Table of Contents
Greener Journal of Agricultural Sciences
ISSN: 2276-7770
Vol. 16(2), pp. 106-114, 2026
Copyright ©2026, Creative Commons Attribution 4.0 International.
https://gjournals.org/GJAS
DOI: https://doi.org/10.15580/gjas.2026.2.061726089
1, 5Department of Biotechnology, Faculty of Science, University of Delta, Agbor, Delta State, Nigeria;
2Department of Statistics, Faculty of Science, University of Delta, Agbor, Delta State, Nigeria;
3Department of Microbiology, Faculty of Science, University of Delta, Agbor, Delta State, Nigeria;
4Aradhe Grammar School, College Road, KwaleOzoro Road, Delta State, Nigeria
Sustainable nutrient recovery from human urine offers a desirable biotechnology approach for improving soil fertility, microbial activity, and crop productivity in low-input farming systems. This study evaluated the effects of 17-day stored human urine on soil–plant–microbe interactions and the reproductive performance of Talinum fruticosum. A completely randomized pot experiment was conducted using two urine application rates (360.10 mL and 620.10 mL) and an untreated control. Soil samples collected before urine application and after harvest were analyzed for bacterial and fungal populations, while reproductive parameters, including days to first flower bud initiation, number of buds, flowers, and fruits, were monitored for 30 days. The 360.10 mL treatment resulted in the earliest flower bud initiation (13 days) and produced the highest numbers of buds, flowers, and fruits, indicating optimal nutrient availability and microbial stimulation for reproductive development. Although the 620.10 mL treatment enhanced reproductive performance compared with the control, its reproductive output was lower than that of the 360.10 mL treatment, suggesting that excessive nutrient input may reduce reproductive efficiency. Microbial populations increased following urine application, with the highest bacterial and fungal counts recorded under the 620.10 mL treatment. Predominant bacterial genera included Bacillus, Flavobacterium, Klebsiella, and Pseudomonas, while Aspergillus, Penicillium, and Rhizopus were the dominant fungal genera. The findings demonstrate that moderate application of stored human urine can enhance soil microbial activity, flowering, and fruit production, highlighting its potential as an affordable and environmentally sustainable biofertilizer for nutrient-efficient agricultural production.
Type: Research
Full Text: PDF, PHP, HTML, EPUB, MP3
DOI: 10.15580/gjas.2026.2.061726089
Accepted: 20/06/2026
Published: 22/06/2026
AKPOROBARO Uyoyou Agnes
E-mail: agnesuyoyou@gmail.com
Keywords: Stored human urine; Soil microorganisms; Reproductive growth; Sustainable agriculture; Nutrient recycling
1. INTRODUCTION
Human excreta reuse, particularly the application of stored or raw human urine has gained attention as a sustainable and affordable strategy for improving soil fertility. Although several studies have examined its nutrient contributions and agronomic effects, very few investigations have identified the specific bacterial or fungal species present in soil before urine application to determine whether stored human urine could enhance these microbial populations and subsequently increase leafy fruit yields. Literature search found no peer-reviewed studies that simultaneously identified the microbial species listed in this research study in soil prior to urine application and assessed whether urine influences their abundance or activity. Consequently, some literature review synthesizes the known ecological and plant-growth-promoting roles of the listed microorganisms based on existing microbiology and soil science.
Bacillus cereus enhances seedling establishment through enzyme secretion, nutrient mineralization, and stress-alleviating ting activities (Zhao et al., 2024). Bacillus circulans produces indole-3-acetic acid and metabolites that stimulate root development and improve disease tolerance (Qin et al., 2021). Bacillus mycoides, a rhizosphere colonizer, aids nutrient turnover through extracellular enzyme production. Flavobacterium denitrificans and Flavobacterium vireti contribute to carbon and nitrogen cycling by decomposing organic substrates. Nitrogen-fixing Klebsiellaoxytoca and Klebsiella pneumoniae enhance cereal and legume growth through atmospheric nitrogen fixation and production of growth-promoting volatiles (Liu et al., 2018; Yu et al., 2025). Micrococcus luteus and related species participate in organic matter decomposition and help maintain microbial resilience in soil ecosystems. Micrococcus denitrificans, though less documented, likely contributes to similar nutrient-cycling processes. Among plant-growth-promoting rhizobacteria, Pseudomonas fluorescens is well recognised for siderophore production, phosphate solubilization, pathogen suppression, and overall biomass improvement (Lally et al., 2017; Garcia-Seco et al., 2015),Pseudomonas putida supports nutrient availability, degrades organic pollutants, and reduces plant stress, while nitrogen-transforming Pseudomonas stutzeri includes strains that enhance plant nutrition and resilience.
The fungal species identified also play important roles in soil productivity and plant health. Aspergillus flavus, although known for aflatoxin production, influences soil ecology and crop establishment Shenge et al., 2019; Ngwenya et al., 2025). Aspergillus nidulans contributes to nutrient mineralization and soil enzyme pools (Agnihotri et al., 1964). Aspergillus niger, Penicillium species, including P. bissettii, P. chrysogenum, and P. Expansum, P. Chrysogenum, Penicillium janthinellum, Penicillium ortum, Penicillium radiatolobactum, Rhizopusoryza,and Rhizopus stolonifer remain under-studied with respect to plant-yield enhancement in agricultural soils. Trichoderma harzianum is particularly well documented for phosphate solubilization, phytohormone production, biocontrol activity, and induction of systemic resistance, all of which contribute to stronger plant growth and yield (Altomare et al., 1999).Few previous studies have shown that bacterial species and Rhizopus stolonifer play central roles in nutrient cycling, decomposition, pathogen suppression, and root-stimulation processes that form the biological basis of soil fertility. Their populations and activities could theoretically be influenced by nutrient-rich inputs such as stored human urine. However, the current lack of studies identifying these specific microorganisms before urine application leaves a significant research gap. Addressing this gap will require experimental work that couples controlled urine-amendment trials with microbial identification and count to determine whether stored human urine can enhance beneficial microbial populations and improve crop productivity.
2. MATERIALS AND METHODS
Fig. 1: Area of field work
2.1 Description of study site
Field investigations were carried out within a rural settlement situated close to an urban center in the Niger Delta region, governed by a local administrative authority in the southern part of the country.
2.2 Origin of biological samples
Reproductive plant materials were obtained from a non-commercial horticultural site overseen by an academic professional in a low-density countryside environment.
2.3 Materials and equipment used
Plant growth vessels of fixed capacity were employed alongside a mass-determination instrument, a volumetric apparatus for dispensing preserved biological fluid, and untreated supply liquid served as the reference condition.
2.4 Sampling and handling of growth medium
Substrate material was obtained from the upper surface layer of an agricultural zone adjacent to a private plot within a southern settlement with bulk quantities transported using large-capacity sacks.
2.5 Donor origin and processing of liquid amendment
A single donor supplied biological liquid (urine) gathered at dawn (between 5:30 and 6:00 a.m) over consecutive days using sealed polymer containers. The accumulated sample underwent seventeen days controlled retention before predefined aliquots were separated into marked vessels, while an additional unit containing untreated liquid functioned as a baseline. Acidity–alkalinity status was evaluated using an indicator reagent, yielding a pale blue response.
2.6 Filling and arrangement of cultivation units
Cultivation units were loaded with a standardized mass of growth medium and arranged according to specified vertical depth and planar spacing measurements.
2.7 Amendment delivery to treatment containers
Allocated treatments received defined volumes of the retained biological liquid, whereas comparison units were supplied solely with untreated liquid at an equivalent quantity.
2.8 Source, origin, and identification of seeds material
Plant material (Talinum fruticosum) used in this study originated from a non-commercial single-family home garden. The origin of the seeds was traced to edible water leaves purchased from Baleke Market, Agbor, Delta State, Nigeria. After consumption of the fresh stems and leaves, residual plant materials containing fruits with embedded seeds were discarded within the corresponding author’s home garden, where natural germination occurred. The resulting plants matured, produced fruits, and seeds harvested from these fruits were used for the experiment. The cultivated water leaves produced during the study were also consumed by the corresponding author, who holds a doctoral degree in Plant Physiology and Biochemistry and is a lecturer in plant- and biotechnology-related courses in the Department of Biotechnology, Faculty of Science, University of Delta, Agbor, Nigeria. Plant identification was carried out by the corresponding author based on professional expertise. No voucher specimen was deposited. Plant material was obtained exclusively from a privately managed home garden, and no sampling from wild populations was undertaken. Consequently, no collection permits or licences were required. All procedures complied with local and professional regulations governing experimental research on cultivated plants.
2.9 Direct introduction of propagules into containers
Following moisture application, paired propagules extracted from mature reproductive structures were introduced into each container. Subsequent emergence was monitored, after which excess juvenile growth was removed to maintain a single individual per unit.
2.10 Layout and allocation strategy
A randomized allocation framework governed the study, ensuring uniform exposure conditions across all growth units.
2.11 Climatic conditions during cultivation period
Propagation commenced in early July under seasonal precipitation conditions, with supplemental hydration administered at scheduled evening intervals using measured liquid volumes across multiple weeks.
2.12 Evaluation of reproductive development
Developmental timing and reproductive output parameters, including initiation events and organ counts, were systematically monitored for all specimens.
2.13 Documentation of structural characteristics
Structural characteristics were visually captured and systematically recorded across treatments, with particular attention directed toward yield-related traits.
2.14 Isolation and characterization of heterotrophic bacteria and fungi from the experimental soil samples: before application of 17-day stored human urine and after plant harvest.
The soil microbial analysis was conducted in Quality Analytical Laboratory Services Ltd. Km 8, Benin/Lagos Express Road, Opposite Konkon Petrol Station, Evbumare Quarters, Benin City, Edo State, Nigeria. The below steps were followed:
2.14.1 Isolation of bacteria and fungi using serial dilution
Soil samples were collected from experimental pots before the application of the 17-day stored human urine and after plant harvest to investigate the total bacteria and fungi species population present. The procedures were used to determined bacterial and fungal activities. All media were prepared according to manufacturer instruction. The media used in the analysis include nutrient agar (NA) and potato dextrose agar (PDA). Test tubes, petri dishes and other equipment were washed and sterilised at high temperature of 160oC. Dilution plating method was applied, test tubes labeled serial number 1 to 10 were used, 5 g from the soil sample was poured in a 20 mL test tube labeled 1 and 5 mL of distilled water was added making it 10 mL, 1.0 mL from the test tube labeled 1was pipetted and added to another test tube labeled 2 containing 9 ml of distilled water, making it 10 ml, then 1 mL was pipetted from the test tube labeled 2 and added to test tube labeled 3 containing 9 ml of distilled water making it 10 ml and 1mL was pipetted and added to test tube labeled 4 containing 9 ml of distilled water, making it 10 ml, then 1 mL was pipetted from the test tube labeled 4 and added to test tube labeled 5 containing 9 ml of distilled water and 1 ml was pipetted and added to another labeled test tube by following the same trend until it get to test tube labeled 10 and 1ml was pipetted from test tube labeled 10 and discarded. The test tube labeled 2, 4 and 6 were poured in9.5 cm in diameterpetri dishes labeled 2, 4, and 6. The culture petri dishes were swirled and allowed to solidify and the petri dishes were incubated at 37oC for 48 hours, NA for isolation of bacteria and while fungi petri dishes were incubated at 30oC for 3 days, PDA was used for the isolation of fungi. After incubation, observed discrete colonies were enumerated.Unique bacterial colonies were purified and identified according to their morphological, cultural, and biochemical characteristics using the identification criteria (Cheesbrough, 2000) while unique fungi colonies were identified by mycelium growth (Grasser and Donaghue, 1999; Gonzalez, 1996).
2.14.2 Identification and morphological bacteria isolates test
Smears of the isolates were prepared and heat fixed on clean grease free slides. The smears were stained for 1 minute with crystal violet. This was washed out with distilled water. The slides were flooded with dilute iodine solution for 1 minute.This was washed off with distilled water and the smears were decolorized with 95% alcohol for 30 seconds and rinsed off with distilled water. The smears were then counter stained with saffranin solution for 1 minute. Finally, the slides were washed off with distilled water, air dried and observed under oil immersion objective. This is a test to detect the presence or absence of catalase enzyme. The catalase enzyme catalyses the breakdowns of hydrogen peroxide to release free oxygen gas and the formation of water. A few drops of freshly prepared 3% hydrogen peroxide were added onto the bacteria isolates smeared on a slide (Olutiola et al., 1999). There was production of gas bubble which indicated catalase enzyme positive [14]
2.14.3 Oxidase test
A piece of filter paper was wet with a few drops of the dilute (1%) solution of oxidase reagent (tetramethyl-pphenylenediaminedihydrochloride) which was prepared by standard procedure. A bit of growth from the nutrient agar slant was obtained using sterilized platinum wire loop and smeared on the wet piece of paper (Holt et al., 1994). Development of an intense purple color by the cells within 30 seconds was observed which indicates a positive oxidase test (Cheesbrough, 2005).
2.14.4 Coagulase test
Clean slide was divided into two sections, to one section of the slide the test organism was smeared on it using a sterile wire loop while a drop of distilled water was added to the other section which serves as control. Then human plasma was added to both sections and the slide was rock gently for some minutes. A clumping/agglutination of the plasma were used to indicate the presence of coagulase.
2.14.5 Urease test
The bacterial isolates were inoculated into slants of urea medium and incubated at 37 0C for 48 hours (Cheesbrough, 2005).
2.14.6 Indole test
This test was used to determine which of the isolates has the ability to split indole from tryptophan present in buffered peptone water. The test was used as an aid in the differentiation of gram-negative Bacilli spp. especially those of the enterobacteriacene. Peptone water was prepared and about 3 mL of it was dispensed in bijou tubes using a sterile pipette. Then, fresh sterile loops were used to pick a well-isolated colony of bacteria and inoculated into bijou tubes, thereafter, the tubes were incubated at 37 0C for 48 hours. After incubation period, 0.5 ml of Kovac’s Indole Reagent was added to the inoculated bijou tubes. The tubes were subjected to gentle shaking and examined for red color in the surface layer within 10 minutes (Cheesbrough, 2000).
A red ring on top of the tube was observed which indicated indole positive reaction.
2.14.7 Citrate utilization test
This test was based on the ability of some organisms to utilize citrate as a sole source of carbon. This was carried out by inoculating the test organism in test tube containing Simon’s citrate medium and this was incubated at 37 0C for 48 hours. There was a development of deep blue color after incubation which indicates a positive result.
2.14.8 Sugar fermentation test
Each of the isolates was tested for its ability to ferment a given sugar with the production of acid and gas. Since most bacteria especially Gram negative bacteria utilize different sugars as source of carbon and energy with the production of either acids and gas or acid only, the test was used as an aid in their differentiation. The growth medium used was peptone water and the peptone water was prepared in a conical flask and the indicators; phenol red was added. The mixture was dispensed into test tubes containing Durhams tubes. The tubes with their content were sterilized by autoclaving at 121 0C for 15 minutes. 1% solution of the sugar was prepared and sterilized separately at 115 0C for 10 minute. It was then aseptically dispensed in 5 ml volume into the tubes containing the peptone water and indicator. The tubes were inoculated with young culture of the isolates and incubated at 37 0C. Acid and gas production was observed after about 24 hours of incubation. Acid production was indicated by the change of the medium from light green to yellow color while gas production was indicated by the presence of gas in the Durham’s tubes.
2.14.9 Identification of fungi isolates
The fungi isolates were identified microscopically using lactophenol cotton blue test. The identification was achieved by placing a drop of the stain on clean slide with the aid of a wire loop, where a small portion of the mycelium from the fungal cultures was removed and placed in a drop of lactophenol. The mycelium spread rapidly on the slide with the aid of the wire loop. A cover slip was gently applied with little pressure to eliminate air bubbles. The slide was then mounted and observed with x10 and x40 objective lenses respectively (Grasser and Donaghue, 1999; Gonzalez, 1996).
2.14.10 Total bacterial count
Twenty-eight gram (28 g) of nutrient agar powder was dissolved in 1 liter of distilled water in a conical flask covered with cotton wool and aluminium foil paper. It was mixed thoroughly and sterilized by autoclaving at 121 0C for 15 minutes. The medium was cooled to 50 0C and then dispensed aseptically into sterile petri dishes.
2.14.11 Total fungal count
Thirty-nine gram (39.0 g) of potato agar dextrose powder was dissolved in 1 litre of distilled water in a conical flask covered with cotton wool and aluminium foil paper. It was mixed thoroughly and sterilized by autoclaving at 121 0C for 15 minutes. The medium was cooled to 50 0C and then dispensed aseptically into sterile petri dishes.
Although manufacturer procedures for isolating bacteria and fungi were strictly followed, no peer-reviewed published article has provided a step-by-step detailed method for total bacterial and fungal isolation and counting except this present study.
2.15 Data analysis
Data were presented as mean ± standard deviation based on three replicates. Statistical analyses were conducted using one-way analysis of variance (ANOVA) with IBM SPSS Statistics version 29.0 (IBM Corp., Armonk, NY, USA). Where significant differences were detected, treatment means were compared using Duncan’s Multiple Range Test at the 5% level of significance. Differences among means were considered statistically significant at p ≤ 0.05.
Tables 1 and 2 show that microbial populations increased following the application of 17-day stored human urine, with the highest bacterial and fungal counts generally recorded at the 620.10 mL treatment. The two application rates (360.10 and 620.10 mL) were selected to evaluate the effects of moderate and relatively high nutrient inputs on soil microbial communities and reproductive responses of Talinum fruticosum. Stored (fermented) human urine was used because storage promotes urea hydrolysis and nutrient stabilization, reduces the viability of pathogenic microorganisms, and provides a readily available source of nitrogen and other essential nutrients for plant and microbial growth. However, the most favorable reproductive responses were obtained with the 360.10 mL treatment. These observations agree with reports by Zhao et al. (2024), Qin et al. (2021), Lally et al. (2017), Liu et al. (2018), and Yu et al. (2025), who demonstrated that beneficial microorganisms enhance plant productivity through nutrient transformation, nutrient mobilization, and improved rhizosphere functioning. Similarly, fungal taxa, including Aspergillus, Penicillium, Rhizopus, and Trichoderma harzianum, contributed to improved nutrient availability and reproductive performance.
The microbial populations observed provide a biological explanation for the reproductive responses recorded in this study. Bacteria facilitate nutrient mineralization and the production of growth-promoting substances, whereas fungi contribute to decomposition, phosphorus solubilization, and rhizosphere interactions. The higher microbial counts observed under the 620.10 mL treatment suggest that greater nutrient availability favored microbial proliferation; however, the superior reproductive performance recorded at 360.10 mL indicates that moderate nutrient supply provided a more favorable balance between nutrient availability and reproductive processes. These microbial activities help explain the enhanced flowering and fruit production obtained under stored human urine treatments.
This study demonstrates that 17-day stored human urine influences soil microbial communities and reproductive performance in Talinum fruticosum. The 360.10 mL application rate was identified as the optimum treatment for reducing the number of days to first flower bud initiation (Table 3) and increasing flower bud formation, flower production, and fruit set (Tables 4–6; Fig. 2A–2C). The close association between microbial abundance and reproductive responses provides a biological basis for understanding the beneficial effects of stored human urine. Future studies should employ molecular and metagenomic approaches to verify microbial functions and elucidate the mechanisms through which specific microorganisms contribute to the beneficial effects observed under the optimum urine treatment.
Tables 1 and 2 present the microbial counts identified in the experimental soils before the application of different levels of 17-day stored human urine (Soil 1), and after 30 days of growth of Talinum fruticosum in the control (0 mL) treatment (Soil 2), the 360.10 mL treatment (Soil 3), and the 620.10 mL treatment (Soil 4).
Table 1: Microbial species and counts in experimental soil
Values = sample mean ± standard deviation; n = 3, mean with different superscript alphabets in a row are significantly different at p ≤0.05.
Table 2: Microbial species and counts in experimental soil
Table 3: Effect of 17-day stored human urine on days to first flower bud initiation in Talinum fruticosum
Table 4: Effect of 17-day stored human urine on number of flower buds in Talinum fruticosum at 18, 25, and 30 days after sowing
Values are mean ± standard deviation; n = 3, mean with different superscript alphabets in a row are significantly different at p ≤0.05.
Table 5: Effect of 17-day stored human urine on number of flowers in Talinum fruticosum at 25 and 30 days after sowing
25
Values are mean ± standard deviation; n = 3. mean with different superscript alphabets in a row are significantly different at p ≤0.05.
Table 6: Effect of 17-day stored human urine on number of fruits (terreria) formed in Talinum fruticosum at 30 days after sowing
Fig. 2:Thirty days reproductive growth of Talinum triangulare grown in 0mL (A) untreated-soil, 360.10 mL (B), and 620.10 mL (C).
The application of 17-day stored human urine significantly improved the reproductive performance of Talinum fruticosum and increased soil microbial populations. The 360.10 mL treatment produced the earliest flower bud initiation and the highest numbers of buds, flowers, and fruits, demonstrating that moderate nutrient enrichment provided the most favorable conditions for reproductive development. Although the 620.10 mL treatment generated the highest bacterial and fungal populations, reproductive performance was lower than that achieved with the moderate application rate. The study demonstrates that stored human urine can function as an effective, low-cost biofertilizer capable of enhancing soil biological activity and supporting sustainable crop production. Future studies should employ molecular approaches to verify microbial functions and further investigate the physiological mechanisms underlying plant responses to stored human urine.
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this manuscript.
Authors’ contributions
Uyoyou Agnes Akporobaro provided the 17-day-stored human urine and waterleaf (Talinum fruticosum) seeds used for planting, conceived and designed the study, and wrote the original draft of the manuscript. Uyoyou Agnes Akporobaro and Esiwo Precious Akporobaro conducted the field experiment. Rita NnekaNwaka performed the data analysis. Uyoyou Agnes Akporobaro, Odimegwu Agholor, Joy Anwuli Obiaigwe, andEsiwo Precious Akporobaro conducted the microbial analyses in the laboratory, while Esiwo Precious Akporobaro and Joy Anwuli Obiaigwe wrote the literature review in the introduction section. All authors provided financial support for the laboratory analyses, reviewed, and approved the final manuscript.
Funding information
This study was self-funded by the authors, with no financial support from any organisation.
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Akporobaro, UA; Agholor, O; Nwaka, RN; Chiedu, E; Obiaigwe, JA; Akporobaro, EP (2026). Sustainable nutrient recovery from human urine: a soil–plant–microbe interaction study for enhanced agricultural productivity of Talinum fruticosum L (Juss.). Greener Journal of Agricultural Sciences, 16(2): 106-114, https://doi.org/10.15580/gjas.2026.2.061726089.
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