Evaluation of nitrogenous bio-inoculants through nitrogen substitution for improving productivity and juice quality of sugarcane in calcareous soil of indo Gangetic plains of India

Introduction

Sugarcane (Saccharum officinarum L.) is one of the most important crops globally, playing a critical role in the production of sugar and biofuels. As a key source of raw materials for various industries, including food and bioenergy, sugarcane cultivation has expanded significantly over recent decades (Kumar et al. 2024a) [12] . In India, the second-largest producer of sugarcane in the world, the crop occupies about 4.6 million hectares of land, contributing nearly 370 million tonnes of sugarcane annually, the increasing demands on production systems, along with environmental concerns, have prompted researchers and farmers to explore sustainable agricultural practices, particularly in nutrient management (Singh et al., 2024) [20] . The use of nitrogenous fertilizers has been a cornerstone of conventional agriculture, yet their overuse poses several risks to soil health and the environment (Meena et. al., 2024; Kumar et al., 2025b) [20,21,16] .

Nitrogen is a vital macronutrient for plant growth and is required in large quantities, especially for sugarcane, which is a high-demand crop (Singh et al., 2025) [26] . However, nitrogen management in sugarcane cultivation is challenging due to the crop's prolonged growing period and substantial nutrient requirements (Sinha et al., 2024; Shraddha, et al., 2026) [27,31] . Traditionally, fertilizers, such as urea or ammonium nitrate, are applied to ensure optimal yields. Unfortunately, a significant portion of nitrogen applied to soils is lost through processes like volatilization, leaching (Kumar et al., 2024b) [17] , and denitrification. These losses not only efficiency of nitrogen use but also contribute to environmental issues such as groundwater contamination, eutrophication and the release of greenhouse gases. Moreover, in calcareous soils which are prevalent in many sugarcane-growing regions, nitrogen availability is often compromised due to high pH levels (Kumar, 2015; Singh et al., 2022; Singh et al., 2023) [9,23,22] . The alkaline nature of calcareous soils can inhibit nutrient uptake by plants, further reducing the effectiveness of nitrogen fertilizers (Kumar et. al., 2023a) [10,24] . Thus, the development of strategic nitrogen use efficiency (NUE) in these soils is crucial for improving sugarcane productivity while minimizing environmental impacts through carbon sequestration (Kumar et al. 2024a) [12] . Bio-inoculants as a Sustainable Source in the recent years, there has been growing interest in the use of bio-inoculantsmicroorganisms that promote plant growth and improve soil fertility-as an alternative to chemical fertilizers (Kumar and Paswan., 2015) [9] . Bio-inoculants such as nitrogen-fixing bacteria can supplement for nitrogen management. These microorganisms, including Azotobacter and Acetobacter, have been shown to enhance plant growth by fixing atmospheric nitrogen and making it available to plants in a more sustainable manner (Kumar et al., 2024d) [13] . In sugarcane, bio-inoculants have been reported to improve uptake, increase crop yield and enhance juice quality (Kumar et al., 2026a) [30] . Azotobacter and Acetobacter: Key Nitrogen-Fixing Bio-inoculants Azotobacter and Acetobacter are endophytic free-living nitrogen-fixing bacteria that play an important role in sustainable agriculture by contributing to the nitrogen cycle in soil. Azotobacter, a well-studied bio-inoculant, is known for its ability to fix atmospheric nitrogen into a form that is available for plant uptake, promoting growth even in soils with low nitrogen content. Additionally, Azotobacter produces growthpromoting substances, which further enhance crop development.

Similarly, Acetobacter, particularly Gluconacetobacter diazotrophicus, is another bacterium that has been widely studied for its symbiotic relationship with sugarcane (Kumar and Paswan., 2014) [8] . Acetobacter is capable of fixing atmospheric nitrogen and contributing significantly to the nitrogen the plant without requiring the energy inputs associated with chemical fertilizers. Research indicates that Acetobacter can thrive in the root zones of sugarcane plants, where it improved growth and yield. Furthermore, both Azotobacter and Acetobacter have been found to enhance the uptake of other essential nutrients, such us and potassium, thereby improving overall plant health.

Nitrogen Substitution for Sustainable Production

The substitution of chemical nitrogen fertilizers with nitrogenous bio-inoculants aligns with the principles of sustainable agriculture, which emphasize reducing reliance on synthetic inputs while maintaining or enhancing crop productivity (Kumar., 2024) [1,2] . By using bio-inoculants as partial replacements for chemical fertilizers, farmers can reduce the environmental footprint of agriculture, where nitrogen losses are prevalent. The integration of bioinoculants into nutrient management strategies has been shown to enhance nitrogen use efficiency, promote soil health, and mitigate environmental impacts associated with excessive fertilizer use (Meena et. al., 2023) [21] . In calcareous soils, where nutrient availability is often limited by high pH levels, the role of bio-inoculants becomes even more critical. Studies have demonstrated that bio-inoculants can improve nutrient availability and uptake in such soils by altering the soil microbiome and enhancing soil structure. This, in turn, leads to better crop growth, higher yields, and improved quality of produce, such as sugarcane juice.

Bio-inoculants in Improving Sugarcane Juice quality

In addition to their role in enhancing sugarcane growth and yield, bio-inoculants have been shown to positive the quality of sugarcane juice. Juice quality parameters such as Brix (a measure of sugar content), Pol (polarization or the amount of sucrose in the juice), and juice purity are critical for determining the commercial value of sugarcane. Research suggests that the application of nitrogenous bio-inoculants can lead to improvements in these parameters, likely due to the enhanced nutrient uptake and better overall plant health by bio-inoculants. In the present study, the application of Azotobacter and Acetobacter is expected to result in significant improvements in sugarcane juice quality. Preliminary data indicate that treatments in corporation of bio-inoculants yield higher Brix and Pol values compared to conventional nitrogen fertilizer applications (Sinha et. al., 2024) [27] . This enhancement in juice quality not only benefits farmers by increasing the commercial value of their produce but also aligns with consumer preferences for highquality sugarcane juice products.

Significance of the Study

This research is particularly significant in the context of global efforts to promote sustainable agriculture and reduce the environmental impacts of intensive farming practices (Kumar et. al., 2014) [15] . The use of bio-inoculants as a substitute for chemical nitrogen fertilizers represents a promising approach to improving crop productivity while minimizing the negative effects of excessive fertilizer use. By focusing on sugarcane, a crop of major economic importance, this study has the potential to influence agricultural practices on a broad scale, particularly in regions where calcareous soils pose challenges to nutrient management (Kumar et. al., 2018) [11] . The findings from this research will provide valuable insights into the practical applications of bio-inoculants in sugarcane cultivation, offering farmers an environmentally friendly and economically viable alternative to chemical nitrogen fertilizers.

Materials and Methods

Experimental Site/Latitude/Longitude/Climate

The field experiment was conducted at Kalyanpur Farm, RPCAU, Pusa, the farm is situated at 52.0 m above mean sea level and 25°95' N latitude, 85°77' E longitude. The experimental site had a subtropical climate with distinct wet and dry seasons. The experimental plot was well drained, medium upland and had uniform topography. The experimental site is belongs to a region characterized by calcareous soils, which are high in calcium carbonate and present challenges for nutrient uptake due to their alkaline nature. The site was chosen for its typical representation of soil conditions found in major sugarcane-growing regions of Bihar, which require improved nutrient management strategies.

Plant Material

The sugarcane genotype used for the experiment was CoP-21438, which is well-adapted to local growing conditions and commonly cultivated in the region for its desirable agronomic traits, such as good juice quality and high yield potential.

Experimental Design

The experiment followed a Randomized Block Design (RBD) with seven treatments and three replications. The plot size for each treatment was 6 x 5.4 meters, with six rows of sugarcane per plot. Adequate spacing was maintained between plots to avoid interference between treatments. Treatments: The treatments were designed to test different combinations of nitrogen substitution using bioinoculants (Azotobacter and Acetobacter) in addition to the recommended dose of fertilizers (RDF). The following seven treatments were applied: T 1: RDF 100% (150:85:60:: N: P₂O₅:K₂O)-Control treatment with no bio-inoculants; T 2: RDF 100% + Azotobacter @ 5 kg/ha.; T 3: RDF 100% + Acetobacter @ 5 kg/ha; T 4: RDF 100% + Azotobacter @ 5 kg/ha + Acetobacter @ 5 kg/ha; T 5: RDF 75% (Nitrogen) + 100% (P₂O₅ and K₂O) + Azotobacter @ 5 kg/ha; T 6: RDF 75% (Nitrogen) + 100% (P₂O₅ and K₂O) + Acetobacter @ 5 kg/ha; T 7: RDF 75% (Nitrogen) + 100% (P₂O₅ and K₂O) + Azotobacter @ 5 kg/ha + Acetobacter @ 5 kg/ha.

Soil Preparation and Crop Establishment

The experimental field was ploughed and prepared according to standard agronomic practices. Soil testing was conducted before the experiment to determine the baseline nutrient status, particularly nitrogen, phosphorus, potassium and pH levels. Based on the soil test results, the required quantities of chemical fertilizers were calculated and applied as per treatment specifications. Planting: Sugarcane setts were planted manually at a row spacing of 90 cm with a depth of about 8-10 cm. The setts were treated with appropriate fungicides to prevent any initial infections.

Bio-inoculant Application

The bio-inoculants Azotobacter and Acetobacter were applied in their respective treatments at a rate of 5 kg/ha. The inoculants were mixed with organic carriers and applied to the soil near the root zones of the plants during planting. The inoculants were used from a bio-fertilizer production unit, of sugarcane research institute, Pusa and their quality was tested before use to ensure viability and effectiveness. The bio-inoculants were applied both as a seed treatment and through soil application. In the seed treatment method, sugarcane setts were soaked in slurry of bio-inoculants before planting.

Fertilizer Application

Chemical fertilizers were applied in the form of urea, single super phosphate (SSP) for phosphorus and muriate of potash (MOP) for potassium. These were applied in split doses, with nitrogen applied at planting and in two top dressings at 45 and 90 days after planting. Phosphorus and potassium were applied as basal doses at the time of planting.

Irrigation and Crop Management

The crop was irrigated through a surface irrigation system at intervals depending on weather conditions. Weeds were controlled through manual weeding. Pest and disease management practices were followed using integrated pest management (IPM) strategies, ensuring minimal pesticide use to avoid interference with microbial bio-inoculants.

Observations and Data Collection

Observations were made at critical growth stages and data were collected on plant growth parameters, yield attributes and juice quality as follows: A. Plant Growth Parameters, Number of tillers per hectare at 120 DAP, Plant height (cm) at 180 DAP, Cane girth (cm) at the maturity stage. B. Yield Attributes and Cane Yield, Number of millable canes (NMC) at maturity, Single cane weight (kg) at harvest, Total cane yield (t/ha) at harvest. C. Juice Quality Parameters, like Brix (%): A measure of total soluble solids, indicating sugar content; Pol (%): A measure of sucrose content in the juice; Juice purity (%): Calculated as the ratio of Pol to Brix.

Statistical Analysis

The collected data were analyzed statistically using analysis of variance (ANOVA) to determine the significance of treatment effects on plant growth, yield attributes, and juice quality. The least significant difference test was applied at a 5% probability level to compare treatment means. The standard error of the mean (SEm) was calculated to assess the variability within treatments. All data were presented in tabular form to provide clarity on the effects of bioinoculants on sugarcane productivity and juice quality.

Soil and Environmental Monitoring

Soil samples were taken from each plot before planting and after harvest to assess changes in nutrient levels and microbial activity. Key soil parameters measured included pH, organic carbon content, nitrogen, phosphorus, potassium, and microbial populations (Azotobacter and Acetobacter counts). These measurements were important for understanding the influence of bio-inoculants on soil health and nutrient dynamics. [27,3,12] . The results clearly demonstrate that the application of nitrogenous bio-inoculants, either singly or in combination, significantly enhanced sugarcane growth, yield attributes, and cane yield. The treatments involving both Azotobacter and Acetobacter (T 7 ) consistently performed better across all parameters, showing the synergistic effect of these bioinoculants. The increase in tiller count, plant height, and cane girth can be attributed to the improved nitrogen availability and enhanced nutrient uptake facilitated by bioinoculants, particularly in calcareous soils (Kumar et al., 2022) [4,5] where nutrient availability is often limited due to high pH. The ability of Azotobacter and Acetobacter to fix atmospheric nitrogen and promote root development likely contributed to better crop growth (Kumar and Maurya, 2006) [34] . Furthermore, the increase in single cane weight and overall cane yield highlights the potential of bioinoculants to partially substitute chemical nitrogen fertilizers without compromising productivity. This is especially relevant for sustainable agriculture, as reducing chemical fertilizer inputs can mitigate environmental risks while maintaining high yields (Singh et al., 2026) [28,29] .

Results and Discussions

Juice

Purity, the ratio of Pol to Brix, was relatively consistent across treatments, with no significant differences observed between the control and bio-inoculated treatments. All treatments showed purity levels around 89%. The work is in conformity with the work of Kumar et al., 2023 [10,24] , Kumar et al., 2024 [2] ; Kumar et al., 2025a [16] . Kumar and Singh (2022) [23] ; Singh et al., (2026) [28,29] , highlighted the impact of climate change on soil microorganisms, emphasizing its role in maintaining soil health and plantmicrobe interactions (Kumar et al., 2026) [30] . This perspective aligns with our study, as soil microbial activity significantly influences nutrient availability, which in turn affects juice quality and sugar yield. The observed variations in sugar concentration and juice purity could be attributed to changes in soil microbial dynamics, as suggested by previous research The application of nitrogenous bio-inoculants significantly improved cane juice quality, particularly in terms of Brix and Pol values. The higher Brix percentages observed in treatments T 4 and T 7 suggest that bio-inoculants not only enhance crop yield but also improve the concentration of sugars in the juice, which is a critical factor for sugarcane producers. The increase in Pol values further supports the positive impact of bio-inoculants on juice quality. The improved sucrose content in bio-inoculated treatments can be attributed to better nutrient uptake and the enhanced physiological functioning of the plants due to the nitrogenfixing capabilities of Azotobacter and Acetobacter. While the purity percentage did not show significant differences between treatments, the overall improvement in Brix and Pol values highlights the potential of bio-inoculants to enhance the commercial quality of sugarcane juice. These findings suggest that integrating bio-inoculants into nutrient management practices could benefit both sugarcane farmers and sugar processors by increasing the overall sugar yield per hectare.

Conclusion

The present study on the "

Recommendations based on the results

The adoption of bio-inoculants as part of integrated nutrient management practices offers several advantages: Improved Productivity: Enhanced plant growth parameters and higher cane yield can contribute to increased profitability for sugarcane farmers. Enhanced Juice Quality: Improved Brix and Pol values indicate better sugar recovery, benefiting sugar mills and the overall supply chain. Environmental Sustainability: The reduction in chemical nitrogen fertilizer usage helps mitigate environmental risks such as soil degradation, water contamination and greenhouse gas emissions. Overall, this study provides evidence that nitrogenous bio-inoculants can play a key role in promoting sustainable sugarcane production while maintaining or even improving yield and quality. Future research should focus on long-term field trials and the economic feasibility of large-scale bio-inoculant application to validate these findings further and promote widespread adoption among sugarcane farmers.

Acknowledgement

AcknowledgementAuthors are thankful to technical and supporting staff of Department of Soil Science, Sugarcane Research Institute, RPCAU, Pusa, for valuable help during the course of study.

References

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