Azotobacter Species Research Articles

Isolation, Selection, and Biological Evaluation for Bacteria that Fix Nitrogen and Produce Indole-3-Acetic Acid from Paddy Soils in Vietnam

Introduction/Background Acidic soils are limiting the production of crops and indirectly harming the environment due to the use of nitrogen (N) chemical fertilizer. Therefore, the current study aims to isolate a promising N2-fixing candidate to solve this issue in some communes of Phu Luong District, Thai Nguyen Province, Vietnam. Materials and Methods The bacteria were isolated using the Ashby medium. The IAA generation and nitrogen fixation were assessed using spectroscopy. The selected bacteria were tested at temperatures ranging from 28°C to 42°C and pH levels of 4.0 to 9.0. As a result, the Box-Behnken model yielded a growth equation. The API kit test was used to measure the biochemical properties of the selected bacteria. Results The result demonstrated that there were ten nitrogen-fixing bacteria (NFB) isolates with capacities of N2 fixation and IAA production at 2.35–24.61 and 0.00–119.13 µg/ml. Among them, the NL3 strain was the best strain. The optimum condition to grow the NL3 strain was calculated as 5.44 days at pH 7.10 and temperature of 32.18°C to reach a bacterial density of 9.77929×108 CFU/ml. In addition, the NL3 strain was identified as an Azotobacter species. The genomic analysis revealed that the genome of Azotobacter sp. NL3 was nearly 5.4 Mb long and contained 45 N2 fixation-relating genes and 7 IAA production-relating genes. Conclusion Not only can this study provide insight into the features of Azotobacter spp., but it also introduces a potent candidate that can improve soil health and crop yield by fixing N2 and producing IAA in order to limit the use of N chemical fertilizer for a sustainable agriculture. Therefore, the selected bacterial strain should be further tested under the local field conditions and subsequently commercialized as a biofertilizer.

A Pangenomic Analysis of the Diversity and Biological Functioning of the Genus Azotobacter

Azotobacter is a diverse genus of free-living soil bacteria that fixes atmospheric nitrogen and act as a biocontrol agent for different phytopathogens. This study utilized the genomic sequences from four different Azotobacter species, comprising thirty strains, to construct the genus pangenome. Through comparative genomics and pangenomics analysis, we elucidated the genomic diversity and functional relationship between and within the species of the Azotobacter genus. The selected Azotobacter strains exhibited an ANI of ≥ 85%, with core genes constituting up to 9% and a large proportion of 55% of unique genes. This indicated the sharing of a highly open pangenome of the Azotobacter genus. The COG analysis revealed that core, unique, and accessory genes play a significant role in the Azotobacter’s metabolic and information storage processes. The KEGG enrichment evaluation showed general functioning, amino acid transport and metabolism, as the major biological processes of the Azotobacter’s genomic content. Furthermore, the predominant BGCs present in the genomic structure of Azotobacter genus exhibited a 27% participation in the production of NRP-metallophores, 17% for the NI-siderophores and 17% for type III polyketide synthase (T3PKS). The genomic content analysis of the core genome revealed various genes that encoded for acid and universal stress proteins, playing a role in Azotobacters adaptation. For the biological functioning attributes, it was found that alginate production, riboflavin transporters, nitrogen-responsive regulatory flavoprotein, nitrogen-related phosphotransferase, and molybdenum cofactor genes were involved in its core genome for the major functioning of its nitrogen fixation.

Growth response of tomatoes to application of bacterial-coated NPK fertilizer in a pot experiment

Nitrogen-fixer and phosphate-solubilizer bacteria increase the chemical-fertilizer efficiency and soil health. Bacterial-coated NPK fertilizer is a novel approach for intensifying biofertilizer application in vegetable production. The study aimed to observe the compatibility between two Azotobacter species and two Bacillus species, their population on two formulations of bacterial-coated NPK fertilizer (BCN), and the effect of BCN dose on growth, N and P content, and their uptake in tomato shoots. The compatibility test was performed using the streak method. The population of Azotobacter and Bacillus were counted for two formulations of NPK fertilizer coated by solid biofertilizer (5% and 10%) and zeolite (1% and 5%). The pot experiment was conducted in a randomized block design with four treatments and six replications. The treatments were 100% recommended doses of conventional NPK fertilizer (700 kg/ha) and 100%, 80%, and 60% doses of BCN. The results showed four bacterial species were compatible, indicated by synergistic growth on the plate agar. The BCN formula using 5% liquid inoculant and 5% zeolite has higher cell viability. The BCN enhanced stem thickness and leaves number but did not change the plant height, dry weight, N and P content, and their uptake in shoots. Applying 60% of BCN caused greater stem thickness and leaf number. Despite being insignificantly different from another treatment, that dose increased the biomass and the shoot uptake of N and P. The NPK fertilizer coated by Azotobacter and Bacillus has the potency to increase tomato growth and NPK fertilizer dose.

Extraction and Profiling of Antifungal Compounds Produced by Azotobacter Species

Food loss and wastage occur in large quantities globally every year and it occurs in the entire supply chain right from the production to the processing stage. The loss of food is due to various factors like adopting traditional cultivation practices, low investment in the food sector, and more loss from poor pests and disease management of agricultural crops. The most important and major cause is due to microbial spoilage; fungi are most harmful to the consumers and also to the agriculture sector. Synthetic chemical strategies can prevent fungal growth and may reduce wastage but still causes accumulation of chemical substances in the environment and food chain in a long run. For these reasons, the use of bio-control technologies can be a great solution to agriculture and food sector as well. In view of this, the present study has been conducted using an efficient Azotobacter species, which belongs to the PGPR group. In this study, antifungal compounds produced by Azotobacter have been extracted by following solvent extraction protocols and identified using GCMS methods. The antifungal compounds were tested against the major fungal pathogens viz., Aspergillus, Fusarium, and Penicillium species. The metabolites produced by Azotobacter species were efficient in controlling the growth of the fungal species. These compounds can be used as a potential bio-preservative in the food sector instead of synthetic chemicals. Thus, these compounds can further be analyzed and tested on the food sample, having a great scope in the future to replace the chemical preservatives.

Biochemical characterization of bacteria isolated from Rhizosphere soils of sugarcane grown in Uttar Pradesh

Rhizospheric soil samples of Sugarcane fields were collected from distinct regions of U.P. Further, these soil samples were used for the isolation and characterization of bacteria to identify their maximum PGPR traits and biochemical potential. A total of ten bacterial isolates were collected from the soil samples and sub-cultured in the nutrient agar plates and characterized through Biochemical test. One bacterial strain was found negative and the other 9 isolates were found positive for gram staining. All bacterial isolates were positive for citrate test. 3 isolates were found MR positive and 2 positives for VP test. 5 isolates were positive for gelatinase test and 9 isolates were positive for nitrate reductase test. 2 isolates were positive for starch hydrolysis test, while 7 isolates were found positive for phosphate solubilization test and 3 isolates were positive for siderophore production test. These results showed that the bacterial isolates from soil sample were Azotobacter Species, Azospirillum Species, Pseudomonas species, Bacillus Species, Rhizobium Species, Serratia Species and E. coli. These bacterial strains followed the maximum PGPR traits. After proper validation, these bacterial strains could be used for the bio-fertilizer formation for crop production enhancement by improving soil fertility.

Effects of C and N application on Azotobacter and nitrogen cycle in farmland soils of central Guizhou, China

Carbon sources and nitrogen sources are two important groups of substances in analyses of the effects of exogenous additives on soil microorganisms. Glucose (present in soil) and urea (extensively used in agricultural production) are substances often used to study the specific effects of carbon and nitrogen addition on microorganisms. Azotobacter, a non-symbiotic nitrogen-fixing bacterium, has been characterized in the laboratory and applied to soil in numerous studies. However, soil microorganisms display considerable diversity, and the effects of in exogenous substances stimulation on various microorganisms are uncertain. The potential effects of forced exogenous substance stimulation on Azotobacter are not well understood. Here, the effects of C and N application on Azotobacter growth and nitrogen cycle metabolism in farmland soil in central Guizhou, China, were studied through analysis of four treatment groups: control (CK), glucose treatment (C), urea treatment (N), and glucose + urea treatment (CN). The results showed that the relative abundances of the Azotobacter genus and relevant species were increased in group C, indicating promotion of Azotobacter growth (P < 0.001). The relative abundances of the Azotobacter genus and relevant species in group CN were significantly different from the abundances in group N and CK (P < 0.05). Furthermore, the relative abundances of nif genes (i.e., nifH, nifD, and nifK) and nirD were significantly increased in group C. However, the relative abundances of the aforementioned four nitrogen cycle-related genes did not significantly differ between group CN and groups CK and N. The main source species of the aforementioned four nitrogen cycle-related genes were Azotobacter species. The total nitrogen and alkali-hydrolyzed nitrogen contents in soil did not significantly differ in a comparison between group C and groups N and CN. Piecewise structural equation modeling analysis revealed that bacterial α-diversity, Azotobacter, and nitrogen cycle genes had significant direct effects on the alkali-hydrolyzed nitrogen content in soil and had negligible direct effects on the total nitrogen content in soil. These findings improve the broader understanding of Azotobacter and provide theoretical support for reduced Azotobacter utilization in soil.Graphical

Effect of pH on Growth and Nitrogen Fixation of &lt;i&gt;Azotobacter spp.&lt;/i&gt;

Three species of Azotobacter were isolated from prawn-cum-paddy fields of Kerala. These strains were found to have the optimum pH for growth and nitrogen fixation near or slightly above neutrality, i.e., in the pH range of 7.0 to 8.5

Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.

Isolation and characterization of Azotobacter sp. for plant growth promotion and abiotic stress tolerance in Telangana's agro-climatic regions: Towards sustainable development

Current study was done to understand and improve plant survival and growth under biotic and abiotic stress tolerance was considered. The primary goal of this work was to isolate azotobacterial species from indigenous places where the agriculture community has been practicing traditional methods along with regular places, also examined the areas of stressed agriculture areas which are seasonally cultivated and environments in their capacity to enhance drought stress tolerance brought from different crop fields such as Maize, sweet Sorghum, Sugarcane, corn, and wheat to increase the yield.These crops soil samples were taken from few drought areas tolerant places, i.e. under different temperature, salinity, pH and moisture conditions of those particular areas in Telangana. The different soil samples isolated for azotobacter strains from different regions in Telangana were collected and analyzed for growth in media to get the high yield for the agriculture purpose which will benefit the agricultural community and leads to sustainable development. Morphological, biochemical, and molecular characteristics were used to evaluate the Azotobacter species. Azotobacter beijerinckii strain and Azotobacter tropicalis strain were identified by biochemical testing and 16s rRNA sequencing. Azotobacter beijerinckii strain (BKPOU06TS) and Azotobacter tropicalis strain (BKPOU08TS) 16S rRNA sequences were submitted to GenBank with accession numbers OP536202 and OP536206, respectively.

Microbial Characterization of Azotobacter sp Isolates From West Kalimantan in Nitrogen Fertilizer Efficiency in Maize Plants (Zea mays L.) on Peatlands

This study aims to characterize the microbial species Azotobacter sp from peat soil in West Kalimantan, which can bind N, dissolve nutrients P, K and produce indole compounds. This research was carried out in the plant pest and disease laboratory, agronomy laboratory and greenhouse, Faculty of Agriculture, Tanjungpura University, Pontianak. This study used a non-factorial Randomized Block Design (RBD) with four levels of treatment. Inoculation of Azotobacter sp isolates was without inoculation of Azotobacter sp isolate (a0), inoculation of WH.3.2.d (a1) isolate, inoculation of Azotobacter sp isolate WH.2.4 (a2), inoculation of Azotobacter sp WH.3.1.l (a3). The study was carried out from September to November 2021. The results showed that Azotobacter sp inoculation significantly affected the parameters of vigor index, growth synchronously, primary root length, wet weight of normal sprouts, and plant height in the greenhouse. From the bioassay test results on seeds and growth testing for 15 days in a greenhouse, the two best isolates were obtained, namely WH.2.4 and WH.3.1.l isolates.

Antifungal potential of Azotobacter species and its metabolites against Fusarium verticillioides and biodegradation of fumonisin.

In the study, seven Plant Growth Promoting Rhizobacteria (PGPR) Azotobacter species were screened against three strains of Fusarium verticillioides to test its antifungal activity. Azotobacter strains were tested for the degradation of fumonisin produced by F. verticillioides. Secondary metabolites were isolated and characterized from the Azotobacter strains for the first time. Potential seven Azotobacter species antifungal activity was tested following the dual culture assay against three strains of Fusarium verticillioides namely FVM-42, FVM-86 and MTCC156 estimating the substantial zone of inhibition. Azotobacter species AZT-31 and AZT-50 strains significantly inhibited the growth of F. verticillioides recording drastic growth enhancement of maize under in-vitro conditions by calculating the infection incidence, vigour index and germination percentage. As confirmation, dereplication studies were conducted for the reconfirmation of Azotobacter strains by isolating from rhizoplane. Azotobacter strains played a key role in the degradation of fumonisin produced by F. verticillioides reporting 98% degradation at 2h of incubation with the pathogen. Furthermore, in the study first time, we have tried to isolate and characterize the secondary metabolites from the Azotobacter strains exhibiting six compounds from the species AZT-31 (2) and AZT-50 (4). Preliminary in-vitro experiments were carried out using the compounds extracted to check the reduction of infection incidence (90%) and increase in germination percentage upto 50 to 70% when compared to the test pathogen. Azotobacter strains referred as PGPR on influencing the growth of plant by producing certain substances that act as stimulators on inhibiting the growth of the pathogen. The future perspective would be the production of an active combination of carboxamide compound and Azotobacter species for preventively controlling the phytopathogenic fungi of plants and crops and also towards the treatment of seeds.

Growth response of Basella alba. Linn to Azotobacter species isolated from marine sediments and garden soil

Growth response of Basella alba. Linn to Azotobacter species isolated from marine sediments and garden soil

Response of Bread Wheat Cultivars Inoculated with Azotobacter Species under Different Nitrogen Application Rates

A field trial was conducted to investigate the productivity of three bread wheat cultivars, namely Giza-168, Shandawel-1, and Misr-2, under different fertilization treatments, i.e., azotobacter inoculation, 25% nitrogen (N) + azotobacter, 50%N + azotobacter, 75%N + azotobacter, and 100%N of the recommended level (180 kg/ha). The treatments were laid in a split-plot design, and each was replicated three times. The findings showed that wheat cultivars examined in the two seasons exhibited significant variations (p ≤ 0.05) in plant height (PH, cm), number of tillers m−2 (NTM), number of spikelets per spike (NSS), 1000-grain weight (TGW, g), spike length (SL, cm), biological yield (BY, ton ha−1), grain yield (GY, ton ha−1), straw yield (SY, ton ha−1), harvest index (HI, %), protein content (PC, %), days to 50% heading (DTH), and chlorophyll content (CC, SPAD). As a result, Giza-168 had a higher GY (14%), HI (27%), and TGW (10%) than any of the other two cultivars in both growing seasons. Furthermore, Misr-2 exhibited the highest PH (16%), NTM (26%), NSS (28%), SL (10%), BY (30%), SY (46%), and CC (3%). The application of the two treatments of 100%N and N75% + azotobacter exhibited high and statistically similar performance, resulting in an increase in all studied traits by greater than 30–50% compared to the other three treatments. According to the findings of the current investigation, the application of N fertilizer combined with azotobacter increased wheat yield more than either solely azotobacter or N application. We concluded that the application of nitrogen combined with azotobacter reduced the quantity of applied nitrogen by 25%.

Characterization and diversity of native Azotobacter spp. isolated from semi-arid agroecosystems of Eastern Kenya.

Declining food production in African agroecosystems is attributable to changes in weather patterns, soil infertility and limited farming inputs. The exploitation of plant growth-promoting soil microbes could remedy these problems. Such microbes include Azotobacter; free-living, nitrogen-fixing bacteria, which confer stress tolerance, avail phytohormones and aid in soil bioremediation. Here, we aimed to isolate, characterize and determine the biodiversity of native Azotobacter isolates from soils in semi-arid Eastern Kenya. Isolation was conducted on nitrogen-free Ashby's agar and the morphological, biochemical and molecular attributes evaluated. The isolates were sequenced using DNA amplicons of 27F and 1492R primers of the 16S rRNA gene loci. The Basic Local Alignment Search Tool (BLASTn) analysis of their sequences revealed the presence of three main Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris and Azotobacter tropicalis. Kitui County recorded the highest number of recovered Azotobacter isolates (45.4%) and lowest diversity index (0.8761). Tharaka Nithi County showed the lowest occurrence (26.36%) with a diversity index of (1.057). The diversity was influenced by the soil pH, texture and total organic content. This study reports for the first time a wide diversity of Azotobacter species from a semi-arid agroecosystem in Kenya with potential for utilization as low-cost, free-living nitrogen-fixing bioinoculant.

Effect of Azospirillum and Azotobacter Species on the Performance of Cherry Tomato under Different Salinity Levels

Effect of Azospirillum and Azotobacter Species on the Performance of Cherry Tomato under Different Salinity Levels

Plant growth promotional studies of novel PGPR strains isolated from the rhizosphere of Neolamarckia cadamba (Roxb.)Bosser plantations in Narasipuram, Tamil Nadu

Forests of Indian subcontinent are one of the biodiversity hot spots of the world. They are the second-largest inland use next to agriculture, yet possess a high degree of endemism. Plantation forestry in India was initiated mainly for the production of industrial raw materials as well as fuelwood and fodder from exotic species. The increase in demand for industrial raw material has spurred the rise in plantations of fast-growing native tree species. Neolamarckia cadamba (Roxb.) Bosser is a native tree species whose wood has been largely used in the pulpwood and pencil industry for making boards, plywood, packing cases, tea-boxes, carving and turnery articles. Supplementation of these native trees with effective bio inoculants will improve and maintain the soil fertility and sustainability in the natural soil ecosystem besides providing economic benefits. In the present study, the diversity of PGPR was assessed in the rhizosphere of N. cadamba plantations in Narasipuram, Coimbatore, Tamil Nadu. 33 PGPR isolates from N. cadamba rhizosphere were isolated viz. four species of Azotobacter, three species of Azospirillum and six different PSB species. The isolates were studied for their plant growth promotional abilities such as Indole Acetic Acid (IAA) production and phosphate solubilization.

Impact of Azotobacter in Different Doses of Dichlorvos

Azotobacter is free living, non–symbiotic, heterotrophic bacteria capable of fixing an average of 20kg N / hector year. Azotobacter species are Gram negative, free–living, aerobic soil dwelling, oval or spherical bacteria that form thick–walled cysts (means of asexual reproduction under favorable condition). Out of six species of Azotobacter, some of them are motile by means of peritrichous flagella while others are not. They are typically polymorphic and their size ranges from 2–10µm long and 1–2µm wide. A. chroococcum is the first aerobic free– living nitrogen fixer. These bacteria utilize atmospheric nitrogen gas for their cell protein synthesis. Dichlorvos is an organophosphate widely used as an insecticide to control household pests, in public health, and protecting stored products from insects. In present study of dichlorvos in Azotobacter through different doses are effected of seed germination and observed the toxicity and harmful effect of dichlorvos solution.

Nitrogen Fixing Azotobacter Species as Potential Soil Biological Enhancers for Crop Nutrition and Yield Stability.

Biological nitrogen fixation (BNF) refers to a microbial mediated process based upon an enzymatic “Nitrogenase” conversion of atmospheric nitrogen (N2) into ammonium readily absorbable by roots. N2-fixing microorganisms collectively termed as “diazotrophs” are able to fix biologically N2 in association with plant roots. Specifically, the symbiotic rhizobacteria induce structural and physiological modifications of bacterial cells and plant roots into specialized structures called nodules. Other N2-fixing bacteria are free-living fixers that are highly diverse and globally widespread in cropland. They represent key natural source of nitrogen (N) in natural and agricultural ecosystems lacking symbiotic N fixation (SNF). In this review, the importance of Azotobacter species was highlighted as both important free-living N2-fixing bacteria and potential bacterial biofertilizer with proven efficacy for plant nutrition and biological soil fertility. In addition, we described Azotobacter beneficial plant promoting traits (e.g., nutrient use efficiency, protection against phytopathogens, phytohormone biosynthesis, etc.). We shed light also on the agronomic features of Azotobacter that are likely an effective component of integrated plant nutrition strategy, which contributes positively to sustainable agricultural production. We pointed out Azotobacter based-biofertilizers, which possess unique characteristics such as cyst formation conferring resistance to environmental stresses. Such beneficial traits can be explored profoundly for the utmost aim to research and develop specific formulations based on inoculant Azotobacter cysts. Furthermore, Azotobacter species still need to be wisely exploited in order to address specific agricultural challenges (e.g., nutrient deficiencies, biotic and abiotic constraints) taking into consideration several variables including their biological functions, synergies and multi-trophic interactions, and biogeography and abundance distribution.

Biosorption of lead ions by exopolysaccharide producing Azotobacter sp.

Aim: Removal of lead from wastewater using Azotobacter species and optimisation of various parameters to maximise the adsorption of lead by response surface methodology as a tool. Methodology: The bacterial isolate UBI-7 recovered from sewage water irrigated soil was examined for its biosorption potential towards lead. The lead removal efficiency of Azotobacter salinestris was studied with respect to metal concentration (50-250 mg l-1), contact time (24-120 hrs), and pH (4-8).Using response surface methodology, these factors were optimized and R2 value obtained was 0.9710 for lead ions, which indicates the validity of the model. Observation with Fourier Transform Infrared (FTIR), Scanning Electron Microscope imaging (SEM) and Energy Dispersive X-ray Spectroscopic analysis (EDX) were carried out to confirm lead biosorption by Azotobacter salinestris. Results: The lead tolerant bacterium isolated from sewage water irrigated soil (UBI-7) was recognized as Azotobacter salinestris by 16S rRNA based gene sequence analysis. The highest removal percentage of Pb (61.54) was 50 mg l-1 in 72 hrs equilibration period. Interaction effect between different levels of Pb and different contact time of the solution were found to be significant. Lead biosorption by the organism was confirmed by the changes in stretching intensities of functional groups as well as appearance of strong OH stretching at 3291.69 cm-1. Images obtained from Scanning Electron Microscope and Energy Dispersive X-ray Spectroscopic studies of the bacteria (UBI-7) before and after biosorption clearly indicated lead adsorption. Interpretation: Current study proves that the functional groups of Azotobacter salinestris are involved in lead biosorption from aqueous solution which was confirmed through FTIR.EDX analysis also elucidated the lead absorption by the bacterial cells. Hence, this could be effectively utilized for decontamination of lead from the polluted environment. Key words: Azotobacter salinestris, Biosorption, Lead, Response surface methodology

Effect of CaCO&lt;sub&gt;3&lt;/sub&gt; Form on Soil Inherent Quality Properties of Calcareous Soils

Four soil samples different in there CaCO3 forms contents (total, active and active /total CaCO3) were taken from tillage horizon 0-30 cm depth to study the impacts of CaCO3 form on some soil inherent quality indicators in calcareous soils. Result revealed that the total CaCO3 forms have a great influence on particle size distribution to make it coarser in general comparing with the absence of CaCO3 with soil particles. The increase of active /total (A/T CaCO3%) till 50% has a positive effect on increasing soil electrical conductivity E.C ds.m-1, while increment of A/T CaCO3 over 50% till 70% detrimentally reduced soil E.C ds.m-1 hundred fold. The elevation of (A/T CaCO3%) has inverse correlation with soil cation exchange capacity (CEC Cmole.kg soil). the elevation of (A/T CaCO3%) has inverse correlation with soil cation exchange capacity (CEC Cmole.kg soil) the (A/T CaCO3%) ratio over 56% till 69% significantly improves the mean weight diameter and macro aggregate. In general the optimum total CaCO3 for micro aggregate formation may not exceed 20%. The best Total and (A/T CaCO3%) for better aggregate stability is between 20 to 48% . The proper total calcium carbonate for better total heterotrophic aerobic bacterial activity is around 20% . The best growth of the Azotobacter species which is belonging to non-symbiotic nitrogen fixing bacteria is obtained when total CaCO3 ranges between 10-20% or between 48-56% of (A/T CaCO3%).