Research Article: Production of a potential liquid plant bio-stimulant by immobilized Piriformospora indica in repeated-batch fermentation process

Date Published: May 25, 2017

Publisher: Springer Berlin Heidelberg

Author(s): Nikolay Vassilev, Bettina Eichler-Löbermann, Elena Flor-Peregrin, Vanessa Martos, Antonia Reyes, Maria Vassileva.


Piriformospora indica, a mycorrhizal-like fungus able to establish associations with roots of a wide range of plants, supporting plant nutrition and increasing plant resistance and tolerance to stress, was shown to solubilise phosphate applied in the form of animal bone char (HABO) in fermentation systems. The process of P solubilisation was caused most likely by proton extrusion and medium pH lowering. The fungal mycelium was successfully immobilized/retained in a polyurethane foam carrier. Further employment of the immobilized mycelium in repeated-batch fermentation process resulted in at least 5 cycles of P solubilization. The concentration of soluble P increased during the experiment with 1.0 and 3.0 g HABO l−1 and at the end of the 5th batch cycle reached 40.8 and 120 mg l−1, respectively. The resulting final liquid product, without or with solubilized phosphate, was found to significantly increase plant growth and P plant uptake. It can be used as a biostimulant containing microbial plant growth-promoting substances and soluble P derived from renewable sources (HABO) thus supporting the development of sustainable agro-ecosystems.

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Chemical fertilizers and pesticides are used for fertilizing crops and controlling pests thus successfully increasing production capacity of farm systems. However, the massive and long-term use of these chemical products has negatively affected the environment (soil quality/yielding capacity, biodiversity, underground water) and public health (Hazell and Wood 2008). The practice of intensive production methods and indiscriminate application of agrochemicals also provoked a decrease in plant resistance to biotic and abiotic stress factors. One of the most attractive alternatives of the intensive chemical-based production methods that make a positive contribution to environmentally safe sustainable agriculture is the use of plant beneficial microorganisms (Vessey 2003; Gray and Smith 2005; Singh et al. 2011). The beneficial microbial effects include promotion of plant growth, biological control of diseases, increases in crop yield, and quality improvement. In recent years, studies are carried out to produce biofertilizers as efficient formulated products in order to substitute for the chemical fertilizers. Beneficial microbial inoculants in agriculture are mainly plant growth-promoting bacteria and fungi that according to their function are grouped in biofertilizers and biocontrol agents. In fact, they are formulated products containing one or more microorganisms that enhance the nutrient status and health of the plants by either replacing soil nutrients and/or by making nutrients more available to plants and/or by increasing plant access to nutrients or by producing specific metabolites (Malusa and Vassilev 2014). The lack of success of biofertilizers to exert their specific functions reflects problems related with production and formulation of the inocula. The development of a biofertilizer starts with isolation/selection/characterization of an effective microorganism and ends with the main technological steps of the overall biofertilizer production process which are the fermentation mass production process and formulation procedure (Vassilev et al. 2015, 2016). Interestingly, scientific efforts are mainly concentrated on isolation and selection of plant-beneficial microorganisms and their further application in soil–plant system in controlled conditions although immense possibilities exists in developing biotechnological schemes for optimizing/combining mass production and formulation procedure (Vassilev et al. 2015) or direct application of fermentation products avoiding the formulation step.

Plant beneficial microorganisms produced in fermentation conditions, further formulated as bio-inoculants and applied in soil–plant systems, play an important role in sustainable agriculture by improving soil fertility and crop productivity (Bashan et al. 2014; Malusa and Vassilev 2014). The main points for the development of a commercial bio-inoculant include isolation, selection and characterization of suitable microorganism, large-scale production of biomass/spores, formulation, and testing in soil–plant systems (Vassilev et al. 2016). One of the important constrains for the large application of plant beneficial microorganisms is the fact that very few products are found to be promising (Bashan et al. 2014). This is particularly true for arbuscular mycorrhizal fungi (Faye et al. 2013), which, in addition, are difficult to be produced without host plant (Vassilev et al. 2005). During the last years, mycorrhizal-like fungi like P. indica, a symbiont root endophytic fungus that infests roots of a broad range of mono- and di-cotyledonous plants (Varma et al. 1999), are actively studied as they can be easily grown in axenic cultures on various complex and minimal substrates. Different techniques were studied to formulate P. indica including the production of cell-free products such as culture filtrates (Bagde et al. 2011; Kumar et al. 2012), The efficacy of P-enriched filtrates was recently reported for other fungi (Mendes et al. 2017). In this work however, the filtrate was produced applying the immobilized-cell technology that was widely used to study soil microorganisms (Vassilev et al. 2001). Moreover, the final product was enriched by soluble phosphate solubilized along the repeated-batch process. The results of this study are not surprising. Freely suspended mycelium of P. indica was recently reported to solubilize calcium phosphate and rock phosphate in fermentation processes in shake-flasks in Pikovskaya medium (Ngwene et al. 2016). However, this is the first report on solubilization of insoluble inorganic phosphate (animal bonechar) by immobilized P. indica. The reticulated structure of the polyurethane foam particles seems an ideal biomass carrier, as proposed originally by Atkinson et al. (1980). In this work, it efficiently enabled entrapment of biomass as reported for other, including P-solubilizing, fungal microorganisms (Vassilev et al. 1997, 2012). In the repeated batch process, we found no lag time for P solubilization at the first moments of each batch, indicating that the immobilized cells were metabolically active throughout the experiment. By this reason, we can explain the effectiveness of the shorter batch cycles and the enhancement of the volumetric productivity with each batch. HABO was recently reported as an excellent P-bearing source with a number of advantages compared to the finite, non-renewable rock phosphate (Vassilev et al. 2013). Its microbially based solubilization was also proved possible employing organic-acid producing filamentous fungi (Vassilev et al. 2012, 2013).