Single-shot LIBS: A rapid method for in situ and precise nutritional evaluation of hydroponic lettuce

Abstract

Hydroponic farming has emerged as a promising method that can enable year around crop production, particularly in regions with non-arable land. Ensuring precise control over nutrient levels and growing conditions is imperative for optimizing crop quality and nutritional value. However, the existing state-of-the-art nutrient assessment methods demand tedious sample preparation and often prove to be either destructive or offline, lacking in options for in situ monitoring. Previous approaches to nutritional evaluation using laser-induced breakdown spectroscopy (LIBS) utilized multiple laser shots or labor-intensive sample preparation to achieve enhanced sensitivity. In this context, we propose a single-shot LIBS system with a custom-made optical collection unit coupled to spectrograph to improve sensitivity and reduce sample damage by employing low excitation energy levels (~ 1.5 mJ). This study demonstrates in situ nutrient monitoring of hydroponically grown lettuce leaves and roots using single-shot LIBS analysis, paving the way for enhanced crop cultivation practices and improved agricultural productivity. Additionally, we discuss energy optimization strategies aimed at improving sensitivity and achieving a high signal-to-background ratio, which are essential for effective and safe nutrient monitoring and analysis in hydroponic farming systems. The results and analysis reveal that highly reproducible and sensitive LIBS spectra can be obtained directly from lettuce plants without any prior sample preparation.

Graphical Abstract

Introduction

Hydroponics, as a farming technique, involves cultivating plants in a soil-free medium with mineral nutrients dispersed as solution [1]. This approach presents numerous benefits, including efficient resource utilization, improved crop quality, reduced dependence on arable land, and minimized risk of soil-borne diseases. With the potential to facilitate year-round production of fresh and nutritious crops, hydroponics plays a crucial role in addressing global food security challenges [2]. To facilitate this, continuous assessment and feedback on the nutritional content of crops becomes essential. Additionally, hydroponic farming platforms offer an excellent method for screening stress-tolerant inbred lines due to their consistent growth conditions [3]. They also serve as an optimal setup for investigating typically concealed plant parts such as length and biomass of root, which are directly relevant to various abiotic stress factors [4]. Plant root plays a pivotal role as it anchors the plant in place and helps plants uptake of water, oxygen, and nutrients as shown in Fig. 1. A mixture of essential nutrients, including potassium (K), calcium (Ca), magnesium (Mg), nitrogen (N), molybdenum (Mo), phosphorous (P), boron (B), sulphur (S), and iron (Fe) combined in right proportions is necessary for optimal plant growth [5]. For example, potassium is a vital macronutrient crucial for various physiological processes, such as photosynthesis, enzyme activation, and osmoregulation [6]. Adequate potassium availability is essential for promoting plant growth, improving stress tolerance, and enhancing the overall yield and quality of crops. Calcium is also crucial for various physiological processes in plants, including cell wall development, membrane stability, and enzyme activation [6]. Ensuring adequate calcium availability is important for maintaining plant health and supporting optimal growth. Hence, the monitoring of nutritional status of leaf and root of such hydroponic crops is essential which can indicate the health status of the crops. It also contributes to a universal comprehension of the inherent constituents across different crops at different stages of the crop growth. This understanding aids in overcoming nutritional deficiencies and enables informed cultivation practices for farmers.

Fig. 1

Schematic of nutrient flow in hydroponic cultivation

Current techniques used for measuring the nutritional content of leaf or roots of a plant include optical and mass spectroscopy using inductively coupled plasma (ICP-OES and ICP-MS), atomic level absorption spectroscopy (AAS), gas chromatography—mass spectroscopy (GC–MS), large column liquid chromatography offering high performance (HPLC) or use of ion-selective electrodes [7,8,9]. All of these require digestion of the leaf samples which is invasive in nature. Fourier transform infrared spectroscopy (FTIR), a non-destructive technique is valuable for identifying specific functional groups in plant components, its application to quantify nutrients is challenging due to overlapping absorption bands and the presence of water, which complicates the analysis [10]. In this context, laser-induced breakdown spectroscopy (LIBS) proves to be a powerful technique for directly analyzing agricultural and environmental samples due to its intrinsic analytical capabilities that enable rapid and simultaneous determination of multiple elements [11,12,13,14]. With the tremendous advancement in environmental, biomedical field and agronomy, LIBS has been applied in different types of plant species such as fruit, stem, and leaf. However, most previous studies in nutrient content evaluation using LIBS require sample preparation (e.g., acid digestion and as pellets) and multiple laser shot excitation for sensitive detection which is destructive in nature [15,16,17].

Recently, hydroponic research is advancing rapidly, with remarkable progress being made in several aspects, such as irrigation patterns, illumination wavelengths, nutrient solution formulations, and introduction of new crop varieties [18]. The addition of nanoparticles (NPs) to plants through hydroponic nutrient solution is also initiated and is found to improve stress tolerance in plant species [19]. However, the consumption of such metal nanoparticles beyond a certain percentage can have adverse effects on human health [20]. Gold and silver nanoparticles (AuNPs and AgNPs) are increasingly used in hydroponics to enhance plant growth and stress tolerance [21]. AuNPs have been shown to improve seed germination, root elongation, and overall plant vigor by facilitating better nutrient absorption [22]. AgNPs, on the other hand, are utilized for their antimicrobial properties, reducing the risk of pathogen infections in hydroponic systems. However, concerns regarding the accumulation of these nanoparticles in plants and their potential health risks to consumers require careful monitoring and regulation. For example, excessive exposure to silver can lead to silver toxicity, known as argyria [23]. Argyria can cause discoloration of the skin, mucous membranes, and internal organs, and in severe cases, which may affect neurological function [24]. Therefore, it is essential to regulate the use of silver nanoparticles and ensure that they are used within safe limits to mitigate any potential health risks [13]. Currently, existing laser-based spectroscopy technique utilizing Raman effect is found to be unsuitable for mapping and quantification of such metal nanoparticles in plant parts [25]. Until now, only limited applications of LIBS for the detection of nanoparticles in plants have been reported. Therefore, development of a novel analytical tool is crucial for providing spatial distributions of NPs along the plant parts.

In this context, this manuscript discusses a single-shot LIBS system with an optically modified collection unit directly coupled to a spectrograph, which is developed to improve sensitivity and reduce sample damage by employing low excitation energy levels (order of 1.5 mJ) for in situ plant nutrient monitoring. To integrate the proposed LIBS system into agronomy, optimization of sampling conditions for a non-destructive detection is crucial and require careful consideration. This work also demonstrates the applicability of the developed direct coupled spectrograph-based LIBS system for the in situ nutrient monitoring for hydroponically grown plants to analyze the presence and quantification of nutrient elements in hydroponically cultivated plant leaf and root. We also demonstrate the capability of this system for in situ analysis of different elements present in fresh lettuce roots. Traditionally, LIBS analysis has been primarily conducted on plant leaves, but the extension to root samples provides a more comprehensive understanding of nutrient uptake and distribution within the plant. This allows for a holistic assessment of plant health and nutrient status, enabling targeted interventions to address root-related issues and optimize nutrient uptake efficiency. By monitoring changes in nutrient levels over time, farmers can detect early signs of nutrient deficiencies or imbalances and implement timely interventions to maintain crop health and productivity. The capability of the developed system to detect the presence of nanoparticles in hydroponic crops is also demonstrated. Further, related energy optimization strategies for effective and safe plant nutrient monitoring and analysis using this developed LIBS in hydroponic farming systems are also discussed.

Materials and methods

The experimental setup used for recording the LIBS spectra is illustrated in Fig. 2. A 1064 nm Nd-YAG (Quantel, Q-smart 850) laser beam was focused onto the hydroponic crop parts using a bi-convex lens with a focal length of 50 mm [26]. The emitted plasma was collected in a backward geometry (180° collection scheme) through the same focusing lens (L1). A dichroic mirror reflected the collected emission, which was then focused by a plano-convex lens (L2) onto the entrance slit of a high-resolution Czerny–Turner spectrometer (Kymera 328i, Andor). The spectrometer was equipped with 600 grooves per mm grating, and an intensified charge-coupled device (ICCD) camera (iStar, Andor) captured the dispersed light. To optimize the LIBS signal capture, the acquisition was gated, with the delay between the laser pulse and detection controlled by the timed triggering of the spectrograph using the laser source’s synchronization output signal. The data acquisition software, Andor SOLIS, integrated the laser triggering and spectral collection processes, enabling real-time nutrient analysis of the hydroponic crops. The gate delay was set at 900 ns, and the gate width at 500 ns, ensuring consistency throughout the experimental procedure. The recorded LIBS spectra were analyzed by comparing the emission peaks with the NIST database, and the signal-to-background (S/B) ratios were calculated to evaluate the quality of the spectra obtained. For sample placement, leaf samples were mounted on a sample holder using removable clips, ensuring that the sample surface remained straight and uniform throughout the experiment.

Fig. 2

Experimental setup used for recording LIBS spectra on plant sample

Seeds of green lettuce, Lactuca sativa L. (Mikado, early impulse lettuce) with an average harvest time of 30 days were sown into a sponge and germinated at 20–24 ℃ temperature, and 60–80% relative humidity. The photoperiod was maintained such that the plants received 16 h of light per day. Typically, hydroponic nutrient solutions are stored as a two-part mixture consisting of components A and B. Part A primarily contains calcium (Ca) and nitrogen (N), while part B is a blend of various nutrients such as potassium (K), magnesium (Mg), iron (Fe) and others. The optimal nutrition for lettuce crop growth is achieved by mixing nutrient solution parts A and B to maintain a pH between 5.5 and 6.5, and an electrochemical conductivity (EC) value of 1.8 to 2 mS cm⁻¹. The investigation was carried out in four phases: nutrient monitoring during growth, deficiency treatment, comparative study, and stem infusion of nanoparticles.

In the first phase of the investigation, the crop nutritional status focussing on the nutrient potassium (K) was monitored using the developed system for a duration of 10 days at an interval of two days. During the second phase of the investigation after growing the crops in optimum nutrition (A+B) for 18 days, calcium (Ca) deficiency treatments were induced by adjusting nutrition levels by varying the nutrient part compositions as 0.5A+B, 0.75A+B, and B. The third phase of the investigation deals with a comparative study of the presence of organic compounds (represented by CN bond) in fresh and dry leaves. In the final phase of the study, silver (Ag) nanoplates of average size of 100 nm were introduced into the green lettuce leaves via stem infusion for a duration of 24 h and the spatial distribution of the nanoparticles was measured using the developed system. To facilitate in situ monitoring of nutrient elements, the LIBS data were collected for distinct regions of both the leaves and roots of the hydroponically cultivated green lettuce. In this manuscript, the term “days after planting (DAP)” is used to refer to different stages in the crop life cycle after seeding or being seeded.

Results and discussion

Laser ablation is a powerful technique used in LIBS to generate plasma from the sample surface for elemental analysis. However, excessive laser energy can cause damage to the sample, leading to undesirable effects such as crater formation, surface degradation, and alteration of sample composition. The ablation threshold is the minimum laser energy density required to initiate material removal from the sample surface. For a given material, if the laser energy per pulse is below this threshold, no ablation occurs. Once the threshold is exceeded, material removal begins, and the amount of material ablated per pulse increases with laser energy. The material removal rate, often expressed as the volume or mass of material removed per laser pulse, is directly proportional to the laser fluence (energy per unit area). Mathematically, this can be described as:

$$\text{V}\hspace{0.17em}\propto \hspace{0.17em}\Phi ,$$

where V is the ablated volume and Φ is the laser fluence. However, this proportionality holds true until a saturation point is reached, beyond which increasing the laser energy further does not proportionally increase the ablated volume due to plasma shielding effects [15]. Confocal microscopy images of the ablated sample illustrate the impact of different laser energies on the surface morphology of the leaf, highlighting the formation of distinct craters corresponding to each laser pulse. Higher laser energies result in larger and deeper craters, indicating increased material ablation and plasma generation during LIBS analysis. Figure 3 illustrates the impact of varying laser energy levels on lettuce leaf surfaces through the formation of laser-ablated craters. These images also provide valuable insights into the spatial distribution and severity of laser-induced damage on the leaf surface, facilitating optimization of LIBS parameters for accurate elemental analysis with minimal sample perturbation. A direct correlation is observed between the applied energy and the size of the resulting craters from the confocal microscopy images. As the energy increases, there is a change in crater size, indicating a proportional influence of laser energy on material removal on the lettuce leaf surface. Notably, the figure highlights a specific energy threshold, notably 1.5 mJ to 2.5 mJ, where laser-induced damage to the lettuce leaf is notably minimal. At this energy setting, the lettuce leaf appears to withstand the laser ablation process with limited visible effects, as evidenced by the size and morphology of the craters.

Fig. 3

Confocal microscopy images of laser-induced crater formation on leaf surface at varying laser energies

The dependence of LIBS intensity and signal-to-background ratio (S/B ratio) on the laser energy for the 766.5 nm emission wavelength of potassium (K) is shown in Fig. 4a, b. In each measurement, S/B ratio was calculated by dividing the peak intensity by the average background value. As laser energy increases, the S/B ratio initially improves, indicating that the signal (elemental emission lines) becomes more distinct compared to the background noise. This improvement happens because the higher energy enhances the emission from the elements in the sample, while the background remains relatively constant. However, after reaching an optimal point, further increases in laser energy may lead to a reduction in the S/B ratio [16]. This reduction can be due to excessive background emission from the plasma and potential thermal damage to the sample, which can introduce noise and reduce the clarity of the spectral lines. Figure 4b shows that the optimum laser energy is between 1.5 mJ and 2.5 mJ.

Fig. 4

The variation of a LIBS intensity, b S/B ratio with laser energy on the leaf sample using single-shot analysis

The term “single shot” refers to the use of a single laser pulse per measurement to capture the LIBS spectra. Figure 5a, b illustrates LIBS spectra collected from both plant leaf and root samples across the 350 nm–800 nm spectral range. These emission peaks provide valuable information about the elemental composition of the plant tissues, allowing for the identification and quantification of essential nutrients necessary for plant growth and development. The key observations from the LIBS spectra of lettuce sample (see Fig. 5) include the presence of various macronutrients and micronutrients, particularly calcium (Ca), magnesium (Mg), sodium (Na), and potassium (K).

Fig. 5

LIBS spectra of lettuce a leaf and b root sample recorded in the spectral range 350 nm–800 nm

The ability to monitor potassium levels in plant leaves throughout different growth stages offers valuable insights for farmers. By detecting variations in potassium concentration early on, farmers can identify and address potential nutrient deficiencies or imbalances, optimizing nutrient management practices to ensure healthy plant growth and maximize crop yields. In Fig. 6, the in situ LIBS analysis showcases a consistent increase in potassium concentration from DAP 21 to DAP 31, indicating active potassium uptake by the lettuce plants during this period. Potassium levels increase during each growth stage, especially during rapid vegetative growth, flower and fruit formation, and overall biomass accumulation. This reflects the plant’s heightened demand for this essential nutrient. The increasing potassium concentration within the plant leaves may also reflect the cumulative effect of potassium absorption as the plant matures. However, the stability observed in potassium levels during the last three days of the experiment suggests a balance between potassium uptake and utilization by the plant, reflecting the physiological equilibrium. This highlights the importance of continuous monitoring to understand nutrient dynamics and adjust farming practices accordingly.

Fig. 6

Potassium (K) monitoring in lettuce leaves (as marked in red circle) during growth using in situ LIBS analysis from day 21 to day 31 (potassium variation)

Figure 7 illustrates the S/B ratio obtained from lettuce leaves with varying concentrations of calcium (Ca) in the nutrient solution indicated as different treatments denoted by T1, T2, T3 and T4. Here, T4 contains the highest Ca concentration followed by T3, T2 and T1. The LIBS data represent the emission signals generated by the interaction of a laser beam with the lettuce leaf samples, providing insights into the elemental composition of the leaves. The key observation from the analysis is the variation in the intensity of the Ca emission signal across crops grown in nutrient solution containing different concentrations of Ca. The spectrum from the lettuce leaf grown in a nutrient solution with A+B concentration (denoted by T4) exhibited the highest S/B ratio of Ca emission signal, indicating a higher concentration of calcium compared to other samples (denoted by T1, T2, and T3) in the investigation. Adequate Ca availability is essential for healthy plant growth and development, as evidenced by the higher Ca emission signal intensity observed in lettuce leaves grown in a nutrient solution with higher Ca concentration.

Fig. 7

LIBS analysis of lettuce leaf with different Ca concentrations in nutrient solution

Figure 8 shows the elemental variation observed in dry and fresh lettuce leaves using the developed system. The comparison includes the analysis of CN molecular bands and Ca emission in both fresh and dry leaves. The CN molecular band exhibits higher intensity in fresh leaves as compared to dry leaves. This indicates a higher concentration of carbon and nitrogen molecules in fresh lettuce leaves. The CN molecular band provides insights into the carbon and nitrogen content in plant tissues, which are essential elements for plant growth and metabolism. Higher CN molecular band intensity in fresh leaves suggests a higher concentration of organic molecules, such as proteins, nucleic acids, and carbohydrates, vital for plant growth and development. Conversely, lower CN molecular band intensity in dry leaves may indicate reduced organic content due to dehydration, highlighting the importance of maintaining adequate moisture levels for plant health.

Fig. 8

Elemental variation in dry and fresh leaves using LIBS analysis

Nanoparticles, such as silver nanoparticles, have been studied for their potential to improve plant growth, increase nutrient uptake, enhance stress tolerance, and mitigate disease incidence [27]. The presence of silver nanoparticles in the plant leaf suggests their role in promoting plant health and productivity in hydroponic systems. The detection of silver nanoparticles inside the plant leaf highlights the significance of nanoparticle application in hydroponic cultivation and crop enhancement. Figure 9 shows the LIBS spectra obtained from a plant leaf treated with silver nanoparticles. The LIBS spectra reveal distinct emission lines corresponding to silver (Ag) nanoparticles. These emission peaks provide direct evidence of the presence of silver nanoparticles inside the plant leaf. The appearance of specific emission lines from silver confirms the uptake and accumulation of nanoparticles by the plant. The emission peaks of silver nanoparticles serve as a valuable indicator of nanoparticle uptake by plants, offering opportunities for further research and optimization of nanoparticle-based strategies for sustainable agriculture.

Fig. 9

LIBS spectra of the leaf with silver nanoparticle

The ability to directly monitor nutrient levels in plant tissues using LIBS offers significant advantages for hydroponic farming systems. By analyzing the elemental composition of plant’s leaves and roots in real-time, hydroponic farmers can assess crop health, diagnose nutrient deficiencies or imbalances, and optimize nutrient management strategies to maximize yield and quality. The LIBS spectra obtained from both leaf and root samples can serve as valuable indicators of crop health and nutrient status in hydroponic farming. Additionally, the ability to perform onsite monitoring using LIBS facilitates rapid decision-making and ensures the efficient management of hydroponic farming operations. In future, the developed system can be configured as a flexible probe with inbuilt laser illumination that can be pointed to different parts of crop to perform LIBS-based nutritional status checks.

For accurate quantitative measurements, the system must be calibrated against established techniques for each specific element. Potential sources of variability in the measurements include various environmental and instrumental factors such as pulse-to-pulse fluctuations in laser energy, spectrometer sensitivity, and variability in laser spot position. The variations from these factors were addressed by using a precise translational stage and taking multiple measurements in the area of interest. The proposed system has scalability potential, particularly as a portable unit with the integration of a compact spectrometer and laser unit. Additionally, a probe-based approach could offer a flexible and easily adaptable solution that can form the basis of future work direction.

Conclusion

Rapid in situ monitoring of plant nutritional status has been demonstrated using a developed LIBS system. This direct coupled in situ analysis of nutrient levels in plant tissues using LIBS enables hydroponic farmers to monitor nutrients continuously without the need for sample extraction or laboratory testing. Such real-time feedback allows for timely adjustments to nutrient solutions, ensuring optimal nutrient uptake by plants. By accurately assessing nutrient levels in plant leaves and roots, hydroponic farmers can tailor nutrient solutions to meet the specific needs of their crops at different growth stages. This precision in nutrient management minimizes waste and maximizes nutrient utilization, leading to improved crop yields and quality. The detection of nanoparticle uptake by plants opens avenues for exploring the use of nanoparticles in hydroponic farming for crop enhancement and sustainability. By studying nanoparticle–plant interactions using LIBS, we can also assess the efficacy and potential risks of nanoparticle-based agricultural strategies. The developed method helps to prevent nutrient-related issues from escalating, ensuring sustained crop health and productivity. The application of LIBS in hydroponic farming offers a novel and effective approach to nutrient management and crop health monitoring, contributing to increased efficiency, productivity, and sustainability in modern agriculture.