Hydroponic Lettuce Cultivation with Organic Liquid Fertilizer: Examining Bacterial Inhibition and Phosphate Solubilization

Abstract

This study explores organic hydroponic cultivation as a sustainable alternative to chemical fertilizers amid global supply challenges. With rising costs and the depletion of conventional nitrogen sources, organic liquid fertilizers are gaining attention for their microbial enrichment processes (MEP) that convert organic matter into plant-accessible nutrients. This experiment focuses on lettuce cultivation using two organic liquid fertilizers, Power Fish and POF vol. 2, in controlled environments. The results show that there are significant differences in plant growth parameters such as leaf number, fresh weight, chlorophyll content, and root length across different fertilizer treatments. Key findings include that effective management practices, including pH control and regular nutrient application, are crucial for maintaining microbial activity and optimizing nutrient uptake efficiency. Additionally, Bacillus sp. and Xanthomonas sp. were isolated from these fertilizers and dual-cultured with rot fungi, Pythium sp. and Fusarium sp. This dual cultivation demonstrated inhibitory activity against these pathogens, showcasing the potential of these microorganisms in promoting biofilm-mediated disease resistance in organic hydroponic systems. The study also emphasizes the phosphate solubilization capabilities of isolated bacteria, essential for sustainable nutrient cycling. Overall, organic hydroponic systems present a promising strategy for sustainable agriculture, reducing dependency on chemical inputs while enhancing crop productivity and resilience to environmental stressors.

1. Introduction

There have been significant increases in the cost of chemical fertilizers worldwide due to China’s export restrictions since 2022. Additionally, depletions in natural gas resources in the EU and Asia have led to decreases in ammonia and urea production in the EU. This trend is expected to further drive-up chemical fertilizer costs in the future [1]. To promote sustainable agricultural practices, it is crucial to implement new production systems. For these reasons, organic hydroponic cultivation is gaining considerable interest as a more sustainable alternative to chemical fertilizers to preserve agricultural production [2,3].

Two crucial considerations when utilizing organic liquid fertilizers in hydroponics are the micro enrichment process (MEP) or mineral parallel mineralization (MPM) and fertilizer management. The MEP or MPM relies on microbial activity to convert organic matter into nitrate, which can then be easily absorbed by plants. Plant growth-promoting rhizobacteria (PGPR), a group of microbes, are crucial in this process. Among them, Bacillus sp. stands out for its ability to enhance nitrogen fixation in legumes, solubilize phosphate, increase root surface area, enhance nutrient availability to plants, and provide protection against diseases and insects [4,5].

The second factor is fertilizer management. In organic liquid fertilizer hydroponics, it is crucial to apply fertilizer in amounts that match the crop’s daily absorption capacity, either daily or every few days. Since organic matter acts as the source of fertilizer, changes in inorganic nutrient levels may not have an immediate effect on electrical conductivity (EC). There is a risk of overfertilization, leading to excessive biofilm formation by microorganisms and root acidification, hindering conventional EC management methods. Continuous aeration throughout cultivation is essential to steady aerobic microbial activity [3]. Biofilms can protect plants from airborne diseases common in chemical fertilizer-based hydroponic cultivation [6,7].

Additionally, phosphate fertilizer is a critical element for crop cultivation. However, most phosphate used in fertilizers comes from non-renewable phosphate rocks. Studies show that 90% of the available reserves of phosphate rocks are utilized for fertilizer production and feed [8,9]. It is expected that about half of the world’s current economically viable phosphate resources will be depleted by the end of this century [10,11]. To address this, there is a growing recommendation to shift towards a more circular approach to nutrient use by retrieving phosphorus from local resources [12]. Therefore, there is a need to explore alternative technologies that can provide enough phosphate fertilizer using cost-effective natural resources. The use of organic fertilizers can enhance microbial activity, which is the phosphate solubilization reaction [13].

The primary objective of this study is to promote sustainable agriculture production by addressing the challenges associated with expensive chemical fertilizers, root zone diseases, and significant environmental impact. Previous research on organic cultivation has mainly focused on what kind of organic material and fertilizer is the better for cultivation [14], what kind of hydroponic system is useful [15], or the microorganism culture of plant disease and phosphate solubilization reaction in experiments conducted in individual fields. This study builds on this research by providing a comprehensive analysis of organic hydroponic cultivation with lettuce. To achieve this, there were three objectives as follows: (1) identifying optimal environmental conditions for lettuce cultivation using organic liquid fertilizers, (2) determining the best practices for fertilizer management during cultivation, and (3) isolating microorganisms from organic liquid fertilizers to examine the effectiveness in reducing Pythium sp. and Fusarium sp., as well as their ability to solubilize phosphate.

2. Materials and Methods

2.1. Plant Materials and Growth Parameters

The lettuce used in the experiment was Lactuca sativa cv. Laliqua (Dutch Greenery Co., Ltd., Banklang, Patumthani, Thailand). Each seed was initially sown in polyurethane foam, and after three days, seedlings were transferred to light using chemical fertilizer at EC 1.5 mS/cm for 27 days, following the Enshi nutrient solution (Ca(NO₃)₂·4H₂O: 47.2 g, KNO₃: 40.44 g, MgSO₄·7H₂O; 24.65 g, NH₄H₂PO₄: 7.48 g, FE-EDTA: 67.8 g, H₃BO₃: 8.58 g, MnSO₄·4H₂O: 5.43 g, ZnSO₄·5H₂O: 0.66 g, CuSO₄·5H₂O: 0.24 g, Na₂MoO₄·2H₂O: 0.075 g) (

Table 1. Enshi nutrient solution.

Chemicals	Amounts
	(mmol L)
Ca(NO3)2·4H2O	0.200
KNO3	0.400
MgSO4·7H2O	0.165
NH4H2PO4	0.065
FE-EDTA	0.014
H3BO3	0.002
MnSO4·4H2O	0.005 × 10
ZnSO4·5H2O	0.006 × 102
CuSO4·5H2O	0.002 × 102
Na2MoO4·2H2O	0.006 × 103

). Afterwards, the seedlings were transplanted to containers containing organic liquid fertilizer. Each container housed six plants, with three replications per treatment. Plant height, leaf number, weight, chlorophyll content, and root length were recorded on 7, 14, and 21 days after transplanting (DAT).

2.2. Micro Enrichment Process (MEP) of Organic Liquid Fertilizer

The cultivation of organic liquid fertilizer in hydroponics requires the production of NO₃-N (nitrate nitrogen) through the MEP, which plants absorb from organic matter. The MEP experiment spanned 21 days. On the first day, 50 ppm of sodium thiosulphate (Na₂S₂O₃·5H₂O) was applied as a calcification agent. The water volume in each container (30 L) and the functionality of the pump were verified on the second day. From the third to the seventh day, an organic liquid fertilizer was applied at a dilution ratio of 800 times to set up an EC level around less than 0.5 mS/cm in the root zone [16,17] compared to the water volume, totaling 9.37 mL per day (9.37 mL × 4 days = 37.5 mL) across all containers (size: 124 × 82 cm). Every three days thereafter, samples of the experimental nutrient solution (NS) were taken, and NO₃-N levels were measured until the completion of the MEP.

After the MEP and prior to transplanting, HNO₃ (nitric acid) was added to adjust the pH to 6.5, suitable for lettuce cultivation [14], and maintain it below 7.5, suitable for the lettuce and microorganisms [13], throughout the cultivation period. It was maintained below 7.5 throughout the cultivation period. Additional fertilizer applications began from four days after transplanting (DAT) and continued every three days until harvest, coinciding with sampling times. The total nitrogen (T-N) content of the additional fertilizer was approximately 3.21 mg per plant per day for each container (Figure S1 Supplementary Materials).

2.3. Growth Environment and Conditions

The experiment was conducted in two plant factories with artificial lighting (PFALs) at Mahidol University Kanchanaburi Campus. Each PFAL housed eighteen lettuce plants in individual containers. Plants were provided with two types of organic liquid fertilizers: “Power Fish” from Premier Canning Industry Co., Ltd., (Bang-Boo, Samutprakarn, Thailand) and “Planet Organic Liquid Fertilizer Vol. 2” (POF vol. 2) from Planet Co., Ltd (Toyohashi, Aichi, Japan).

The composition of the fertilizers was as follows:

• “Power Fish”: total nitrogen (T-N): 12 g/L, total phosphorus (T-P): 9 g/L, total potassium (T-K): 20 g/L, total magnesium (T-Mg): 10 g/L.
• “Planet Organic Liquid Fertilizer Vol. 2” (POF vol. 2): total nitrogen (T-N): 12 g/L, total phosphorus (T-P): 7 g/L, potassium (K): 11.5 g/L.

The experiment comprised four distinct conditions (

Table 2. The four conditions in this experiment.

[P1F] treatment	[P1P] treatment	[P2F] treatment	[P2P]treatment
Power Fish	POF Vol.2	Power Fish	POF Vol.2
Room temperature: 26–27 °C		Room temperature: 23 °C
Water temperature: 24–25 °C		Water temperature: 20–21 °C
Humidity: 30–70%		Humidity: 30–70%
Light intensity: 400 μmol−2s−1		Light intensity: 400 μmol−2s−1
Carbon oxidation: 900 ppm		Carbon oxidation: 900 ppm

). P1F and P1P treatments took place in 24 to 25 °C, regarding the appropriate microorganism activity conditions [17]. P2F and P2P took place in 20 to 21 °C in terms of water temperature, regarding the appropriate lettuce cultivation conditions [18] (Figure S2 Supplementary Materials). Throughout the MEP and cultivation phases, a pump was used to ensure continuous aeration.

2.4. Detection of Nitrate

All the collected NS samples were analyzed for nitrate (NO₃) using the sodium salicylate procedure (SSP) as described by Yang et al. (1998) [19].

2.5. Dual Cultivation with Rot Fungi

The bacteria of organic liquid fertilizer were isolated from hydroponic nutrient solution (NS) using serial dilutions of 10⁻⁴ or 10⁻⁵ on nutrient agar (NA) medium, followed by incubated at 35 ± 1 °C for 48 h. Each bacterium was isolated until obtaining a single colony. The colony was then sub-cultured on fresh NS using the streak plate method for two days.

Root rot fungi, specifically Pythium sp. and Fusarium sp., were isolated and cultured in potato dextrose agar (PDA), followed by incubation for one week. The inhibitory activity was assessed using a dual-culture assay on NA medium. A single colony of bacteria was streaked in a line at the edge of a Petri dish and incubated for three days. Subsequently, each root rot fungus was dual-cultured 3 cm away from the bacteria. The percentage inhibition of the Pythium sp. and Fusarium sp. growth was measured when the control condition reached 3 cm. Pythium sp. inhibition was observed after four days, while Fusarium sp. inhibition required ten days.

The bacteria of POF vol.2 and Power Fish were cultured and isolated during the MEP. Three samples of Bacillus sp. (P1 to P3) and one of Xanthomonas sp. (P4) were isolated from POF vol.2. In Power fish, four samples of Bacillus sp. (F1 to F4) were isolated.

2.6. Phosphate Solubilization Reaction

The isolated bacteria were assessed for their ability to solubilize phosphate in Pikovskaya’s agar (PVK) medium. Each isolate was cultured at the center of the medium and incubated at 35 ± 1 °C for nine days. Clear zones around the bacterial colonies were observed and measured on days 3, 5, 7, and 9 after incubation to evaluate phosphate solubilization activity.

3. Results

3.1. Plant Growth

The results of using two different organic liquid fertilizers, Power Fish and POF vol.2, under two distinct environmental conditions, demonstrated significant differences among the four treatments.

Plant height did not exhibit significant differences among the treatments, with an average of approximately 6.7 cm at 21 DAT. Leaf number, however, exhibited significant variations among the treatments. P1F showed slight increases initially but rebounded by 21 DAT. P1P reached its peak leaf production by 21 DAT. P2F also showed slight increases, notably between 0 and 7 DAT and 14 and 21 DAT. Meanwhile, P2P demonstrated gradual increases, with most leaves appearing between 14 and 21 DAT. Notably, the P2P treatment resulted in the highest leaf count. Fresh weight exhibits significant variations among the treatments. P1F showed notably increases between 0 and 7 DAT and reached its peak fresh weight by 14 DAT. P1P showed notably increases at 0, 7, and 14 DAT, and it reached its peak fresh weight by 14 DAT. P2F and P2P showed slight increases until 21 DAT. Chlorophyll contents did not exhibit significant differences among the treatments from 0 to 14 DAT but exhibited significant differences at 21 DAT. Root length exhibited significant variations among treatments. P1F showed slight decreases until 21 DAT. P1P showed slight increases until reaching its peak at 14 DAT. P2F showed slight decreases by 14 DAT and notable increases between 14 and 21 DAT. P2P showed slight increases by 7 DAT, but it rebounded at 14 DAT (Figure 1).

3.2. Change in pH and EC Values from MEP to DAT

The pH decreased from 8.3 to 7.3 during the MEP, but on the days after transplanting (DAT) of lettuce, the pH levels varied: P1F and P2F treatments ranged from pH 6.8 to 7.8, while P1P and P2P treatments ranged from pH 7.2 to 5.4 due to the addition of a small amount of acid (HNO₃) at 21 MEP days before transplanting. The EC value was approximately 0.14 mS/cm when using water only at 3 MEP days to prepare water stocks. Subsequently, the EC value increased from 4 to 7 MEP days. Additionally, the EC value was below 0.5 mS/cm for P2F treatments and below 0.4 mS/cm for P1F, P1P, and P2P treatments during the MEP. All treatments generally showed increasing or nearly constant trends during the MEP but exhibited less consistent patterns during DAT (Figure 2).

3.3. Detected NO₃-N

In the P1F treatment, NO₃-N levels were 1.4 mg/L at 19 MEP days and increased to 1.8 mg/L by 21 MEP days. For P1P, NO₃-N was 2.3 mg/L at 19 MEP days and rose to 3.4 mg/L at 21 MEP days.

Levels of NO₃-N gradually increased after 12 MEP days; however, during DAT, NO₃-N levels mostly decreased in a fluctuating manner. In P1F, NO₃-N was 4.3 mg/L at 1 DAT, decreasing until 7 DAT and fluctuating below 2.0 mg/L thereafter until 23 DAT. P1P started at 5.0 mg/L at 1 DAT, decreasing to 1.8 mg/L by 23 DAT. For P2F, NO₃-N was 7.5 mg/L at 1 DAT, decreasing until 11 DAT and then fluctuating at 3.0 mg/L at 15 DAT and 4.0 mg/L at 23 DAT. In P2P, NO₃-N was 6.1 mg/L at 1 DAT, decreasing to 0 until 7 DAT (Figure 3B).

3.4. Isolation of Bacteria and Their Inhibitory Activities Against Root Rot Fungi

The isolates of Bacillus sp. and Xanthomonas sp. exhibited inhibitory activity, ranging from 33.3 to 51.1% against Fusarium sp. and 79.7 to 87.7% against Pythium sp. In addition, bacteria isolated from Power Fish demonstrated a growth inhibition of Fusarium sp., ranging from 33.3 to 63.3%, and against Pythium sp., ranging from 77.5 to 88.0% (Figure 4 and

Table 3. Inhibition activity of bacteria in organic liquid fertilizer against plant pathogen.

	Bacillus sp.			Xanthomonas sp.
Inhibition (%)	P1	P2	P3	P4
Pythium sp.	84.8 *b	87.7 *a	83.3 *b	79.7 *b
Fusarium sp.	51.1 n.s	50.0 n.s	44.4 n.s	33.3 n.s
	Bacillus sp.
Inhibition (%)	F1	F2	F3	F4
Pythium sp.	77.5 n.s	85.5 n.s	82.6 n.s	88.0 n.s
Fusarium sp.	33.3 *b	43.3 *b	46.7 *b	63.3 *a

Means of the four replications were compared using the Tukey–Kramer test at a significance level of * p < 0.05. n.s indicates no statistical significance.

).

3.5. Ability of Phosphate Solubilization

All isolated bacteria exhibited varying capacities to solubilize phosphate on Pikovskaya’s agar plates. Bacteria isolate F4 demonstrated the fastest phosphate solubilization by day 3, whereas isolates F1 and F2 exhibited activity by day 5 and isolate F3 by day 7. Bacteria isolates P1, P2, and P4 from POF vol.2 demonstrated clear zones of phosphate solubilization by day 5, while isolate P3 showed activity by day 7 (Figure 5 and

Table 4. Reaction of phosphate solubilization activity of the bacteria in organic liquid fertilizer.

	Day 3	Day 5	Day 7	Day 9		Day 3	Day 5	Day 7	Day 9
F1	×	〇	〇	〇	P1	×	〇	〇	〇
F2	×	〇	〇	〇	P2	×	〇	〇	〇
F3	×	×	〇	〇	P3	×	×	〇	〇
F4	〇	〇	〇	〇	P4	×	〇	〇	〇

〇; Reaction of phosphate solubilization activity, ×; No reaction of phosphate solubilization activity.

).

4. Discussion

This experiment demonstrates three key advantages of organic hydroponic cultivation. The first advantage is obtaining results using the micro enrichment process (MEP) and monitoring progress via days after transplanting (DAT). Organic hydroponic cultivation is a sustainable agricultural method that uses microorganisms to mineralize organic matter [6]. It converts complex organic molecules into ionic forms of macro- and micronutrients, allowing them to be easily absorbed by plants in DAT [20]. This study found that the MEP and DAT are effective supplements to conventional chemical hydroponic fertigation.

In both the MEP and DAT, there are four important factors: pH, EC, NO₃-N and the dilution rate of nutrient solution. Crucial to the MEP, pH levels between 7 and 8 are essential to optimize microbial activity, while levels of 5 to 6 are optimal for mineralization and plant growth, which are necessary for DAT [3,13]. Finding have shown that two separate levels of pH management are more suitable for organic cultivation, although specific guidelines have yet to be reported [20]. Similarly, a study on tomato cultivation using organic liquid fertilizer derived from organic waste reported that pH levels were adjusted to 6.3 ± 1.0 using HNO₃ [17]. In this study, pH levels remained consistent at around 8 until 19 MEP days but slightly decreased to around 6.5 between 19 and 21 MEP days due to the addition of HNO₃ in preparation for plant growth. Considering previous research, pH levels in this study were adjusted to between 6 and 7.5 to promote plant growth and improve the mineralization activity of microorganisms by adding additional fertilizer every three days (Figure 2B).

In all treatments, EC levels showed slight increases between 3 and 8 MEP days (Figure 2C). Additional organic fertilizer was applied every four days. After 8 MEP days, all treatments remained relatively stable until 21 MEP days. Comparing with chemical hydroponics, EC levels are regularly at around 1.5 to 2.0 mS/cm [18]. Previous studies generally have not focused on the absorption by a single plant per day, whereas organic hydroponics focus on measuring absorption per plant daily. Therefore, it is possible to achieve values below 0.5 mS/cm, as shown in our results (Figure 2C). On the other hand, the results during DAT showed that EC levels slightly decreased between 0 and 7 DAT before increasing again until 23 DAT (Figure 2D). However, previous research has reported that EC levels during DAT tend to stay relatively stable [17]. Thus, further research is needed on the number of lettuce leaves and the more frequent application of fertilizer.

In the P2F and P2P treatments, NO₃-N was not detected until 12 MEP days but showed notable increases by 19 MEP days. In contrast, NO₃-N in the P1F and P1P treatments was not detected until 16 MEP days, with increases also observed by 19 MEP days after approximately two weeks. Following previous research, a waiting period of around two to three weeks was implemented before transplanting for mineralization [3]. Comparatively, all treatments exhibited decreases in NO₃-N during the DAT period in contrast to studies reporting that NO₃-N is typically undetected after two to three weeks during DAT [3].

The second advantage is plant protection against pathogens and pest via bacteria. The root zone bacteria offers significant protection against pathogens [20]. Notably, Bacillus sp., a beneficial bacterium, enhances plant resistance by producing antibiotics and forming protective biofilms around the roots, mitigating diseases such as Fusarium wilt [4,6,21,22,23,24]. The ability of Bacillus sp. and Xanthomonas sp. to form protective biofilms around plant roots not only enhances plant health but also provides an effective strategy for managing soil-borne diseases in sustainable agriculture.

Additionally, Bacillus sp. promotes plant growth by increasing the number of lateral roots through the production of phytohormones and plant growth regulators [23]. In this experiment, the Gram staining of microorganisms isolated from organic liquid fertilizer revealed three species of Bacillus sp. in POF Vol. 2 and four species in Power Fish (Table 3). These isolates demonstrated resistance to Fusarium sp. by Bacillus sp., indicating their effectiveness in disease management. These results also suggest that cultivating with organic liquid fertilizers containing Bacillus sp. is well suited for lettuce cultivation, as supported by the data presented in Figure 4 and Table 3. This reinforces the potential of incorporating beneficial microorganisms into organic hydroponic systems for the future research to enhance plant growth and health.

Bacillus sp. and Xanthomonas sp. are classified as phosphate-solubilizing microorganisms (PSMs) [13,25,26]. In response to root exudates, a PSM moves toward the root zone, affecting the composition of the root zone microorganism’s system [13]. Phosphate availability is essential for plant composition, function, and metabolism; deficiency symptoms can occur when phosphate concentrations fall below 0.8% of dry weight (DW) and 0.2% in leaves [13]. The phosphate solubilization observed in all microorganisms isolated from Power Fish and POF Vol. 2 in this study can potentially contribute to promoting plant growth (Figure 1 and Figure 5 and Table 4), even considering the limited nutrient availability indicated by changes in the EC levels during transplantation. Furthermore, the F4 microorganisms showed the fastest phosphate solubilization reaction, occurring within three days of dual culture, underscoring their potential role in enhancing nutrient availability for plants.

The third advantage is the implementation of sustainable fertilization practices. Organic hydroponic systems minimize dependency on chemical fertilizers, which are subject to supply chain disruptions, such as those experienced during the COVID-19 pandemic and geopolitical issues [1]. Utilizing organic liquid fertilizers encourages the formation of microbial biofilms that enhance nutrient uptake and provide protection against pathogens. This lowers overall costs and lessens the environmental impact. These practices promote sustainable agriculture by incorporating renewable organic inputs, contributing to a more resilient and eco-friendly farming system [27]. By fostering a healthy microbial community and utilizing organic resources, organic hydroponic systems can maintain productivity while supporting ecological balance.

5. Conclusions

In conclusion, organic hydroponic cultivation presents a sustainable approach that leverages microbial processes for nutrient mineralization, protects plants via microorganisms, and serves as a viable alternative to chemical fertilizers. These advantages highlight its potential to contribute to resilient and environmentally sustainable agricultural practices.

For future research, it is important to focus on efficiently reducing the MEP period and determining the appropriate planting density and fertilizer amount for each plant. Cultivating organic liquid fertilizers will empower food producers to develop sustainable business models.