred. A fast rate of freezing enables the formation of small crystals, which are more desirable, as large crystals may damage the fragile cell membranes. This can be achieved by snap freezing the product in liquid nitrogen, which is then loaded into the freeze dryer.
Primary drying is when frozen moisture is removed by sublimation of the ice crystals to water vapour. This is achieved by reducing the chamber pressure below the vapour pressure of the ice in the product. The sublimation front starts at the surface and moves towards the centre of the sample. Whilst frozen water is still present in the product, collapse and melting are possible if the product temperature increases above the so-called collapse temperature (T_c). At this temperature, the water molecules can re-enter into a highly mobile liquid state. This can cause loss of structure and/or damage the product and affect the final quality of the product. Therefore, the temperature of the sample must remain below T_c.
During the secondary drying, the residual (bound) water is removed by desorption from the sample. At this stage, the product may still contain 10 – 35% of bound water which is trapped within the solid matrix and therefore can take time to remove. It is very important to realise that the samples submitted to fast freezing produce numerous small crystals with a large surface area, which favours water desorption. Whereas a slow freezing rate results in a small surface area (large crystals) resulting in a slow drying rate during the secondary drying. The endpoint of the secondary drying can be determined by analysis of the residual moisture remaining in the freeze-dried product.

There are unlimited parameters within the freeze-drying process that can affect the quality of the final product and the drying time. These parameters should be carefully considered or/and tested prior to large scale production. Just to mention one example, different freezing and drying rates may result in different freeze-dried products. Considering the different material suspensions which have varying collapse or eutectic/glass transition points should also not be omitted.
One must remember that also design of an adequate freeze dryer is required to obtain the best quality of the final product.
Therefore, an interdisciplinary approach will ensure a better understanding of the product, the instrumentation, and the further agricultural applications.

Final thoughts

A strong interest in sustainable agricultural technologies opens new opportunities for many promising bacterial strains to enter the market. Below and above the ground, there is a fascinating life and a gentle technology such as freeze-drying can provide successful results which benefit people and respect nature.

For more information please contact SD Freeze Drying at info@sdfreezedrying.com

Literature

Compant, S., et al. (2019). A review on the plant microbiome: Ecology, functions, and emerging trends in microbial application. Journal of Advanced Research, 19, 29 – 37.
Berendsen, R. L., et al. (2012). The rhizosphere microbiome and plant health. Trends in Plant Science, 17, 8, 478 – 486.
Berninger, T., et al. (2018). Maintenance and assessment of cell viability in formulation of non-sporulating bacterial inoculants. Microbial Biotechnology, 11 (2), 277 – 301.
Prakash, O., et al. (2013). Practice and prospects of microbial preservation. Federation of European Microbiological Societies, 339, 1 – 9.
Glick, B. R. (2012). Plant growth – promoting bacteria: Mechanisms and applications. Scientifica, 1 – 15.
Malusá, E., et al. (2012). Technologies for beneficial microorganisms inocula used as biofertilizers. The Scientific World Journal, 2012, 1 – 12.
Morgan, C. A., et al. (2006). Preservation of micro-organisms by drying; A review. Journal of Microbiological Methods, 66, 183 – 193.
John, R. P., et al. (2011). Bio-encapsulation of microbial cells for targeted agricultural delivery. Critical Reviews in Biotechnology, 31 (3), 211 – 226.