An Environmentally Friendly Engineered Azotobacter Strain That Replaces a Substantial Amount of Urea Fertilizer while Sustaining the Same Wheat Yield

Abstract

In our endeavor to improve upon nitrogen fixation efficiency of a soil diazotroph that would be unaffected by synthetic nitrogenous fertilizers, we have deleted a part of the negative regulatory gene and constitutively expressed the positive regulatory gene in the chromosome of CBD15, a strain isolated from the local field soil. No antibiotic resistance gene or other foreign gene was there in the chromosome of the engineered strain. Wheat seeds inoculated with this engineered strain, which we have named HKD15, were tested for three years in pots and one year in the field. Yield of wheat was enhanced by ∼60% due to inoculation of seeds by HKD15 in the absence of any urea application. Ammonium only marginally affected acetylene reduction by the engineered strain. When urea was also applied, the same wheat yield could be sustained by using seeds inoculated with HKD15 and using ∼85kg less urea (∼40kg less nitrogen) than the usual ∼257 kg urea (∼120 kg nitrogen) per hectare. Wheat plants arising from the seeds inoculated with the engineered strain, exhibited far superior overall performance, had much higher dry weight and nitrogen content and assimilated molecular N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. IAA production by the wild type and the engineered strain was about the same. Inoculation of the wheat seeds with HKD15 did not adversely affect the microbial population in the field rhizosphere soil.IMPORTANCE Application of synthetic nitrogenous fertilizers is a standard agricultural practice to augment crop yield. Plants, however, utilize only a fraction of the applied fertilizers, while the unutilized fertilizers cause grave environmental problems. Wild type soil diazotrophic microrganisms cannot replace synthetic nitrogenous fertilizers, as these reduce atmospheric nitrogen very inefficiently and almost none at all in presence of added nitrogenous fertilizers. If the nitrogen fixing ability of soil diazotrophs could be improved upon and sustained even in the presence of synthetic nitrogenous fertilizers, then a mixture of the bacterial and reduced quantity of chemical nitrogenous fertilizers could be employed to obtain the same grain yield, but at much reduced environmental cost. The engineered that we have reported here has considerably enhanced nitrogen fixation and excretion abilities and can replace ∼85 kg of urea per hectare, but sustain same wheat yield, if the seeds are inoculated with it before sowing.

Molecular Biology in the Improvement of Biological Nitrogen Fixation by Rhizobia and Extending the Scope to Cereals

“…Inoculation of crops with plant growth-promoting bacteria has been studied to enhance yield and production with reduced reliance on chemical N fertilizers [ 160 ]. The free-living diazotrophs, e.g., Azotobacter , Beijerinckia , and Clostridium , with the ability to fix nitrogen have been explored in cereals [ 161 , 162 ]. A mutation in the nifL gene in Azotobacter vinelandii allowed nitrogen fixation at higher ammonium concentration [ 163 ].…”

“…Manipulations of the glutamine synthetase promoter and the nifL gene of this diazotroph not only enabled secretion of high amounts of ammonium but also showed strong proliferation of microalgae and promoted growth in cucumber plants in the absence of added N fertilizer [ 164 ]. The deletion in the negative regulatory region of nifL genes and inclusion of positive regulatory gene nifA in another species of Azotobacter enhanced its capability of nitrogen fixation and reduced the reliance on N fertilizer under field conditions of wheat cultivation [ 162 ]. The engineered bacterium enhanced the yield by 60% compared to unfertilized controls.…”

Biological nitrogen fixation in maize: optimizing nitrogenase expression in a root-associated diazotroph

“…Furthermore, expressing nifA under the control of a nitrogen-independent promoter could decouple nitrogenase biosynthesis from the PII protein regulatory cascade ( Fig. 2a ) ( Bageshwar et al , 2017 ). Secondly, truncation of the GlnE protein to delete its adenylyl-removing (AR) domain would lead to constitutively adenylylated GS, limiting ammonium assimilation by the microbe and leading to ammonium build up and release ( Fig.…”

“…Additionally, nitrogen-fixing bacteria efficiently assimilate fixed nitrogen into microbial biomass, preventing it from being released to the rhizosphere ( Batista and Dixon, 2019 ). While several plant-associated diazotrophs with altered ammonium assimilation pathways have been shown to support the growth of algae ( Ortiz-Marquez et al , 2014 ; Barney et al , 2015 ) and plants ( Pankievicz et al , 2015 ; Ambrosio et al , 2017 ) in lab and greenhouse settings, few have been applied in the field ( Bageshwar et al , 2017 ), probably due to a lack of fitness in the rhizosphere ( Colnaghi et al , 1997 ). Recently, synthetic biology has been applied to refactor the regulation of genes encoding the nitrogenase enzyme complex (known as the nif cluster), enabling more control over nitrogenase expression in a range of species ( Temme et al , 2012 ; Wang et al , 2013 ; Smanski et al , 2014 ).…”

Nitrogen Fixation in Cereals

“…For example, ammonium excreting Azospirillum exhibited enhanced nitrogen supply to wheat plants ( Van Dommelen et al, 2009 ). Similar mutants of Azospirillum , Kosakonia , Pseudomonas , and Azotobacter ( Zhang et al, 2012 ; Setten et al, 2013 ; Geddes et al, 2015 ; Ambrosio et al, 2017 ; Bageshwar et al, 2017 ) proved capable of stimulating plant growth. We would recommend obtaining ammonium-excreting mutants of Paraburkholderia , Herbaspirillum , or Azoarcus as well, to test if they also improve plant growth through nitrogen fixation.…”