Alternative Nitrogen Sources Research Articles

Enhancing Food Security in an Era of Rising Fertilizer Prices: Evaluation of an Intervention Promoting Mungbean Adoption in Nepal

The improved food security in Asia that has facilitated the region’s development progress depends on nitrogenous fertilizers. Rising prices and shortages of imported fertilizer have prompted countries to explore alternative sources of crop nitrogen, including diversification with legumes. We evaluate an intervention in Nepal that promoted mungbean adoption. Our doubly robust impact evaluation approach accounts for nonrandom patterns of adoption related to livestock rearing, participation in agricultural cooperatives and training, and greater irrigated land use. Adopters growing mungbean for the 2-year study period showed an average increase of 20 kilograms (kg) in their annual consumption of mungbean-based foods, applied almost 40[Formula: see text]kg per hectare (ha) less fertilizers to their rice crops, and obtained an additional 280[Formula: see text]kg in rice yield per ha. Hence, agricultural innovations that use legumes such as mungbean can help promote sustainable intensification of cereal-based production systems, while enhancing food security and reducing balance-of-payments issues for the countries dependent on fertilizer imports.

Carbon and Nitrogen Sources Influence Parasitic Responsiveness in Trichoderma atroviride NI-1.

Parasitic species of Trichoderma use hydrolytic enzymes to destroy the host cell wall. Preferent carbon and nitrogen sources suppress the expression of genes related to parasitism. Here, different nutrients were evaluated in the parasitic isolated NI-1, which was identified as Trichoderma atroviride. The genes cbh1 and chb2 (cellobiohydrolases), bgl3.1 (endoglucanase), and pra1 and prb1 (proteinases) were poorly expressed during the interaction between NI-1 and Phytophthora capsici on PDA. However, gene expression improved on minimal medium with preferent and alternative carbon sources. Dextrin and glucose stimulated higher transcript levels than cellulose, sucrose, and glycerol. Also, ammonium stimulated a stronger parasitic responsiveness than the alternative nitrogen sources. During interaction against different phytopathogens, NI-1 detects their host differentially from a distance due to the cbh1 and cbh2 genes being only induced by P. capsici. The pra1 and ech42 genes were induced before contact with Botrytis cinerea and Rhizoctonia solani, while when confronted with P. capsici they were stimulated until contact and overgrowth. The prb1 and bgl3.1 genes were induced before contact against the three-host assayed. Overall, T. atroviride prefers to parasitize and has the capacity to distinguish between an oomycete and a fungus, but nutrient quality regulates its parasitic responsiveness.

РОСТСТИМУЛИРУЮЩАЯ АКТИВНОСТЬ БАКТЕРИЙ ENTEROBACTER LUDWIGII BLK ПРИ ОПТИМИЗАЦИИ СРЕДЫ КУЛЬТИВИРОВАНИЯ

A method for optimizing the cultivation of Enterobacter ludwigii BLK and the treatment of wheat plants using a preparation based on this bacterium is proposed. By reducing the concentration of peptone and partially replacing it with an alternative nitrogen source – yeast extract – in the culture medium, the cost of producing the drug was reduced without reducing its effectiveness. It was found that the preparation, produced in a modified environment, activated root growth and promoted the accumulation of chlorophyll in bread wheat leaves. The stimulating effect was more pronounced with a combination of pre-sowing and post-emergence treatment.

UTILIZATION OF SOYBEAN POWDER AS AN ALTERNATIVE MEDIA FOR THE GROWTH OF ANAEROBIC BACTERIA

Background:Culture media or microbial growth media is a material consisting of a mixture of nutrients used by microorganisms for growth and reproduction. Anaerobic bacteria can thrive by requiring little or no oxygen for growth and metabolism. Purpose: The purpose to determine the potential of soybean powder as an alternative nitrogen source in Streptococcus mutans bacterial growth media and determine the optimum concentration of soybean powder in alternative media for Streptococcus mutans bacterial growth. Methods: The research method is a laboratory experiment to test soybean powder as an alternative medium for the growth of Streptococcus mutans bacteria using the Candle jar method. The population used is soybean powder with the brand soy flour, while the sample used is Streptococcus mutans bacteria. Results: The results obtained can be known that the average value of colony growth of Streptococcus mutans bacteria in alternative media of soybean powder with a concentration variation of 2 grams was 30 colonies, a concentration variation of 3 grams was 54 colonies, a concentration variation of 4 grams was 40 colonies, and a concentration variation of 5 grams was 72 colonies. From the average value, it can be seen that the growth of Streptococcus mutans on alternative media of soybean powder can grow well, but the most optimum growth is in the concentration variation of 5 grams. Conclusion:Based on the observations obtained, it shows that soybean powder (Glycine max (L.) Merr) can be used as an alternative medium for the growth of Streptococcus mutans bacteria.

Utilizing Alfalfa as an Alternative Nitrogen Source in Improving Bermudagrass Grazing Systems in the Southeastern United States

AbstractThe environmental conditions of the Coastal Plains region of the United States provides livestock producers with unique forage production opportunities that may not be experienced nationwide. While the environmental conditions are favorable for forage production, and longer growing seasons, the rising cost associated with livestock production has producers seeking alternative ways to increase productivity while also reducing external input costs, specifically those associated with feeding livestock. Earn 0.5 CEUs in Crop Management by reading this article and taking the quiz at https://web.sciencesocieties.org/Learning‐Center/Courses.

Solid and Liquid Fraction of Digestate as an Alternative Mineral Nitrogen Source: Two-Year Field Research in Croatia

Solid and Liquid Fraction of Digestate as an Alternative Mineral Nitrogen Source: Two-Year Field Research in Croatia

Microbial Bioherbicides Based on Cell-Free Phytotoxic Metabolites: Analysis and Perspectives on Their Application in Weed Control as an Innovative Sustainable Solution.

Weeds cause significant agricultural losses worldwide, and herbicides have traditionally been the main solution to this problem. However, the extensive use of herbicides has led to multiple cases of weed resistance, which could generate an increase in the application concentration and consequently a higher persistence in the environment, hindering natural degradation processes. Consequently, more environmentally friendly alternatives, such as microbial bioherbicides, have been sought. Although these bioherbicides are promising, their efficacy remains a challenge, as evidenced by their limited commercial and industrial production. This article reviews the current status of microbial-based bioherbicides and highlights the potential of cell-free metabolites to improve their efficacy and commercial attractiveness. Stirred tank bioreactors are identified as the most widely used for production-scale submerged fermentation. In addition, the use of alternative carbon and nitrogen sources, such as industrial waste, supports the circular economy. Furthermore, this article discusses the optimization of downstream processes using bioprospecting and in silico technologies to identify target metabolites, which leads to more precise and efficient production strategies. Bacterial bioherbicides, particularly those derived from Pseudomonas and Xanthomonas, and fungal bioherbicides from genera such as Alternaria, Colletotrichum, Trichoderma and Phoma, show significant potential. Nevertheless, limitations such as their restricted range of action, their persistence in the environment, and regulatory issues restrict their commercial availability. The utilization of cell-free microbial metabolites is proposed as a promising solution due to their simpler handling and application. In addition, modern technologies, including encapsulation and integrated management with chemical herbicides, are investigated to enhance the efficacy and sustainability of bioherbicides.

Enhancing docosahexaenoic acid production from Schizochytrium sp. by using waste Pichia pastoris as nitrogen source based on two-stage feeding control

To reduce the cost of docosahexaenoic acid (DHA) production from Schizochytrium sp., the waste Pichia pastoris was successfully used as an alternative nitrogen source to achieve high-density cultivation during the cell growth phase. However, due to the high oxygen consumption feature when implementing high-density cultivation, the control of both the nitrogen source and dissolved oxygen concentration (DO) at each sufficient level was impossible; thus, two realistic control strategies, including “DO sufficiency-nitrogen limitation” and “DO limitation-nitrogen sufficiency”, were proposed. When using the strategy of “DO sufficiency-nitrogen limitation”, the lowest maintenance coefficient of glucose (12.3 mg/g/h vs. 17.0 mg/g/h) and the highest activities of related enzymes in DHA biosynthetic routes were simultaneously obtained; thus, a maximum DHA concentration of 12.8 ± 1.2 g/L was achieved, which was 1.58-fold greater than that of the control group. Overall, two-stage feeding control for alternative nitrogen sources is an efficient strategy to industrial DHA fermentation.

Comparative analysis of prokaryotic microbiomes in high-altitude active layer soils: insights from Ladakh and global analogues using In-Silico approaches.

The active layer is the portion of soil overlaying the permafrost that freezes and thaws seasonally. It is a harsh habitat in which a varied and vigorous microbial population thrives. The high-altitude active layer soil in northern India is a unique and important cryo-ecosystem. However, its microbiology remains largely unexplored. It represents a unique reservoir for microbial communities with adaptability to harsh environmental conditions. In the Changthang region of Ladakh, the Tsokar area is a high-altitude permafrost-affected area situated in the southern part of Ladakh, at a height of 4530m above sea level. Results of the comparison study with the QTP, Himalayan, Alaskan, Russian, Canadian and Polar active layers showed that the alpha diversity was significantly higher in the Ladakh and QTP active layers as the environmental condition of both the sites were similar. Moreover, the sampling site in the Ladakh region was in a thawing condition at the time of sampling which possibly provided nutrients and access to alternative nitrogen and carbon sources to the microorganisms thriving in it. Analysis of the samples suggested that the geochemical parameters and environmental conditions shape the microbial alpha diversity and community composition. Further analysis revealed that the cold-adapted methanogens were present in the Ladakh, Himalayan, Polar and Alaskan samples and absent in QTP, Russian and Canadian active layer samples. These methanogens could produce methane at slow rates in the active layer soils that could increase the atmospheric temperature owing to climate change.

Valorization of black carrot pomace and pea powder for co-production of polygalacturonase and pectin lyase

AbstractPectic enzyme groups, particularly polygalacturonase and pectin lyase, are vital components of a high-value microbial enzyme category widely employed in applications within the fruit juice and wine industries. The exploration of alternative carbon and nitrogen sources remains crucial for enhancing enzyme production while reducing costs. This study evaluates the impact of carbon (black carrot pomace) and nitrogen (pea protein) loading on fermentable sugar content, protein content, and enzyme activities during both batch and fed-batch cultivation. Additionally, three distinct valorization techniques—thermal (steam), thermochemical (steam assisted with dilute acid), and microwave-assisted with dilute acid pretreatments—were assessed for their effectiveness in hydrolyzing black carrot pomace as a carbon source. The findings indicate that microwave-assisted dilute acid, coupled with enzymatic saccharification, resulted in the highest fermentable sugar production (0.493 g/g), achieving an 87.3% conversion yield. Pea protein demonstrated more favorable outcomes with the highest polygalacturonase activity (20.50 ± 0.52 U/L) and pectin lyase activity (46.44 ± 3.45 U/L) compared to whey protein and yeast extract used as nitrogen sources. Meanwhile, the highest polygalacturonase and pectin lyase activity, along with the highest total protein content (52.25 ± 0.06 mg/L), was recorded under the same culture conditions, reaching 164.34 ± 2.26 and 188.22 ± 1.72 U/L, respectively, after 72 h, representing approximately 1.18- and 1.34-fold increases from the batch system. Consequently, these results prove that fed-batch cultivation, utilizing black carrot pomace hydrolyzate as a feeding substrate and pea protein as a nitrogen source, significantly increases polygalacturonase and pectin lyase activity compared to batch cultivation.

Effects of dietary urea supplementation on performance of selected insect species.

Feeding costs of farmed insects may be reduced by applying alternative nitrogen sources such as urea that can partly substitute true proteins. The aim of this study was to examine the effects of different nitrogen sources on body weight (BW) and survival rate (SR) of the Jamaican field cricket (JFC, Gryllus assimilis), the house cricket (HC, Acheta domesticus), yellow mealworm larvae (YM, Tenebrio molitor) and superworm larvae (SW, Zophobas morio). Crickets were either housed individually or in groups, and larvae were group-housed. Six isonitrogenous feeds composed of 3.52% nitrogen were designed forall four insect species using four independent replicates with micellar casein: urea proportions of 100-0%, 75-25%, 50-50%, 25-75%, 0-100% and 100% extracted soybean meal. All selected insect species were able to utilise urea. However, urea as the only nitrogen source resulted in low final BW. Inthe HC, the JFC, and the YM on nitrogen basis urea can replace 25% of micellar casein without having any negative effects on BW and SR in comparison to the 100% micellar casein group. In the SW, a 25% urea level did not have a significant effect on final BW, but SR decreased significantly.

The Nitrogen Content in the Fruiting Body and Mycelium of Pleurotus Ostreatus and Its Utilization as a Medium Component in Thraustochytrid Fermentation.

Following the idea of a circular bioeconomy, the use of side streams as substitutes for cultivation media (components) in bioprocesses would mean an enormous economic and ecological advantage. Costly compounds in conventional media for the production of the triterpene squalene in thraustochytrids are the main carbon source and complex nitrogen sources. Among other side streams examined, extracts from the spent mycelium of the basidiomycete Pleurotus ostreatus were best-suited to acting as alternative nitrogen sources in cultivation media for thraustochytrids. The total nitrogen (3.76 ± 0.01 and 4.24 ± 0.04%, respectively) and protein (16.47 ± 0.06 and 18.57 ± 0.18%, respectively) contents of the fruiting body and mycelium were determined. The fungal cells were hydrolyzed and extracted to generate accessible nitrogen sources. Under preferred conditions, the extracts from the fruiting body and mycelium contained 73.63 ± 1.19 and 89.93 ± 7.54 mM of free amino groups, respectively. Cultivations of Schizochytrium sp. S31 on a medium using a mycelium extract as a complex nitrogen source showed decelerated growth but a similar squalene yield (123.79 ± 14.11 mg/L after 216 h) compared to a conventional medium (111.29 ± 19.96 mg/L, although improvable by additional complex nitrogen source).

Improved Cordycepin Production by Cordyceps Militaris Using Corn Steep Liquor Hydrolysate as an Alternative Protein Nitrogen Source.

Cordycepin production in the submerged culture of Cordyceps militaris was demonstrated using hydrolyzed corn processing protein by-products, known as corn steep liquor hydrolysate (CSLH), as an alternative nitrogen source. The growth, metabolism, and cordycepin production of Cordyceps militaris were evaluated under various concentrations of CSLH induction. The results demonstrated that CSLH addition had positive effects on the growth and cordycepin production with various C. militaris strains. The optimum strain, C. militaris GDMCC5.270, was found to effectively utilize CSLH to promote mycelium growth and cordycepin production. Low concentrations of CSLH (1.5 g/L) in the fermentation broth resulted in 343.03 ± 15.94 mg/L cordycepin production, which was 4.83 times higher than that of the group without CSLH. This also enhanced the metabolism of sugar, amino acids, and nucleotides, leading to improved cordycepin biosynthesis. The increase in key amino acids, such as glutamic acid, alanine, and aspartic acid, in the corn steep liquor hydrolysate significantly enhanced cordycepin yield. The corn steep liquor hydrolysate was confirmed to be a cost-effective accelerator for mycelium growth and cordycepin accumulation in C. militaris, replacing partial peptone as a cheap nitrogen source. It serves as a suitable alternative for efficient cordycepin production at a low cost.

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences.

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (β-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while β-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. These results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

Efficient Production of Succinic Acid from Sugarcane Bagasse Hydrolysate by Actinobacillus succinogenes GXAS137

Sugarcane bagasse (SCB) is an abundant agricultural waste, rich in cellulose and hemicellulose, that could be used as an ideal raw material for succinic acid (SA) production. A two-step chemical pretreatment, involving alkali extraction and alkaline hydrogen peroxide treatment, was utilized to treat SCB, followed by multi-enzyme hydrolysis to obtain a reducing sugar hydrolysate mainly composed of glucose and xylose. Optimization of the multi-enzyme hydrolysis of pretreated SCB resulted in a final reducing sugar concentration of 78.34 g/L. In order to enhance the bioconversion of SCB to SA and to reduce the production costs, the initial reducing sugar concentration, nitrogen source, and MgCO3 content were further optimized. The results demonstrated that the inexpensive corn steep liquor powder (CSLP) could be utilized as an alternative nitrogen source to yeast extract for the production of SA; and the optimal concentrations of initial reducing sugar, CSLP, and MgCO3 were 70 g/L, 18 g/L, and 60 g/L, respectively. When fed-batch fermentation was conducted in a 2 L stirred bioreactor, approximately 72.9 g/L of SA was produced, with a yield of 83.2% and a productivity of 1.40 g/L/h. The high SA concentration, yield, and productivity achieved in this study demonstrate the potential of SCB, an agricultural waste, as a viable alternative substrate for Actinobacillus succinogenes GXAS137 to produce SA. This lays a solid foundation for the resource utilization of agricultural waste and cost-effective industrial-scale production of SA in the future.

Waste valorization through acetone-butanol-ethanol (ABE) fermentation

BackgroundBiobutanol, produced through acetone-butanol-ethanol (ABE) fermentation, has been proposed for use as a transportation fuel and for the development of butanol-based materials. However, the economic viability of ABE fermentation is heavily dependent on the cost of raw materials. MethodsThis study assesses various carbon and nitrogen sources for ABE fermentation. Carbon sources examined include local molasses (M1), Thai molasses (M2), Taiwan sugar agricultural molasses (M3), and Taiwan sugar edible molasses (M4). Nitrogen sources considered are corn steep liquor (CSL), acid-hydrolyzed black soldier fly larval meal (IA), and enzyme-hydrolyzed black soldier fly larval meal (IE). Significant findingsOur findings reveal that M2CSL, a combination of Thai molasses and corn steep liquor, serves as a cost-effective and efficient medium for ABE fermentation, resulting in a butanol concentration of 7.4 ± 3.5 g/L. Furthermore, our research identifies variations in molasses and their impact on sucrose consumption inhibition. Notably, this study underscores the potential of insect peptone derived from enzyme-hydrolyzed black soldier fly larval meal (IE) as an alternative nitrogen source, while cautioning against the use of insect peptone obtained from acid-hydrolyzed black soldier fly larval meal (IA) due to its inhibitory properties.

Utilization of Nostoc piscinale as Potential Biofertilizer to the Growth and Development of Oryza sativa L.

<em>Nostoc</em> is a blue-green cyanobacteria that produce their food through photosynthesis and nitrogen fixation. These organisms undergo nitrogen fixation and provide a potential nitrogen source for growth and development. Since rice is known as one of the world’s staple foods, especially in Asia, this study aims to determine the utilization of <em>Nostoc piscinale </em>as a potential biofertilizer for planting rice crops. <em>N. piscinale</em> was inoculated into three subcultures and incubated for 87 to 170 days, and then analyzed for nitrogen-fixing activity and rice plant development. Growth of cyanobacteria showed a significant increase in chlorophyll <em>a</em> starting from day 30 up to day 170 while nitrogen-fixing activity remained constant from day 4. On the other hand, the growth and development of rice treated with cyanobacteria showed correlated trends with commercial fertilizer (CSF) in terms of root and shoot (growth and fresh weight) and chlorophyll <em>a</em> content with no statistical differences (p-value ≥ 0.05). Nitrogen tests indicate the utilization of ammonia produced by <em>N. piscinale</em> and the change in soil pH. After harvesting the samples at 20 days and measuring the soil pH, the cyanobacterial samples were seen to lower the soil pH before planting, which is significantly different from the untreated and CSF-treated samples. The utilization of nitrogen for the growth and development of <em>Oryza sativa</em> subsp. <em>indica </em>proved that <em>N. piscinale</em> would be a positive alternative source of nitrogen due to the results obtained from the soil nitrogen composition and soil pH.

Proteomics of Paracoccidioides lutzii: Overview of Changes Triggered by Nitrogen Catabolite Repression.

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MSE proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, β-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection.

Effect of Calcium Cyanamide as an Alternative Nitrogen Source on Growth, Yield, and Nitrogen Use Efficiency of Short-Day Onion

Effective nitrogen (N) management in agriculture is vital to optimize crop growth and yield while minimizing environmental impact. Conventional nitrogen (N) sources, such as urea, have limitations in promoting growth and reducing N leaching. A two-year field experiment was carried out to investigate the effects of calcium cyanamide (CaCN2) as a slow-release N source on short-day onion growth, yield, and N use efficiency (NUE). Six types of N sources were administered: (i) an initial application of 80 kg ha−1 N in the form of CaCN2 before planting; (ii) an initial application of 80 kg ha−1 N in the form of CaCN2 before planting, followed by a topdressing of 50 kg ha−1 N in the form of limestone ammonium nitrate (LAN); (iii) an initial application of 80 kg ha−1 N in the form of CaCN2 before planting, followed by a topdressing of 50 kg ha−1 N in the form of urea; (iv) an initial application of 80 kg ha−1 N in the form of LAN before planting, followed by a topdressing of 50 kg ha−1 N in the form of LAN; (v) an initial application of 80 kg ha−1 N in the form of urea before planting, followed by a topdressing of 50 kg ha−1 N in the form of urea; and (vi) control (0 kg ha−1 N). Preplant CaCN2 (80 kg ha−1 N) outperformed the standard fertilizers used in onion as an N source (urea and LAN) by improving growth and yield, and reducing N leaching. Preplant CaCN2 topdressed with either LAN or urea led to a significant increase in plant growth and total yield compared to using LAN or urea alone. The application of CaCN2, followed by topdressing with either LAN or urea, decreased onion bolting by 1.6% and 1.83%, respectively, compared to the control. The study suggests that applying LAN or urea as a topdressing to preplant CaCN2 enhances N utilization efficiency, leading to increased onion bulb yield and quality while reducing N leaching. This approach can help mitigate farm-level environmental pollution and provide valuable insights for improving onion production and sustainable agriculture practices in South Africa.

Azolla Fertilizer as an Alternative Organic Nitrogen Source for Malabar Spinach Production

The spread of organic farming has led to the need for identifying alternative sources of high-quality organic fertilizer. Azolla is a genus of floating aquatic ferns which has high nitrogen and organic matter content due to its fast growth and symbiotic relationship with nitrogen-fixing bacteria (Anabaena-azollae). However, little research has been done on the use of Azolla as an organic fertilizer for vegetable production. The study evaluated the effects of different levels of Azolla fertilizer on the growth and yield of Malabar spinach (Basella alba). The experiment included 6 treatments: Without application – as a control (T1); 1 ton ha-1 soybean meal (T2); 16 tons ha-1 cow manure (T3); 12 tons ha-1 Azolla fertilizer (T4); 16 tons ha-1 Azolla fertilizer (T5); and 20 tons ha-1 Azolla fertilizer (T6). The results showed that the application of Azolla fertilizer significantly increased shoot length, number of leaves, leaf size, dry matter, leaf area index (LAI), and SPAD of Malabar spinach compared to the control or cow manure. The application of Azolla fertilizer at the rates of 12, 16, and 20 tons ha-1 significantly increased the yield of Malabar spinach by 150, 192, and 205%, respectively, compared to the control; and by 37, 60, and 67%, respectively, compared to the cow manure treatment. Yields and parameters of harvested vegetables were the highest in treatments with 16 to 20 tons ha-1 Azolla fertilizer. The results suggest that Azolla fertilizer can be used as an alternative organic nitrogen source in organic vegetable production.