High-density spore production of Piriformospora indica, a plant growth-promoting endophyte, by optimization of nutritional and cultural parameters Vinod Kumar, Vikram Sahai, V.S. Bisaria ⇑ Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India article info Article history: Received 9 March 2010 Received in revised form 22 October 2010 Accepted 25 October 2010 Available online 30 October 2010 Keywords: Piriformospora indica Dry cell weight Spore yield Biomass yield Kaefer medium abstract Piriformospora indica is an axenically cultivable root endophytic fungus which exerts plant growth pro- moting effects on its host plants. To enable commercial production of its spores, the medium composition and culture conditions have been optimized in a 14 L bioreactor such that they result in maximum bio- mass during growth phase and in maximum spore yield during subsequent sporulation phase. Maximum spore yields were obtained with modified Kaefer medium using a glucose deprivation strategy. An enhancement of 100% in overall biomass productivity (0.18 g L 1 h 1 ) and reduction of about 70% in the time (60 h) required to achieve the maximum spore yield (9.25  10 7 spores/mL) was achieved in comparison to the original Kaefer medium. The high spore yield obtained in the present study seems to be economical for commercial production of P. indica. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Piriformospora indica, a root endophytic fungus, was isolated from the rhizosphere of the woody shrubs Prosopsis juliflora and Zizyphus nummularia growing in desert interior of Rajasthan, India (Verma et al., 1998). It exhibits most of the beneficial characteris- tics of arbuscular mycorrhizal fungi (AM fungi). Like AM fungi, it has a broad and diverse host spectrum and exerts plant growth- promoting effects on its host plants (Singh et al., 2000). But the most important advantage of P. indica over AM fungi is that it is a facultative symbiont and can be easily cultivated axenically on a variety of synthetic media (Varma et al., 2001). P. indica belongs to the division of basidiomycota. It has simple septum with dolip- ores and continuous, straight parenthosomes. It promotes the growth of plants and improves their productivity, increases the drought tolerance of host plants, delays the wilting of leaves, pro- longs the ageing of callus tissue and protects the plants from the attack of pathogens (Waller et al., 2008; Zuccaro et al., 2009). It en- hances the phosphate uptake by the plants (Yadav et al., 2010). The biological hardening of tissue culture-raised plants with P. indica protects them from transplantation shock and increases their sur- vival rate to 90–100%. The stress conditions caused due to acidity, desiccation and heavy metal toxicity are relieved by P. indica (Kumari et al., 2004). The fungus also induces systemic disease resistance by enhancing the concentration of antioxidants, ascor- bate and glutathione, in the plant body to cope up with the oxida- tive stress caused by pathogens (Waller et al., 2005; Vadassery et al., 2009). Thus P. indica shows tremendous potential to be used as a biological agent for plant growth promotion and control of plant root disease and a tool for biological hardening of microprop- agated plants. Another advantageous feature of the fungus is that it produces a large number of thick walled, pear-shaped spores called chlamydospores having longer shelf life (Wall and Lewis, 1980; Lewis and Papavizas, 1983). These spores, rather than fungal myce- lia, are to be used as bioinoculant for agricultural crops. The spores can be produced easily, can survive unfavorable conditions and germinate on the onset of favorable conditions, which leads again to vegetative growth. All these qualities make spores a good candi- date from application point of view. This is the reason that among the microbes capable of exerting plant growth-promoting effects, which can be used as biological agents against plant pathogens, sporeformers are receiving increasing attention in agriculture as potential alternative to chemicals in the form of biofertilizers and biopesticides (Casula and Cutting, 2002). The fermentation medium influences the nutritional and phys- iochemical environment and therefore directly affects productivity and process economics (Zhang and Greasham, 1999). A suitable medium must thus support vegetative growth and also the subse- quent production of spores. Media optimization is therefore an important consideration in development of bioprocesses that can 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.10.116 ⇑ Corresponding author. E-mail addresses: vsbisaria@hotmail.com, vsbisaria@yahoo.com (V.S. Bisaria). Bioresource Technology 102 (2011) 3169–3175 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech