Low-concentration methanogenic effluent combined with nitrogen-fixing bacteria as a sustainable nutrient solution for hydroponic lettuce production

Abstract

This study evaluated methanogenic effluent combined with nitrogen-fixing bacteria (NFB) strain BSN6 as a sustainable nutrient solution for hydroponic green oak lettuce (Lactuca sativa var. crispa L.) cultivation. A preliminary experiment assessed the NFB effects on germination, while the main experiment tested six effluent concentrations (0, 2.5, 5, 10, 15, and 20%) with and without the NFB using a completely randomized design. The 2.5% effluent combined with the NFB produced optimal growth, achieving 16.66 ± 0.95 cm plant height, 25.60 ± 0.60 leaves, and 39.74 ± 3.21 g fresh weight, which was comparable to commercial AB Mix fertilizer. Higher concentrations (15–20%) caused plant toxicity, with 20% + the NFB resulting in complete mortality. The NFB enhanced nitrogen availability at low concentrations but proved ineffective at higher levels. A preliminary safety assessment showed no detectable copper or chromium in lettuce tissue. This approach offers environmentally sustainable effluent management while reducing synthetic fertilizer dependence, demonstrating significant potential for integrated biogas-agriculture systems.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-52581-w.

Introduction

The global shift toward sustainable development has transformed waste-to-renewable energy (WTRE) technologies into a cornerstone of circular economy systems. Anaerobic digestion (AD) has gained significant attention as a promising WTRE technology over recent decades, contributing to energy security while mitigating waste and greenhouse gas (GHG) emissions^1,2. As a biological process, AD converts organic waste into biogas, primarily consisting of methane (CH₄) and carbon dioxide (CO₂). This technology is particularly relevant in regions experiencing rapid urbanization and population growth, supporting both energy and environmental goals^3,4.

Despite its benefits, AD generates a liquid byproduct known as methanogenic effluent⁵. This effluent forms during the final phase of the digestion process and is produced in large volumes. In Thailand, over 1,700 biogas plants are operating, potentially generating vast quantities of effluent daily that must be properly managed⁶. The management of this effluent remains a critical issue for the biogas industry, as improper disposal poses environmental risks⁷. Therefore, there is a strong need to explore safe and beneficial treatment and valorization strategies for methanogenic effluent, particularly approaches that support sustainable agriculture goals and pursue zero-waste strategies.

One promising application for methanogenic effluent valorization is its use as a nutrient source in hydroponic systems. Recent studies have highlighted the nutrient-rich composition of methanogenic effluent, which contains high levels of essential plant nutrients including nitrogen (N), phosphorus (P), and potassium (K). These characteristics make effluent potentially suitable as a waste-derived nutrient source in hydroponic systems, where crops are cultivated in soilless conditions⁸. Integrating effluent into hydroponic cultivation represents a forward-thinking strategy with significant agronomic and ecological benefits, including effective nutrient recovery, reduced dependence on synthetic fertilizers, and mitigation of environmental burden from effluent discharge. The chemical composition of effluent varies depending on feedstock, digestion process, and operational conditions⁹.

However, significant challenges exist regarding direct application of untreated methanogenic effluent in hydroponics. Its incompletely stabilized nutrient composition, elevated salinity levels, high ammonia concentrations, and fluctuating pH are limiting factors that require attention when using effluent for crop cultivation^10,11. These factors commonly harm plant roots, limit nutrient absorption, and reduce crop yield and quality. Additional pre-treatment processes are necessary to address these issues and ensure suitability for plant growth.

Among the available pre-treatment approaches, dilution represents a simple yet effective method to create a more manageable nutrient solution and lower the concentration of toxic nutrient compounds, making it safer for plant growth. Diluting effluent helps maintain optimal electrical conductivity (EC) and pH levels closer to the ideal range for nutrient uptake in hydroponic systems¹². According to Song et al.¹³, highly concentrated effluent with methanogenic compounds at concentrations between 20 and 40% showed effectiveness comparable to mineral fertilizers. However, effluent concentrations above 40% cause plant stress. Therefore, diluting the effluent could enhance the effectiveness of wastewater as an alternative to commercial fertilizers.

Beyond dilution alone, biological enhancement strategies offer additional potential for improving effluent-based nutrient solutions. To further enhance the agronomic benefits of methanogenic effluent, researchers have investigated plant growth-promoting bacteria, especially nitrogen-fixing bacteria (NFB), which can enrich the nitrogen content of nutrient solutions. These the NFB have been beneficial from the seedling stage to transplanting, improving seed emergence, bolstering seedlings, and enhancing root characteristics¹⁴. These bacteria improve nutrient availability by breaking down organic materials, producing growth hormones, and combating harmful microbes¹⁵. When combined with diluted effluent, the NFB are expected to improve N availability in nutrient solution and promote healthier plant growth.

Despite the potential benefits of methanogenic effluent as a nutrient source and the proven advantages of the NFB in plant cultivation, critical knowledge gaps remain unaddressed in the literature. While previous studies have demonstrated that effluent concentrations between 20 and 40% can be effective, there is insufficient systematic investigation of lower concentration ranges that might provide optimal plant growth without stress observed at higher concentrations. Additionally, although both effluent dilution and the NFB application have been studied independently, the synergistic effects of combining specific concentrations of methanogenic effluent with the NFB have not been comprehensively evaluated. The optimal effluent concentrations for different crops, particularly in combination with the NFB, remain poorly defined for hydroponic systems.

Based on these knowledge gaps and the need for sustainable effluent management solutions, we hypothesized that lower concentrations of methanogenic effluent combined with the NFB would provide superior plant growth and yield compared to higher concentrations, and that the combination would outperform effluent alone. This study evaluated the efficacy of the NFB introduced at two stages of lettuce growth, seedling and transplanting under hydroponic conditions. Seedlings were initially tested to determine optimal conditions for growing healthier seedlings by applying the NFB as a hydroponic germination solution and comparing results to those grown with standard commercial AB mix. In the main experiment, different concentrations of diluted methanogenic effluent (2.5, 5, 10, 15, and 20%), with and without the NFB, were tested during cultivation of green oak lettuce to determine optimal conditions for yield and quality. The findings provide practical guidelines for implementing integrated biogas-agriculture systems that are environmentally sound, economically viable, and scalable.

Materials and methods

Study design and setting

This experimental study was conducted in two phases using a completely randomized design: (1) a preliminary experiment to evaluate effects of the NFB application on germination and early seedling development, and (2) a main experiment to optimize methanogenic effluent concentration and its combination with the NFB for hydroponic green oak lettuce cultivation (Fig. S1). The preliminary experiment was conducted in a greenhouse at the Biohythane Pilot Plant, Khon Kaen University, from December 19 to December 31, 2022. The main experiment was performed at the same location from February 2023 to March 2023. Both experiments were conducted under controlled greenhouse conditions with daily monitoring of environmental parameters.

Plant materials and experimental design

The green oak lettuce seeds used in this study were purchased from Zen Hydroponics, Chiang Mai, Thailand. The plant materials were used in two experiments: first, a preliminary experiment, and second, the main experiment. The details of the plant materials in each experiment are as follows.

For the preliminary experiment (seedling stage), green oak lettuce (Lactuca sativa var. crispa L.) seeds were used with three treatments, each replicated three times, using fifteen seeds per replicate (total n = 135 seeds). To avoid pseudo-replication, the germination percentage and seedling growth parameters from each replicate were calculated as means of the fifteen seeds, and these replicate means (n = 3 per treatment) were used for statistical analysis.

The main experiment (cultivation phase) utilized green oak lettuce with twelve different nutrient solution treatments, each carried out in three replicates with five plants per replicate (total n = 180 plants across 36 experimental units). Each replicate consisted of five plants sharing the same recirculating nutrient solution in an NFT channel. To avoid pseudo-replication, growth parameters were measured for all five plants, and the mean of these five measurements was calculated for each replicate. These replicate means (n = 3 per treatment) served as the experimental units for all statistical analyses.

Bacterial strain and media preparation

The nitrogen-fixing bacterial strain BSN6 was initially isolated from bulk soil and selected based on its ability to grow on nitrogen-free Burk’s medium and fix atmospheric nitrogen. The strain was provided by Dr. Chonticha Mamimin from the Department of Biotechnology, Khon Kaen University. Molecular identification was performed by 16S rRNA gene sequencing; genomic DNA was extracted and the 16S rRNA gene was amplified using universal primers 27F and 1525R, generating a near-complete amplicon (~ 1500 bp). Sanger sequencing was conducted by ATGC Co., Ltd. (Thailand). The resulting sequences (27F: 1,139 bp, avg. Phred Q42.5; 1525R: 1066 bp, avg. Phred Q38.3) were quality-trimmed and aligned against the NCBI nucleotide database (blastn; accessed January 2023), identifying the isolate as putative Klebsiella sp. (strain BSN6) with 98% sequence identity to the top reference sequence. The partial 16S rRNA gene sequences of strain BSN6 have been deposited in NCBI GenBank under accession numbers PZ158854 (27F, 1,139 bp) and PZ158855 (1525R, 1,066 bp). Burk’s medium M707 (Himedia, India) was composed of (in g/L): magnesium sulfate (0.2), dipotassium hydrogen phosphate (0.8), potassium dihydrogen phosphate (0.2), calcium sulfate (0.13), iron (III) chloride (0.00145), sodium molybdate (0.000253), and sucrose (20). Phosphate Buffer Saline (PBS) solution contained (in g/L): sodium chloride (8.5), disodium hydrogen phosphate (1.91), and potassium dihydrogen phosphate (0.38).

As an uncharacterized environmental isolate of unknown taxonomic identity at the time of experimentation, strain BSN6 was handled under precautionary containment practices equivalent to Risk Group 2 (RG2) throughout all experimental procedures, in accordance with institutional biosafety regulations. This is consistent with standard microbiological practice for working with unidentified bacterial isolates. Subsequent molecular identification classified the isolate as putative Klebsiella sp. (see above), a genus classified as RG2 by the Department of Agriculture, Thailand, which confirmed that the precautionary containment level applied was appropriate. No virulence-associated characteristics were observed during the study, and all work was performed under enclosed greenhouse and laboratory conditions with no environmental release. Formal species-level identification and independent biosafety assessment are required prior to any application in open-field or commercial food production settings.

It is important to note that the Klebsiella pneumoniae species complex contains several distinct species, including K. variicola and K. quasipneumoniae, that are frequently misidentified as K. pneumoniae when relying on conventional diagnostics or 16S rRNA sequencing alone^16,17. K. variicola was first described as a novel nitrogen-fixing species isolated from plants such as banana, rice, and sugarcane¹⁸, and has since been extensively characterized as a plant growth-promoting bacterium with nitrogen fixation, indole acetic acid production, and phosphorus solubilization capabilities^19,20. Genomic analysis of the well-characterized endophytic strain Kp342 (now reclassified as K. variicola) demonstrated attenuated pathogenicity in mouse models despite the presence of predicted virulence genes²¹. A recent comprehensive safety review concluded that judicious application of K. variicola strains optimized as biofertilizers does not present undue safety concerns to healthy humans or the environment when proper risk assessment frameworks are applied²². Given that 16S rRNA sequence similarity alone cannot resolve species-level identity within this complex¹⁷, multilocus sequence analysis (MLSA) or whole-genome sequencing is recommended in future work to determine whether strain BSN6 belongs to a plant-associated or clinically relevant lineage.

Nutrient solutions

AB Mix fertilizer was purchased from Agro outlet, Khon Kaen University. Methanogenic effluent produced from the biogas digester located at Khon Kaen University Science Park, produced from molasses in semi-continuous operating mode using anaerobic sludge as methane producer, with operating conditions of 30–35 °C and pH 7.5. The chemical compositions of methanogenic effluent and AB mix fertilizer are shown in Tables S1 and S2.

Bacterial culture preparation

Pure colonies of the NFB strain BSN6 were inoculated into Burk’s medium and incubated at 30 °C for 24 h. Bacteria were grown in 250 ml Erlenmeyer flasks containing 90 ml of Burk’s medium and placed in an incubator shaker at 30 °C and 150 rpm for 24 h. The culture was grown to a final density of 10¹⁴ CFU/mL, as determined by the spread plate technique. Bacterial cells were harvested by centrifugation at 5,000 × g for 10 min and resuspended in sterile PBS solution to serve as the stock culture.

For inoculum preparation, the stock culture (10¹⁴ CFU/mL) was diluted 100-fold with sterile medium to obtain a working suspension of 10¹² CFU/mL. For the preliminary experiment, 250 µL of working suspension was added to each 2.5 L culture, resulting in a final concentration of 10⁸ CFU/mL. For the main experiment, 25 µL of working suspension was added to each 25 L nutrient solution tank, resulting in a final concentration of 10⁶ CFU/mL.

Preliminary experiment: seedling stage

Seeds were sown in wetted germination sponge cubes (2 cm × 2 cm × 2 cm) placed in plastic trays (25 cm × 39 cm × 3 cm) soaked with 2.5 L of the tested solution (see “Plant materials and experimental design” section for sample sizes and replication details). Three treatments were applied:

1. W (AB): Water (days 0–7) followed by AB mix (days 8–14)

2. The NFB (AB): Water containing the NFB (days 0–7) followed by AB mix containing the NFB strain BSN 6 (days 8–14)

3. The NFB (W): Water containing the NFB strain BSN6 throughout the 14 days

Trays were initially covered for optimal germination (3 days), then covers were removed daily between 6:00 and 10:00 am for 4 consecutive days before greenhouse transfer for 7 days of cultivation.

AB Mix solution was prepared by adding 5 mL of solution A to 1 L of tap water, mixing thoroughly, then after 4–6 h adding 5 mL of solution B (application rate: 5 mL/L for each component). The resulting AB Mix solution had an electrical conductivity (EC) of approximately 1.8 mS/cm.

Main experiment: cultivation phase

The experiment followed a completely randomized design with twelve nutrient solution treatments. Each treatment had three independent replicates, with five plants per replicate (total 180 plants across 36 experimental units). Each replicate consisted of five plants growing in the same NFT channel and sharing the same recirculating nutrient solution. Plants within each replicate were treated as subsamples, and the mean of five plants per replicate was calculated for statistical analysis. These replicate means (n = 3 per treatment) served as the experimental units for all statistical tests.

Methanogenic effluent was filtered through a 2 mm sieve to remove solid particles, homogenized, and stored at − 20 °C. The AB Mix solution was prepared as described in Section “Preliminary experiment: seedling stage”. Seeds were sown in sponge cubes (2 cm × 2 cm × 2 cm) in plastic trays (25 cm × 39 cm × 2.5 cm), which were sub-irrigated with 2.5 L of water for days 0–7, then with AB Mix nutrients for days 7–14.

Seedlings with 3–4 true leaves were transplanted into a Nutrient Film Technique (NFT) system with 2 cm nutrient depth. Each replicate used PVC pipes (NFT channels) linked to an independent 50-L tank filled with 25 L of the tested nutrient solution.

Twelve nutrient solution treatments were tested (Table S3):

• Negative control: tap water (TW)

• Positive control: AB Mix (AB)

• Methanogenic effluent at 2.5, 5, 10, 15, and 20% concentrations, each with and without the NFB strain BSN6

Nutrient solutions were changed weekly, and pH was maintained at 5.8–6.0 by adding 0.1 N NaOH or 0.1 M HCl as needed^23,24. Treatments were randomly assigned to experimental units following completely randomized design principles. Plants were harvested 42 days after transplanting.

Environmental monitoring

Environmental monitoring was performed using HTC-1 digital thermo-hygrometer for daily temperature and humidity recording. Greenhouse climate was controlled using a high-pressure fogging system. Electric water pumps were installed for nutrient solution circulation in NFT systems. Daily physicochemical monitoring of nutrient solutions (temperature, pH, and electrical conductivity) was performed using COM-300 pH Meter EC TDS Brand HM Digital.

Chemical analyses

Plant tissue analysis

Plant samples were dried using a hot air oven at 80 °C for 48 h for moisture content determination²⁵. Ash content was determined using a muffle furnace at 550 °C for 15 h²⁶. Moisture content was calculated as [1-(weight of dried sample/weight of sample)] × 100%. Ash content was calculated as [(weight of ash sample−weight of aluminum foil)/(weight of sample)] × 100.

Carbohydrate content was estimated using a standard curve with glucose as standard²⁷. Protein content was evaluated using the technique by Lowry et al.²⁸ with bovine serum albumin as standard. Total lipids were analyzed using the sulfophosphovanillin method with canola oil as standard²⁹.

Nutrient solution analysis

Water samples were filtered through 0.45 μm nylon membrane filters before analysis. Ammonia nitrogen concentration was determined using the phenate technique³⁰. Nitrate nitrogen concentration was determined using UV spectroscopy (i3 UV/VIS Spectrophotometer, Hannan Instruments)³¹. Inorganic nitrogen concentration was calculated as NH₄⁺ − N + NO₃⁻ − N³⁰. Chemical oxygen demand (COD) was determined using close reflux analysis method by heating samples at 150 °C ± 2 °C for 2 h in a heating block³². COD was calculated using the following formula: COD (mg/L) = [(A-B) × N × 8000]/volume of sample (mL), where A is the volume of titrant (ferrous ammonium sulfate) used for the blank (mL), B is the volume of titrant used for the sample (mL), and N is the normality of the titrant.

Heavy metal analysis

Heavy metal analysis for copper (Cu) and chromium (Cr) was performed using Flame Atomic Absorption Spectrophotometry (FAAS) at the Department of Geotechnology, Faculty of Technology, Khon Kaen University. Plant leaf tissue samples were acid-digested with nitric acid and perchloric acid (3:1 v/v) at 150 °C then 220 °C, and filtered through 0.45 μm pore size³³.

Microbial population

Nitrogen-fixing bacterial population was enumerated using the drop plate technique on Burk’s medium with incubation at 30 °C for 24 h³⁴.

Growth parameter measurements

Growth parameters measured included plant height (cm), number of leaves (sheets), leaf length (cm), crown/canopy cover width (cm) on days 7, 14, 21, 28, and 35 after transplantation, plus root length (cm), fresh weight (g/plant), and shoot–root ratio at harvest.

Germination percentage was calculated as (Ni/S) × 100% and germination rate as ΣNi/Di, where Ni = number of seeds germinated, S = total seeds planted, and Di = days from initial sowing³⁵. Seedling measurements were taken on day 14³⁶.

Statistical analysis

All analysis results are presented as mean values with standard deviation. Statistical analysis was performed using Analysis of Variance (ANOVA) and Duncan’s multiple range test at significance level of p < 0.05. SPSS version 28 software was used for all statistical analyses. The observed values were compared to determine differences in plant growth and other parameters between treatments.

Results

Effects of the NFB application on germination and seedling growth

The NFB application during the seedling stage did not result in statistically significant differences in germination percentage and rate, seedling length, or number of leaves (Table 1). However, the W (AB) treatment, which involved water application from days 0–7 followed by AB mix application from days 8–14, demonstrated the highest values for nearly all measured parameters.

Table 1.

Effects of the NFB application on seedling stages solution.

Seedling stage solution	Germination percentage (%)	Germination rate (seeds/day)	Seedling length (cm)	Seedling root length (cm)	Seedling leaves (sheets)
W (AB)	77.33 ± 14.01	10.90 ± 1.85	0.99 ± 0.07	4.47 ± 1.03	3.00 ± 0.00
NFB (W)	64.33 ± 7.51	9.35 ± 1.07	0.73 ± 0.12	2.00 ± 1.06	1.00 ± 0.00
NFB (AB)	62.00 ± 3.46	9.00 ± 0.50	0.92 ± 0.11	2.00 ± 0.59	2.00 ± 0.00

Data are presented as means of three replicates ± standard error (n = 3). Different letters within each parameter indicate significant differences among treatments (Duncan’s multiple range test, p < 0.05). W (AB), water followed by AB Mix; the NFB (AB), water containing the NFB strain BSN6 followed by AB Mix; the NFB (W), water containing the NFB strain BSN6.

The germination percentage ranged from 62.00 ± 3.46% to 77.33 ± 14.01%, with germination rates between 9.00 ± 0.50 and 10.90 ± 1.85 seeds/day. Seedling root length and seedling leaves showed statistically significant differences among treatments. The W(AB) treatment produced significantly longer seedling roots (4.47 ± 1.03 cm) compared to both the NFB(W) and the NFB(AB) treatments (2.00 ± 1.06 cm and 2.00 ± 0.59 cm, respectively). Similarly, seedling leaf number was highest in W(AB) (3.00 ± 0.00), intermediate in the NFB(AB) (2.00 ± 0.00), and lowest in the NFB(W) (1.00 ± 0.00).

The ineffectiveness of the NFB inoculation during the seedling stage can be attributed to the inability of seeds to directly utilize the benefits from non-symbiotic bacteria. As noted by Oliveira et al.³⁷, non-symbiotic bacteria primarily inhabit the rhizosphere, converting atmospheric N₂ into forms usable for plant growth and development. The efficacy of symbiotic interaction between the NFB and plants depends on inoculation method and various abiotic factors, including weather conditions³⁸. The chosen inoculation strategies are crucial for determining microbial establishment and persistence in the rhizosphere, as well as their ability to enhance host plant growth³⁹.

Solution parameters during seedling stage

Electrical conductivity measurements revealed important insights into nutrient availability during early plant development (Fig. 1a). During days 0–7, the W (AB) treatment exhibited the lowest EC compared to the NFB treatments, demonstrating optimal conditions for early seedling development. Essential nutrients, including nitrogen, phosphorus, and potassium exist in organic forms within seeds, and the initial germination stages cannot efficiently absorb nutrients from external sources since root systems remain incompletely developed.

Fig. 1.

Physicochemical properties of nutrient solutions during the seedling stage (days 0–14). (a) Electrical conductivity (EC) measured in weeks 1 and 2 for three treatments: W(AB) (water for days 0–7 followed by AB mix for days 8–14), the NFB(W) (water containing the NFB throughout the 14 days), and the NFB(AB) (water containing the NFB (days 0–7) followed by AB mix with the NFB (days 8–14). (b) pH values showing stability across all treatments throughout the seedling period. (c) Average solution temperature maintained between 24.8 and 25.2 °C. Data represent mean ± standard error (n = 3). W1 = Week 1 (days 0–7); W2 = Week 2 (days 8–14).

After 7 days of germination, stored seed nutrients become depleted, and seedlings begin developing functional roots and leaves⁴⁰. At this stage, external nutrient sources like AB mix become essential for continued growth. Seedlings typically require modest amounts of essential nutrients, particularly nitrogen, phosphorus, and potassium following germination. The W(AB) treatment-maintained EC below 0.5 mS/cm during the first week, remaining within the optimal range for early seedling growth.

pH values remained relatively stable across treatments, ranging from approximately 7.4 to 7.8 with no significant changes between weeks (Fig. 1b). The NFB (W) treatment showed the highest pH of 7.8 among the three solutions. Temperature measurements showed no significant differences across treatments, ranging from 24.8 to 25.2 °C (Fig. 1c). Nutrient solution temperature significantly influences plant growth in hydroponic systems, impacting photosynthesis, respiration, transpiration, root growth, flowering, and seed germination^41,42.

Greenhouse environmental conditions

Greenhouse temperature and humidity were carefully monitored throughout both experimental phases (Fig. 2). During the preliminary 14-day seedling period, morning temperatures ranged from 28 to 33 °C with humidity levels of 60–90%, while afternoon temperatures varied between 32 and 38 °C with humidity declining to 30–40% (Fig. 2a,b). The observed fluctuations in temperature and humidity throughout the experimental periods represent the natural variability in greenhouse conditions.

Fig. 2.

Environmental conditions in the greenhouse during both experimental phases. (a) Morning temperature and humidity during the preliminary seedling experiment (December 19–31, 2022), (b) Afternoon temperature and humidity during the preliminary experiment, (c) Weekly average morning temperature and relative humidity over the 5-week main experiment, (d) Weekly average afternoon temperature and relative humidity over the 5-week main experiment. Temperature fluctuated between 21 and 38 °C across the experimental periods, with morning temperatures typically 28–33 °C and afternoon temperatures reaching 34–38 °C. Humidity varied correspondingly between 30 and 90%. High-pressure fogging systems operated every 45–60 min to maintain optimal conditions. Observed fluctuations represent natural greenhouse environmental variability.

Optimization of methanogenic effluent concentration and the NFB combination

The main experiment demonstrated significant effects of methanogenic effluent concentration and the NFB combination on green oak lettuce growth parameters (Table 2). Lettuce cultivated with 2.5% effluent concentration combined with the NFB exhibited growth performance similar to the positive control (AB mix). Plant height ranged from 7.58 ± 6.57 cm (ME15) to 22.30 ± 1.09 cm (AB), with ME2.5 + the NFB achieving 16.66 ± 0.95 cm. Leaf numbers varied from 0.00 ± 0.00 (ME20 + the NFB) to 27.46 ± 1.53 (AB), with ME2.5 + the NFB producing 25.60 ± 0.60 leaves. Fresh weight measurements showed similar patterns, with ME2.5 + the NFB achieving 39.74 ± 3.21 g compared to 52.32 ± 12.40 g for the positive control (Table 3).

Table 2.

Methanogenic effluent and its combination with nitrogen-fixing bacteria effect on green oak lettuce growth in the hydroponic system.

Treatment	Plant height (cm)	Plant leaves (sheets)	Plant leaf length (cm)	Plant leaf width (cm)	Crown diameter (cm)	Root length (cm)
TW	8.32 ± 0.36ef	13.73 ± 0.76df	8.09 ± 0.28ef	4.87 ± 0.33f.	7.68 ± 0.18 h	15.80 ± 0.20a
AB	22.30 ± 1.09a	27.46 ± 1.53a	21.84 ± 1.13a	17.12 ± 1.02a	28.32 ± 0.33a	11.43 ± 0.34bc
ME2.5	14.05 ± 1.28bcd	20.73 ± 1.10bc	13.62 ± 1.27bcd	9.58 ± 1.17 cd	20.33 ± 1.25bcd	12.17 ± 0.13b
ME2.5 + the NFB	16.66 ± 0.95b	25.60 ± 0.60a	16.28 ± 0.05b	13.03 ± 0.71b	23.66 ± 1.00b	7.09 ± 1.14d
ME5	15.95 ± 0.52b	22.20 ± 0.40abc	15.57 ± 0.47b	10.79 ± 0.28bc	20.61 ± 0.36bcd	10.34 ± 0.34c
ME5 + the NFB	16.18 ± 0.39b	23.60 ± 0.60abc	15.87 ± 0.42b	12.24 ± 0.34b	21.80 ± 0.26bc	12.37 ± 1.00b
ME10	12.06 ± 0.57cde	20.00 ± 1.06bcd	11.65 ± 0.49cde	7.68 ± 1.28de	16.39 ± 0.71def	6.00 ± 0.83d
ME10 + the NFB	10.41 ± 1.50def	14.77 ± 1.68def	9.21 ± 1.69def	5.54 ± 0.48ef	13.85 ± 0.62efg	4.07 ± 0.83e
ME15	7.58 ± 6.57f.	11.66 ± 10.12f.	7.26 ± 6.29f.	4.68 ± 4.08f.	10.10 ± 8.77gh	2.36 ± 2.05e
ME15 + the NFB	14.78 ± 0.97bc	19.11 ± 0.68cde	14.78 ± 1.03bc	10.90 ± 0.67bc	17.81 ± 0.53cde	4.38 ± 0.30f.
ME20	10.70 ± 0.62def	13.25 ± 0.66f.	10.47 ± 0.57def	6.11 ± 0.38ef	12.58 ± 0.55 fg	4.23 ± 0.67e
ME20 + the NFB	–	–	–	–	–	–

Plants in the ME20 + the NFB treatment experienced complete mortality within two days of transplantation; no measurements could be obtained at harvest (day 42). Data are presented as means ± standard error (n = 3 independent replicates). Different letters within each parameter indicate significant differences among treatments (Duncan’s multiple range test, p < 0.05). TW, tap water; AB, AB Mix fertilizer; ME, methanogenic effluent; 2.5–20 (effluent percentages); the NFB, nitrogen-fixing bacteria strain BSN6.

Table 3.

Fresh weight, moisture content, and ash content of green oak lettuce in response to the different concentrations of methanogenic effluent and its combination with the NFB.

Treatment	Fresh weight (g)	Moisture content (%)	Ash content (%)
TW	3.77 ± 0.68e	2.86 ± 0.01 h	0.44 ± 0.07 g
AB	52.32 ± 12.40a	35.91 ± 3.25a	5.32 ± 0.19a
ME2.5	23.70 ± 1.94c	17.05 ± 0.31d	3.05 ± 0.10c
ME2.5 + the NFB	39.74 ± 3.21b	33.87 ± 1.06c	3.74 ± 0.15b
ME5	23.70 ± 1.90c	29.80 ± 0.04c	2.52 ± 0.29 cd
ME5 + the NFB	31.92 ± 3.78bc	31.49 ± 1.25bc	3.93 ± 0.06b
ME10	13.67 ± 3.30d	11.00 ± 0.53e	2.16 ± 0.11de
ME10 + the NFB	7.31 ± 3.74de	7.06 ± 0.09 fg	1.76 ± 0.04ef
ME15	7.34 ± 6.44de	6.09 ± 5.37 g	1.29 ± 1.12f.
ME15 + the NFB	26.56 ± 2.63c	10.26 ± 0.15ef	1.20 ± 0.01f.
ME20	8.69 ± 3.47de	1.43 ± 1.29 h	0.35 ± 0.31 g
ME20 + the NFB	0.00 ± 0.00e	0.00 ± 0.00 h	0.00 ± 0.00 g

Data are presented as means ± standard error (n = 3 independent replicates). Different letters within each parameter indicate significant differences among treatments (Duncan’s multiple range test, p < 0.05). TW, tap water; AB, AB Mix fertilizer; ME, methanogenic effluent; 2.5–20 (effluent percentages); the NFB, nitrogen-fixing bacteria strain BSN6.

The effectiveness of nutrient provision at varying concentrations helped to determine optimal levels for lettuce development. Insufficient or excessive nutrient concentrations impeded lettuce growth, consistent with findings by Ntinas et al.⁴³ who demonstrated that 5% methane effluent/biogas digestate with or without pH correction resulted in significantly higher production and quality of floating hydroponic lettuce. Lettuce growth decreased at higher concentrations (10 and 20%), indicating concentration-dependent toxicity effects.

Growth parameter analysis

Detailed analysis of individual growth parameters revealed distinct concentration–response relationships (Fig. 3). The higher concentrations (10–20%) negatively affected all growth metrics, with 20% effluent + the NFB resulting in complete plant mortality within two days of transplantation. Root length measurements showed interesting patterns, with tap water (negative control) producing the longest roots (15.80 ± 0.20 cm). This response represents a common adaptive strategy where plants extend their root systems to enhance nutrient and water uptake under nutrient-deficient conditions^44,45. Plants typically allocate more energy toward root elongation to compensate for nutrient scarcity in the growing medium. Methanogenic effluent at 2.5 and 5% concentrations combined with the NFB enhanced above-ground growth parameters while maintaining reasonable root development.

Fig. 3.

Growth parameters of green oak lettuce in response to different concentrations of methanogenic effluent with and without nitrogen-fixing bacteria (the NFB). (a) plant height (cm), (b) plant leaves (sheets), (c) plant leaf length (cm), (d) plant leaf width (cm), (e) crown diameter (cm), and (f) root length (cm). Treatments include tap water (TW), AB mix (AB), and five effluent concentrations (2.5, 5, 10, 15, and 20%) with and without the NFB. Data represent mean ± standard error (n = 3 independent replicates; each replicate represented the mean of 5 plants). Different letters within each parameter indicate significant differences among treatments (Duncan’s multiple range test, p < 0.05). Measurements taken at harvest (42 days after transplanting).

Plant morphology and biomass characteristics

Representative images of plant morphology across treatments are shown in Fig. 4. Low concentrations of methanogenic effluent combined with the NFB elicited positive responses, while plants grown in 20% effluent with the NFB failed to survive until harvest. The highest values for fresh weight, moisture content, and ash content were observed in combined treatments of 2.5 and 5% methanogenic effluent with the NFB, excluding the positive control (Table 3). These results suggest that at low concentrations, effluent requires the NFB assistance to regulate nitrogen levels effectively. Higher effluent concentrations (10–20%), both with and without the NFB, exhibited significantly elevated EC values compared to lower concentrations. Electrical conductivity measurements assess nutrient availability within solutions, where low EC can result in nutrient deficiency while elevated EC may hinder nutrient absorption^46,47. Proper, balanced nutrition contributes to substantial biomass development⁴⁸.

Fig. 4.

Visual comparison of representative green oak lettuce growth from each treatment at harvest showing morphological differences among treatments. Each image shows a representative plant from n = 15 plants per treatment (3 replicates of 5 plants each). Top panel: Lettuce grown without the NFB showing (left to right) AB Mix (positive control), tap water (negative control), and methanogenic effluent at 2.5, 5, 10, 15, and 20% concentrations. Bottom panel: Lettuce grown with the NFB addition showing AB Mix, tap water, and effluent at 2.5, 5, 10, and 15% concentrations with the NFB. Note: The 20% + the NFB treatment resulted in complete plant mortality and is not shown. Photographs taken 42 days after transplanting.

Moisture content, which correlates with leaf water content, serves as a key physiological indicator influencing photosynthetic efficiency and biomass yield. Lower moisture content induces reduced relative water content, providing accurate measures of plant water status⁴⁹. Ash content, used to estimate overall mineral content, showed considerable variation, with water intake significantly impacting ash content through potential reduction under water stress conditions⁵⁰.

Nitrogen dynamics in nutrient solution

Nitrogen dynamics in the nutrient solution showed weekly variations in ammonium nitrogen (NH₄⁺ − N) levels across treatments. The highest NH₄⁺ − N concentration (2.58 mg/L) was observed in the AB Mix treatment, while the lowest NH₄⁺ − N (0.25 mg/L) occurred in the negative control during the first cultivation week (Fig. 5a,b).

Fig. 5.

Nitrogen dynamics in nutrient solutions during the 5-week cultivation period. (a) Ammonium nitrogen (NH₄⁺-N) concentrations in treatments without the NFB. (b) Ammonium nitrogen concentrations in treatments with the NFB. (c) Nitrate nitrogen (NO₃⁻-N) concentrations in treatments without the NFB, showing peak levels at week 3. (d) Nitrate nitrogen concentrations in treatments with the NFB, showing variable patterns of nitrate dynamics with the NFB addition. Treatments: TW (tap water), AB (AB Mix), 2.5–20 (effluent percentages). Solutions were changed weekly. Data represent mean values (n = 3) measured at the beginning of each week. Weekly measurements showing variability in nitrogen forms throughout the cultivation period.

In general, NH₄-N concentrations fluctuated over time depending on treatment conditions. Notably, an anomalous drop in NH₄-N concentration to nearly zero was transiently observed at week 4 in the 2.5% effluent treatment, followed by a rebound to more than 3 mg/L at week 5 (Fig. 5a). This pattern deviated from the general temporal trend and was not consistently observed across other treatments.

Nitrate nitrogen (NO₃⁻ − N) patterns showed initial low levels across treatments except for higher doses (15 and 20%), with concentrations peaking at week 3, particularly in high-dose treatments with the NFB addition (Fig. 5c,d). After week 3, NO₃⁻ − N level in most treatments declined, with patterns varying among treatments. The inorganic nitrogen (NH₄⁺ − N + NO₃⁻− N) levels increased across treatments, peaking at week 3 with the highest concentration (~ 2600 mg/L) observed in the 20% effluent treatment, then declining in subsequent weeks.

Chemical oxygen demand and electrical conductivity

The 20% effluent treatment, both with and without the NFB, showed the highest values for NH₄⁺ − N, NO₃⁻− N, and COD (Figs. 5 and 6). COD levels increased with higher effluent concentrations, reaching approximately 2220 mg/L initially (Fig. 6a,b). Higher effluent concentrations (10, 15, and 20%) exhibited significantly higher initial COD values compared to lower concentrations (2.5 and 5%), AB solution, and tap water.

Fig. 6.

Chemical oxygen demand (COD) and electrical conductivity (EC) of nutrient solutions during cultivation. (a) COD levels in treatments without the NFB showing concentration-dependent increases, with initial values reaching 2186.88 mg/L in 20% treatment. (b) COD levels in treatments with the NFB demonstrating similar concentration-dependent patterns. (c) Electrical conductivity in treatments without the NFB, ranging from 0.35 to 8.35 mS/cm. (d) Electrical conductivity in treatments with the NFB showing slightly elevated values compared to non-inoculated conditions. Treatments: TW (tap water), AB (AB Mix), 2.5–20 (effluent percentages). Higher EC values at elevated concentrations correlated with reduced plant growth.

Electrical conductivity ranged from 0.35 to 8.35 mS/cm during the experiment, with higher EC negatively impacting lettuce fresh weight, biomass, and morphological characteristics (Fig. 6c,d). Kappel et al.⁴⁸ found that high nutrient concentrations with EC levels 6–10 mS/cm in root zones affect lettuce production by decreasing stomatal conductance and leaf area. EC increased after transplanting, peaked around weeks 2–3, then slightly decreased. The NFB presence resulted in slightly higher overall EC values, indicating improved nutrient availability. Higher inorganic nitrogen values corresponded to higher EC values, especially with the NFB. This is consistent with the NFB enhancing nitrogen availability through nitrogen fixation, with concurrent nitrification of NH₄⁺ to NO₃⁻ by the resident heterotrophic microbial community further contributing to the EC increase. Because strain BSN6 was selected only for diazotrophic activity on nitrogen-free Burk’s medium and was not independently tested for nitrifying capacity, the nitrification observed here is attributed to background microorganisms originating from the methanogenic effluent, tap water, and greenhouse environment, with a possible minor contribution from BSN6 that cannot be excluded. Lower effluent concentrations (2.5 and 5%) were improved by the NFB, which increased nitrogen availability without excessively raising EC.

Bacterial population dynamics

Bacterial populations in nutrient solutions varied throughout the cultivation period. The ME20 + the NFB treatment showed the highest bacterial concentration (4.20 × 10⁴ ± 2.82 CFU/mL) during week 1, while concentrations fluctuated across treatments in subsequent weeks (Table 4).

Table 4.

The NFB accumulation in the nutrient solution during the cultivation period.

Treatments	Week 1 (CFU/mL)	Week 2 (CFU/mL)	Week 3 (CFU/mL)	Week 4 (CFU/mL)	Week 5 (CFU/mL)
TW	1.65 × 101 ± 2.12	3.4 × 102 ± 8.48	0.00 ± 0.00	6.0 × 101 ± 0.00	0.00 ± 0.00
AB	2.0 × 100 ± 4.94	3.9 × 102 ± 4.24	1.95 × 101 ± 3.53	4.75 × 102 ± 3.53	0.00 ± 0.00
ME2.5	1.20 × 104 ± 2.82	1.05 × 102 ± 6.36	1.70 × 101 ± 7.07	2.50 × 103 ± 7.07	1.80 × 103 ± 2.82
ME 2.5 + the NFB	2.25 × 104 ± 3.53	9.50 × 103 ± 4.94	2.15 × 102 ± 4.94	2.10 × 102 ± 2.82	2.65 × 103 ± 2.12
ME5	1.45 × 104 ± 2.12	3.95 × 102 ± 6.36	3.05 × 102 ± 3.53	1.25 × 103 ± 0.70	2.10 × 103 ± 1.41
ME5 + the NFB	1.20 × 104 ± 2.82	4.10 × 102 ± 2.21	3.50 × 102 ± 2.82	2.00 × 102 ± 2.82	2.40 × 103 ± 1.41
ME10	2.95 × 102 ± 7.77	1.85 × 102 ± 2.12	2.25 × 102 ± 2.12	7.00 × 101 ± 0.00	1.20 × 102 ± 2.82
ME10 + the NFB	1.30 × 104 ± 2.82	5.70 × 102 ± 4.24	2.80 × 102 ± 4.24	2.55 × 102 ± 4.94	1.80 × 102 ± 1.41
ME15	3.85 × 104 ± 2.12	3.15 × 102 ± 3.53	1.15 × 103 ± 2.12	3.20 × 102 ± 2.82	2.00 × 102 ± 4.24
ME15 + the NFB	1.20 × 104 v 2.82	3.60 × 103 ± 7.07	2.10 × 103 ± 8.48	1.80 × 103 ± 2.82	1.80 × 102 ± 5.65
ME20	3.90 × 104 ± 5.65	1.60 × 102 ± 2.82	2.65 × 103 ± 4.94	2.15 × 102 ± 0.70	2.30 × 102 ± 4.24
ME20 + the NFB	4.20 × 104 ± 2.82	1.85 × 104 ± 2.12	5.10 × 103 ± 8.84	2.50 × 102 ± 2.82	2.60 × 102 ± 1.41

Data are presented as means ± standard error (n = 3 independent replicates). Different letters within each parameter indicate significant differences among treatments (Duncan’s multiple range test, p < 0.05). TW, tap water; AB, AB Mix fertilizer; ME, methanogenic effluent; 2.5–20 (effluent percentages); the NFB, nitrogen-fixing bacteria strain BSN6. Bacterial counts in non-inoculated treatments represent background microbial populations from tap water and environmental sources.

Food safety and nutritional quality

Heavy metal analysis of leaf samples from the optimal 2.5% effluent + the NFB treatment showed no detectable heavy metals (Cu and Cr) in plant leaf tissue, despite their presence in 100% effluent concentration. Heavy metal analysis showed no detectable copper or chromium in lettuce tissue from the optimal 2.5% effluent + the NFB treatment. Proximate composition analysis revealed carbohydrate content of 37.01 ± 0.28 mg/100 g, protein content of 17.14 ± 0.16 mg/100 g, and lipid content of 5.33 ± 0.02 mg/100 g, (Table S4) indicating acceptable nutritional quality of effluent-grown lettuce^51–53.

Lipid content reached 5.33 ± 0.02 mg/100 g (Table S4), consisting of various compounds including free fatty acids, glycerols, phospholipids, glycolipids, sterols, waxes, carotenes, terpenes, hydrocarbons, tocols, and chlorophyll pigments⁵⁴. Protein content measured 17.14 ± 0.16 mg/100 g (Table S4), representing essential macromolecules crucial for cellular structure and biological function⁵⁵.

Discussion

Optimal concentration of methanogenic effluent

This study demonstrated that methanogenic effluent at low concentrations (2.5 and 5%) combined with the NFB provides an effective, sustainable alternative to conventional chemical fertilizers for hydroponic cultivation. The optimal combination of 2.5% effluent with the NFB achieved growth performance comparable to commercial fertilizers while maintaining food safety standards. This approach offers significant environmental and economic benefits by converting biogas industry waste into valuable agricultural inputs while reducing dependence on synthetic fertilizers.

At moderate effluent concentrations (2.5 and 5%), the NFB addition was beneficial for plant height, leaf count, leaf length, leaf width, and crown diameter. The 2.5% effluent + theNFB treatment consistently outperformed other concentrations in most measured parameters, suggesting this combination provides optimal nutrient availability and bacterial activity without the toxicity effects observed at higher concentrations.

The concentration-dependent response observed in this study is consistent with previous research on biogas digestate application. The effectiveness of 2.5 and 5% concentrations aligns with findings by Ntinas et al.⁴³, who reported that 5% methane effluent/biogas digestate produced significantly higher lettuce yields. However, our study extends these findings by systematically evaluating the synergistic effects of the NFB addition at different effluent concentrations.

Visual assessment of plant morphology (Fig. 4, showing representative plants) confirmed quantitative measurements (Table 2, Fig. 3), showing superior growth at lower effluent concentrations (2.5 and 5%) while higher concentrations (10–20%) resulted in stunted and inadequate growth.

Role of nitrogen-fixing bacteria

Bacterial population dynamics revealed complex interactions with effluent concentration. Different concentrations of the BSN6 strain may have influenced nitrogen fixation rates, nitrogenase induction periods, and overall nitrogen fixation efficiencies⁵⁶. Generally, effluent treatments maintained higher bacterial concentrations compared to controls, with the NFB-supplemented treatments showing more stable populations over time. This stability may contribute to the sustained nitrogen availability observed in the NFB treatments.

The beneficial interaction between low-concentration effluent and the NFB was demonstrated. The NFB enhanced nitrogen availability while maintaining acceptable electrical conductivity levels. At low effluent concentrations (2.5 and 5%), the NFB supplementation improved plant growth parameters, including height, leaf number, fresh weight, and biomass accumulation. The beneficial effects may be attributed to multiple mechanisms: (1) conversion of atmospheric nitrogen into plant-available forms, (2) improved nutrient solubilization, and (3) potential production of plant growth-promoting substances.

However, the NFB proved ineffective or even detrimental at higher effluent concentrations (15–20%). The complete mortality observed in the 20% + the NFB treatment suggests that the combination of high nutrient concentrations and bacterial activity created unfavorable conditions, likely through excessive EC elevation, osmotic stress, or production of toxic metabolites. This finding emphasizes the critical importance of proper dilution protocols when combining biological amendments with nutrient-rich effluents.

Nitrogen dynamics and plant response

The observed NH₄⁺− N dynamics suggest complex interactions between plant uptake and microbial processes. These results indicate that plant uptake and microbial nitrification by the resident (non-inoculated) community collectively shape the observed NH₄⁺ − N and NO₃⁻ − N dynamics, with nitrification depending on NH₄⁺ − N availability that decreases concurrently with plant uptake¹¹. It is important to stress that strain BSN6 was isolated and characterized solely based on its ability to grow on nitrogen-free Burk’s medium and fix atmospheric nitrogen; it was not tested for ammonia- or nitrite-oxidizing activity in the present study. The nitrification observed here is therefore most plausibly driven by ammonia- and nitrite-oxidizing bacteria introduced with the methanogenic effluent, tap water, and greenhouse air rather than by BSN6 itself. A minor contribution from BSN6 cannot be formally excluded, as some Klebsiella spp. have been reported to perform heterotrophic nitrification; direct testing of the nitrifying capacity of BSN6 is identified as an important direction for future work.

Notably, an abrupt and transient decline in NH₄⁺-N concentration to near zero was observed at week 4 in the 2.5% effluent treatment, followed by recovery at week 5. This anomalous pattern deviates from the general temporal trend and therefore warrants specific consideration. The week-4 value is the mean of n = 3 independent biological replicates; we therefore report it as observed rather than exclude it as an outlier. Several non-mutually exclusive mechanisms may explain this observation. First, a transient increase in plant nitrogen uptake under low-N conditions may have contributed to the temporal depletion of NH₄⁺–N; this mechanism is consistent with the concentration-specificity of the anomaly (confined to ME2.5, where plants experience genuine N limitation) and with the week-5 rebound, which coincided with weekly renewal of the nutrient solution⁵⁷. Second, rapid microbial nitrification or microbial assimilation could also have temporarily depleted the NH₄⁺–N concentration under conditions of low N input and active microbial communities. Third, although variation in effluent NH₄⁺–N or inadvertent dilution during weekly solution renewal cannot be formally excluded, this is unlikely to be the dominant cause, because the renewal protocol was identical across all treatments, yet the week-4 minimum was confined to the 2.5% treatment.

It is important to stress that strain BSN6 was isolated and characterized solely based on its ability to grow on nitrogen-free Burk’s medium and fix atmospheric nitrogen; it was not tested for ammonia- or nitrite-oxidizing activity in the present study. The nitrification observed here is therefore most plausibly driven by ammonia- and nitrite-oxidizing bacteria introduced with the methanogenic effluent, tap water, and greenhouse air rather than by BSN6 itself. A minor contribution from BSN6 cannot be formally excluded because some Klebsiella spp. have been reported to carry out heterotrophic nitrification; direct testing of the nitrifying capacity of BSN6 is identified as an important direction for future work.

The temporal patterns of nutrient availability revealed complex interactions between plant uptake, microbial activity, and nutrient transformations. NO₃⁻ − N levels peaked at week 3 across most effluent treatments (Fig. 5c,d), consistent with nitrification of the accumulated NH₄⁺ − N pool by the resident heterotrophic community. After week 3, NO₃⁻ − N trajectories were strongly modulated by both effluent concentration and the presence of the NFB. Contrary to a simple expectation that NFB inoculation would prolong nitrogen availability, Fig. 5c,d shows that at the higher effluent concentrations (ME15 and ME20) the NFB-inoculated treatments exhibited a faster decline in NO₃⁻ − N between weeks 3 and 5 than the corresponding non-inoculated treatments. A plausible explanation is enhanced microbial assimilation, and possibly denitrification, within an expanded microbial biomass under N-saturated conditions, coinciding with severe plant stress and, in the case of ME20 + the NFB, complete plant mortality, which eliminated further plant uptake. At the lower, more physiologically favourable concentrations (ME2.5 and ME5), NO₃⁻ − N trajectories were variable, and the NFB-inoculated treatments did not consistently retain more NO₃⁻ − N than the corresponding non-inoculated controls. Taken together, these observations indicate that the contribution of the NFB to the nitrate pool of the nutrient solution is concentration-dependent and is shaped by the combined effect of plant uptake, microbial assimilation/denitrification, and solution chemistry, rather than by a uniformly sustained release of fixed N throughout the cultivation period.

However, the relationship between nitrogen availability and plant performance was not linear. High nitrogen levels (NH₄⁺ − N and NO₃⁻ −N) at elevated effluent concentrations (10–20%) correlated with significant reductions in lettuce fresh weight, indicating nitrogen toxicity rather than beneficial effects. This contrasts with optimal nitrogen concentrations reported by¹⁷, who found that N concentrations of 249.71 mg/L enhanced lettuce growth and weight. Our results at 20% effluent concentration (~ 2600 mg/L total inorganic nitrogen at peak) exceeded these optimal levels by more than tenfold, showing that excessive nitrogen availability becomes detrimental to plant growth. This emphasizes the critical importance of maintaining appropriate nutrient concentrations in hydroponic systems, particularly when using nutrient-rich substrates like methanogenic effluent.

Beyond nitrogen dynamics, electrical conductivity (EC) served as a key indicator of overall nutrient concentration and plant stress. EC is influenced by dissolved salts and nutrients, including nitrogen; as nitrogen (NH₄⁺ − N and NO₃⁻− N) is added or produced by the NFB, EC increases due to these nutrients contributing to ion concentration. Biogas digestate contains high concentrations of dissolved ions such as NH₄⁺, NO₃⁻, H₂PO₄⁻, and K⁺, contributing to EC rise¹⁰. Additionally, nitrification processes convert NH₄⁺ into NO₃⁻, further increasing dissolved ion numbers and leading to higher EC levels. Across the five effluent concentrations tested (Fig. 6c,d), EC scaled with effluent percentage and was slightly higher in the NFB-inoculated treatments than in the non-inoculated ones at the same percentage, consistent with the contribution of NFB-mediated fixed N and associated microbial activity to the ion pool. At low effluent concentrations (2.5 and 5%), EC remained below the threshold, commonly considered safe for lettuce and was associated with good plant growth (Fig. 3). At higher concentrations (10–20%), EC approached or exceeded the 6–10 mS/cm range reported by Kappel et al.⁴⁸ as detrimental to lettuce, and this coincided with the strong reduction in fresh weight, leaf number, and crown diameter observed in Figs. 3 and 4, culminating in complete plant mortality in ME20 + the NFB. We emphasize, however, that our system is observational: high residual nitrogen and high EC at elevated effluent concentrations may act both as a cause of impaired plant performance (through osmotic stress and ionic toxicity) and as a consequence of it, because stressed or senescing plants take up less N, so more remains in solution and is further converted to NO₃⁻ by nitrifiers, reinforcing the EC increase. Our design, which did not include unplanted parallel controls at every concentration, cannot fully disentangle these two contributions, and we therefore describe the NFB–EC–biomass relationship as a concentration-dependent correlation rather than a unidirectional causal link.

Seedling stage application

Visual observations on the final measurement day revealed distinct variations in seedling appearance across treatments (Fig. S2). Seedlings treated with AB mix showed visible differences compared to those without it. The W (AB) treatment demonstrated superior growth characteristics, while the NFB (AB) treatment ranked as the second most effective. The application of the NFB combined with AB mix from days 8–14 provided adequate nutrients from both bacterial sources and AB mix. Bacterial growth and activity require essential nutrients including water, a carbonaceous substrate, a nitrogenous substrate, and various inorganic compounds⁵⁸.

However, when comparing treatments with and without the NFB at the germination stage, the preliminary experiment revealed that the NFB application during the germination stage provided limited benefits, with only seedling root length showing significant differences. This finding can be attributed to the biological characteristics of seed germination and early seedling development. Seeds contain sufficient nutrient reserves in cotyledons to support initial growth, and the undeveloped root system cannot efficiently interact with rhizosphere bacteria during the earliest growth stages.

As noted by Oliveira et al.³⁷, non-symbiotic nitrogen-fixing bacteria primarily function in the rhizosphere after root system establishment. The inoculation method and timing are crucial for determining bacterial effectiveness^38,39. Our results suggest that the NFB application is most beneficial after transplanting when root systems are sufficiently developed to support bacterial colonization and nutrient exchange. This is consistent with the enhanced the NFB effectiveness when combined with AB mix from days 8–14, as the nutrient-rich environment supported both plant development and bacterial activity.

Furthermore, the provision of fertilizer during the earliest stages of seedling development, specifically from sowing to the first week following germination, appeared unnecessary due to the presence of cotyledons. These embryonic structures contain sufficient nutrients to support seedling growth⁵⁹. High EC values during initial growth phases indicate excessive fertilization, which can hinder plant development by causing abnormal elongation or deformation⁶⁰. Optimal EC levels vary depending on plant growth stage, with 0.2–0.9 mS/cm considered ideal for most vegetables and leafy greens⁴⁶. These findings support our experimental design of delaying nutrient supplementation until after the initial germination period.

Environmental and economic implications

With over 1700 biogas plants operating in Thailand⁶, the potential for effluent valorization through hydroponic agriculture represents a substantial opportunity for circular economy implementation. The successful utilization of methanogenic effluent at 2.5% concentration could reduce both the environmental burden of effluent discharge and the economic costs associated with synthetic fertilizer purchase.

Environmental control was essential for optimizing plant growth in this study. The main experiment recorded morning temperatures around 28 °C and afternoon temperatures ranging from 34 to 36 °C (Fig. 2). High-pressure fogging systems operated at regular intervals (every 45–60 min for 10 min) to maintain optimal greenhouse microclimate conditions and prevent heat stress that could affect nutrient uptake and plant performance. While this environmental control contributed to successful plant cultivation, it also represents a significant operational cost that must be considered in the economic feasibility of using methanogenic effluent in commercial hydroponic systems.

Biogas plants currently face disposal challenges for methanogenic effluent, as our data showed COD levels exceeding 2000 mg/L in undiluted effluent, which would violate wastewater discharge standards⁶¹. High COD levels indicate the presence of organic compounds, which can decrease dissolved oxygen levels in receiving water bodies⁶². Thai wastewater discharge regulations require COD concentrations below 80 mg/L for standard A and below 200 mg/L for standard B⁶¹. Our results demonstrate that dilution to 2.5–5% simultaneously reduces COD to manageable levels while providing adequate nutrition for lettuce cultivation comparable to commercial fertilizer, exemplifying principles of industrial ecology and circular economy. While this dilution ratio (20–40 folds) may initially appear economically unfavorable compared to using concentrated fertilizer solutions, several factors suggest potential cost advantages: (1) biogas plants currently bear disposal costs for untreated effluent that violates discharge standards, (2) the dilution process requires minimal infrastructure beyond water input, which is already necessary for hydroponic systems, and (3) the approach converts a waste disposal liability into a nutrient resource. However, evaluating the cost-effectiveness of this approach requires consideration beyond just nutrient provision. Comprehensive economic analysis comparing dilution and processing costs, disposal cost savings, and commercial fertilizer expenses would be necessary to fully assess the practical viability of this approach at different scales. Future research should include detailed cost–benefit analyses and life cycle assessments to determine under what conditions effluent-based hydroponic systems provide economic advantages over conventional fertilizer use.

Food safety considerations

Heavy metal analysis for copper and chromium showed no detectable levels in lettuce tissue from the optimal 2.5% effluent + the NFB treatment. Even though this is encouraging, comprehensive food safety assessment would require analysis of additional priority heavy metals (lead, cadmium, arsenic, mercury, nickel) and potential microbial contaminants before definitive food safety conclusions can be drawn for commercial application.

Beyond heavy metal safety, the nutritional composition of lettuce grown with methanogenic effluent was comparable to conventionally grown lettuce. Protein analysis is important for nutritional labeling, as well as for characterizing the biological activities and functional aspects of foods⁶³. Although lettuce has relatively low protein content compared to other vegetables, the protein content measured in this study (17.14 mg/100 g) indicates that lettuce leaves grown with diluted effluent maintain suitable nutritional quality for consumption⁶⁴. Carbohydrate and lipid contents were also within acceptable ranges for lettuce, indicating that effluent-based cultivation maintains product quality comparable to conventional production systems without compromising nutritional value.

It should be noted that lipid content and metabolism in plant leaves can be influenced by multiple factors including variety, light quality, nutritional status, drought stress, and cold stress⁶⁵. Despite these potential variables, our findings demonstrate that methanogenic effluent-grown lettuce maintains acceptable nutritional quality. Combined with the absence of detectable heavy metals, these results indicate that lettuce cultivated with diluted methanogenic effluent is safe for consumption under the conditions tested.

Limitations and future directions

Several limitations of this study should be acknowledged. First, only one lettuce variety was tested under specific greenhouse conditions, limiting the generalizability of findings to other crops and environments. Second, the 42-day cultivation period represented a single growth cycle, and long-term effects of repeated effluent application remain unknown. Third, the molecular identification of strain BSN6 remains preliminary, identifying the isolate as putative Klebsiella sp. based on 98% 16S rRNA sequence similarity. As noted above, species within the K. pneumoniae complex share > 97% 16S rRNA similarity and cannot be reliably differentiated without genomic approaches such as MLSA or whole-genome sequencing^16,17. While recent literature supports the agricultural safety of plant-associated Klebsiella lineages^19,22, this genus is classified as Risk Group 2, and formal biosafety assessment and regulatory clearance are required before any application in commercial food production systems can be recommended. The partial 16S rRNA gene sequences have been deposited in GenBank (see “Materials and methods”); however, species-level confirmation requires further genomic analysis. The present study represents a controlled proof-of-concept investigation only. These limitations should be addressed in future work through completion of species-level identification via MLSA or whole-genome sequencing, and independent biosafety validation before any large-scale or food production application is considered. Fourth, food safety assessment was limited to two heavy metals (copper and chromium). Comprehensive safety evaluation for human consumption should include additional priority heavy metals (lead, cadmium, arsenic, mercury, and nickel) and microbial quality testing to comply with food safety standards. Fifth, volatile fatty acid (VFA) concentration in the methanogenic effluent was not measured in this study. Future studies should include VFA analysis as it is an important parameter for understanding organic acid content and its effects on plant nutrition. Sixth, our design is observational: in the planted system used here, residual dissolved nitrogen and EC in the nutrient solution at a given time reflect the net balance of plant uptake, microbial nitrogen transformations, and ion release from the effluent, and the design did not include unplanted parallel controls at every effluent concentration. Consequently, the observed positive association between high effluent concentration, high EC, and reduced plant biomass should be interpreted as a correlation that is consistent with—but does not prove—a unidirectional causal effect of high EC and high N on plant performance. Seventh, strain BSN6 was selected only for diazotrophic activity on nitrogen-free Burk’s medium and was not tested for ammonia- or nitrite-oxidizing activity. Future work should therefore include unplanted controls replicated at each concentration and direct screening of BSN6 for nitrifying activity, to separate the contributions of plant uptake, strain BSN6, and the background microbial community to the nitrogen and EC dynamics of effluent-based hydroponic systems.

Future research should focus on: (1) extending these findings to other crop species and varieties, (2) investigating long-term effects of effluent-based cultivation on soilless media and system components, (3) developing automated systems for optimal concentration management based on real-time monitoring, (4) conducting economic analysis comparing production costs with conventional systems, and (5) performing life cycle assessments to quantify environmental benefits comprehensively.

Conclusion

This study successfully demonstrated that 2.5% methanogenic effluent combined with nitrogen-fixing bacteria represents a sustainable alternative to conventional hydroponic fertilizers for green oak lettuce cultivation. The optimal treatment achieved plant performance comparable to commercial AB Mix, with 16.66 ± 0.95 cm plant height, 25.60 ± 0.60 leaves per plant, and 39.74 ± 3.21 g fresh weight. Higher effluent concentrations (15–20%) proved toxic, with 20% + the NFB causing complete plant mortality within two days of transplantation, emphasizing the critical importance of proper dilution. The synergistic relationship between low-concentration effluent and the NFB was clearly established, with the NFB enhancing nitrogen availability while maintaining acceptable electrical conductivity levels. Heavy metal analysis showed no detectable copper or chromium in lettuce tissue, though comprehensive safety assessment including additional priority contaminants and microbial testing is required before commercial application. This approach can offer significant environmental and economic benefits by converting biogas industry waste into valuable agricultural inputs while reducing synthetic fertilizer dependence. With over 1700 biogas plants operating in Thailand, the potential for effluent valorization through hydroponic agriculture represents a substantial opportunity for circular economy implementation. The findings provide practical guidelines for implementing integrated biogas-agriculture systems that are environmentally sound, economically viable, and scalable. Future research should extend these findings to other crop species, conduct long-term economic analysis to facilitate commercial adoption, and develop automated concentration management systems. The integration of effluent management with hydroponic agriculture represents a promising approach for sustainable intensification of food production systems while addressing waste management challenges in the renewable energy sector.

Author contributions

Oktaviani, R: Investigation, Methodology, Formal analysis, Writing-original draft. Sukitprapanon, T: Supervision, Conceptualization, Methodology, Funding acquisition, Visualization, Writing—review and editing. Plangklang, P: Data curation, Writing—review and editing. Sittijunda, S: Visualization, Data curation, Writing-review and editing. Reungsang, A: Conceptualization, Methodology, Resources, Visualization, Writing—review and editing, Resources, Funding acquisition, Project administration.

Funding

This research was supported by the National Research Council of Thailand (NRCT) (Grant Number: N42A670487), the Fundamental Fund of Khon Kaen University (Grant Number: 203181), and the KKU Scholarship for Association of Southeast Asian Nations and Greater Mekong Sub-region countries’ personnel.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.