Nitrogen fixing biofertilizer for carbon storage

Abstract

Nitrogen is primarily required by plants for their growth and dinitrogen as it exists has very stable triple bond which requires relatively high amount of energy to cleave making it unusable by plants. Therefore, despite its availability in large excess; are still unavailable for the crops and has to undergo biological nitrogen fixation so as to be absorbed by them. To meet the nutritional demand of plants nitrogen (N) is employed in the form of fertilizers in agricultural field. Long-term use of synthetic chemical fertilizers has resulted in soil acidification, poor soil aggregate stability and low levels of micronutrients. Further, it aggravates the environmental pollution through denitrification (N2 and N2 O) and volatilization (NH3 ) potentially contributing in global warming, stratospheric ozone layer depletion and acid deposition. Hence, alternative for these are sought in the form of biofertilizer that minimizes the potential hazardous effects to environment. Further, steep increase in production and utilization of polymers has resulted in significant burden on solid waste management followed by rapid depletion of fossil fuels used in production of such polymers. Moreover, synthetic biodegradable plastics are actually not truly biodegradable as they are labelled imposing harsh impacts on ecological degradation. Therefore, green alternative for these are bioplastics and biocomposites which when discarded in soil and landfills are usually acted upon by microbes such as bacteria, fungi, algae generating CO2 , CH4 , cellular components and other products. PHB in particular is polymer of industrial importance and has found to contain properties like biodegradability, thermoplasticity along with other traits similar to petrochemical plastics of recent usage. The preliminary identification of isolates via biochemical tests were followed by molecular characterization that involved PCR amplification, gel electrophoresis and DNA sequence analysis. When subjected to PCR amplification, the universal primer used yielded an amplicon of the expected size ~1500 bp. The sample A17 showed 98% similarity with the gene cluster sequence of Azotobacter species available from GenBank database while P8 depicted 99% concurrence with Pseudomonas species. The sequence obtained by DNA sequencing was further characterized by multiple sequencing alignment. The clustering approach called neighbor joining (NJ) method was used for the reconstruction of phylogenetic trees that yielded unrooted tree. The isolates of A. vinelandii and P. fluorescens were presented as nitrogen fixing species spectrophotometrically and as PHB producer using Flow cytometry that utilizes FlowJo software to quantify the PHB. The amount of fixed nitrogen was found to be as high as 21.8395 gm/L for A17 while the value of 1.8199 gm/L was achieved for P8 for 144 hours of incubation. PHB was quantified under different condition and maximum production was recorded at nitrogen limiting condition. The bacterial PHB content can directly be correlated with median fluorescence intensity expressed in AFU, Arbitrary Fluorescence Units. For P8 maximum value of 688.7505 AFU has been achieved with median 57.4 where 99.9% of the population produced PHB. Similarly for A17 maximum value of 545.1923 AFU of PHB was obtained with the median value of 30.0 where 93.6% of the total population were actively producing PHB. The isolates which possess the capability to fix the nitrogen along with its ability to store the carbon source as PHB was obtained. The amount of fixed nitrogen and PHB produced by the isolates established them as potent candidate for both biofertlizer as well as biopolymer producer. Keywords: Biofertilizer, biopolymer, nitrogen fixation, PHB, spectrophotometry, Flow cytometry