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Journal of Energy and Environmental Sustainability Qualitative in Vitroevaluation of Plant Growth Promoting Activity of Electrog Journal of Energy and Environmental Sustainability, 1 (2016) 47-51 47 An official publication of the International Society for Energy, Environment and Sustainability (ISEES) Journal of Energy and Environmental Sustainability Journal homepage:www.jees.in Qualitative in vitro Evaluation of Plant Growth Promoting Activity of Electrogenic Bacteria from Biohydrogen Producing Microbial Electrolysis Cell towards Biofertilizer Application C.Nagendranatha Reddy, K.Arunasri, Dileep Kumar Yeruva, K.Vamshi Krishna, S.Venkata Mohan* Bioengineering and Environmental Sciences (BEES), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad-500 007, India ARTICLE INFO ABSTRACT Received : 31 August 2015 DBio-fertilizers confer a sustainable alternate to chemical fertilizers through their plant growth promoting Revised : 07 November 2015 activities and contribute significantly to enhance soil fertility and crop yields. The present study identifies Accepted : 10 November 2015 five bacterial strains (K7, K8, K9, K11 and K18) isolated from a different microbiome (Microbial Electrolysis Cell; MEC) to have nitrogen fixing capability. Consequently, the isolates were identified by 16S ribosomal RNA gene sequencing analysis as Bacillus safensis strain K7, B. nealsonii strain K8, B. nealsonii strain K9, Leifsonia shinshuensis strain K11 and Streptomyces griseoviridis strain K18. Since the Keywords: isolates exhibited nitrogen fixation property which is one of the important characteristics of plant Biofertilizer; Bacterial Isolates; growth promoting activity, these strains were further evaluated for other biofertilizer properties viz. Siderophores; Indole Acetic Acid; phosphate solubilization, indole acetic acid (IAA) production, siderophores activity and anti-fungal Antifungal Activity activity. The results showed that siderophore activity was observed in both K7 and K18 strains while IAA production and antifungal activity was observed in K7 and K18 strains respectively. Phosphate solubilization was observed only in K9. The results illustrate that Streptomyces griseoviridis strain K18, Bacillus safensis strain K7 and B. nealsonii strain K9 hold promise to be used as a biofertilizer with potential plant growth promoting activity. © 2016 ISEES, All rights reserved 1. Introduction to the living soil and does not add any pollution to nature like chemical Chemical based fertilizers in agriculture have imposed a negative fertilizers. Efficient strains of nitrogen fixing, phosphate solubilizing, impact on the overall environment and soils. Thus, bio-fertilizer, which is siderophores activity, IAA productivity and antifungal activity in ecofriendly and have no adverse effects on environment and living beings combination will provide good scope for production of biofertilizers as well are in great demand (Khan et al., 2009). Biofertilizers accelerate having plant growth promoting properties (Shahab et al., 2009). certain microbial processes in the soil by increasing the extent of availability In the present study, an attempt was made to evaluate the biofertilization of nutrients in a form easily assimilated by plants and are extremely abilities of the isolates from Microbial electrolysis Cell (MEC). Previous advantageous in enriching soil fertility and fulfilling plant nutrient reports on microbial diversity in MEC have shown that the microbial requirements by supplying the organic nutrients through microorganism populations are rich in electrogenic bacteria having biohydrogen and their by-products (Rajasulochana and Krishnamoorthy., 2014). They production capacity (Venkata Mohan and Lenin Babu., 2011; Venkata add nutrients to the soil through natural processes of atmospheric nitrogen Mohan et al., 2007; Kannaiah Goud et al., 2012). The microbial diversity fixation, phosphate solubilization and mobilization, translocate minor studies have also demonstrated that the enriched electrogenic bacteria are elements like zinc, and copper, secreting plant growth promoting substances mostly anaerobic or microaerophilic having the ability to form biofilm or like Indole acetic acid (IAA), siderophores, vitamins and amino acids colonize on biotic (plant roots) or abiotic (electrode) surfaces (Puente et etc., and also shun plant pathogenic fungi, thereby improving the soil al., 2004). These electrogenic bacteria has exhibited the ability to colonize health and crop production. Symbiotic integration of plant and plant roots and utilize root exudates formed by plant rhizosphere as microorganisms is synergistic in nature as plants supply root borne carbon substrate for their metabolic activities and simultaneously release electrons, compounds, that can be metabolized for bacterial growth and bacteria protons and microbial metabolites to the external environment provide soluble nutrients in return (Perez et al., 2007). Use of biofertilizers enumerating the positive function of rhizosphere (Chiranjeevi et al., 2012). is one of the important components of integrated nutrient management, So, it was assumed that the isolated strains might also exhibit plant growth as they provide eco-friendly organic agro-input and restore the soil’s promoting activity. However, so far no reports are available on using natural nutrient cycle and build soil organic matter for sustainable electrogenic bacteria for evaluating plant growth promoting and agriculture (Mohammadi and Sohrabi, 2012). Knowledge of using organic biofertilizer activity. Thus in the present study, the bacterial isolates from biofertilizers dates back to history with production and usage of compost. MEC were initially screened for diazotrophy and later on for other Bio-fertilizers do not contain any synthetic chemicals which are detrimental biofertilizer capabilities. * Corresponding Author: E-mail: [email protected]; Tel/Fax: +91-40-27191765 © 2016 ISEES All rights reserved 48 Reddy et al. / Journal of Energy and Environmental Sustainability, 1 (2016) 47-51 2. Experimental Methodology 2.6 Phosphate solubilization The phosphate solubilisation activity of the isolated strains was 2.1 Bioreactor evaluated using PVK medium (1000 ml; Glucose-10 g, tri-calcium phosphate-5 g, ammonium sulphate-0.5 g, sodium chloride-0.2 g, A single chambered MEC is used to produce Bio-H2 by electro fermenting the volatile fatty acid (VFA) effluents (4000 mg/l and 8000 magnesium sulphate-0.1 g, potassium chloride-0.2 g, yeast extract-0.5 g, mg/l) generated from acidogenic fermentation with simultaneous waste manganese sulphate-0.002 g, ferrous sulphate-0.002 g and agar powder- treatment is used for isolation of potent organisms which can withstand 15 g) (Pikovskaya., 1948) . The dissolved media was subjected to autoclaving for 15 min prior to preparation of petri plates. The petriplates the potential of 0.6 V. Maximum cumulative H2 production (CHP) and hydrogen production rate (HPR) of 39.35 ml and 0.057 mmol/h was filled with PVK medium was spread with serially diluted samples and observed at 0.6 V respectively and maximum amount of VFA utilization incubated for 48-72 h. The halo and colony diameters were measured as substrate is observed to be 68% at 0.6 V (Annie Modestra et al., 2015). after 5 days of the incubation where halo size was calculated by deducting colony diameter from total halozone diameter (Nguyen et al., 1992). 2.2 Bacterial strains Cultivation 2.7 Screening of bacterial isolates for Siderophore Activity Five bacterial strains viz., K7, K8, K9, K11 and K18 screened from MEC system were used in the study to evaluate the biofertilizer activity. Simple succinate media (SSM; 1000 ml, ammonium sulphate- 1 g, Cultures were grown on nutrient agar plates by streak plate method for 48 dipotassium phosphate-6 g, magnesium sulphate-0.2 g, potassium hours at 37oC and the representative colonies, according to their dihydrogen phosphate-3 g, succinic acid- 4 g; pH 7) was used to detect morphologies and nitrogen fixing ability, were selected for further siderophore activity colorimetrically. Autoclaved media after inoculation characterization. Colonies were re-streaked on nutrient agar plates until a with test strains were subjected to incubation at room temperature for 48 pure culture was obtained. A single colony was picked from these plates h. Fully grown culture was centrifuged (9000 rpm; 12 min) and absorbance and transferred to 50 ml of nutrient broth and incubated for growth at was recorded at wavelengths between 350 and 450 nm. The presence of 37OC (150 rpm). Overnight grown cultures were used for various peaks at these particular wavelengths signifies the presence of siderophores biofertilization assay procedures unless until mentioned. (Ines et al., 2012). 2.3 Characterization and Identification of isolates 2.8 Antifungal Activity Following the selection of pure isolates, 5 isolates were identified The antifungal activity of the strains was assessed by performing based on grams staining and microscopy. Colony morphology of all the germination test (Hernández-Albíter et al., 2007). Fine ground nut (Arachis isolates grown on nutrient agar plates was also studied. Sequencing of hypogaea) seeds were used to determine the effectiveness of the isolated 16S rRNA gene was done to identify the bacteria. Colony PCR was used strains. Plastic trays were spread with wet sterilized cotton to provide to amplify the 16S rRNA gene using universal primers 27F (5'- moisture content for the seeds to germinate and the whole setup was AGAGTTTGATCMTGGCTCAG-3') and 1525R (5' autoclaved prior to experiment. In control, the seeds were treated with
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