1. Introduction
Crop plants are important sources of macro- and micronutrients for a healthy human diet [
1]. However, our world will confront two main challenges in the future: climate change and a global population growth. It was estimated that by 2050, the world would require 60% more food than it does today, with around 80% of this increase coming from land already under cultivation [
1]. The significant rise in food demand must be met while reducing agriculture’s global environmental footprint, and this must be done at a time when agriculture is already under pressure due to climate change [
2]. To fulfil contemporary food demand, agriculture employs agrochemicals (synthetic fertilizers and pesticides), intensive tillage, and over-irrigation [
3,
4].
The uncontrolled and excessive use of synthetic chemical inputs to enhance agricultural productivity has been degrading the soil and threatening the environment, with it being estimated that 40% of global arable land undergoes decreasing productiveness [
5]. Recently, some eco-friendly alternatives to chemical fertilizers have been applied and seaweed can help in this endeavor [
6]. These seaweed extracts contain cytokinins, auxins, betaines, gibberellins, carbohydrates, vitamins, polysaccharides, alginates, amino acids, and trace elements (Fe, Cu, Zn, Co, Mo, Mn, and Ni) that are involved in plant growth and regulation. The mechanisms of action of these seaweed compounds are complex, and it is unknown why they are effective; nonetheless, it was likely that when these molecules are combined, they work synergistically, promoting plant growth and well-nurtured plants [
7,
8,
9]. Seaweeds are marine resources that produce a wide range of primary and secondary metabolites that have a significant positive impact on agricultural crops [
10]. Quite a few studies have shown that different seaweed extracts can improve seed germination, plant growth, and development [
11,
12,
13,
14,
15,
16,
17]. Furthermore, it has been demonstrated that seaweed extracts can support plants in coping with biotic and abiotic stresses (such as herbivory, deleterious microorganisms such as fungi and bacteria, and drought and salinity), while also improving crop nutritional profile, mainly in mineral and vitamin concentrations [
16,
18,
19,
20,
21].
Saccharina latissima, known as sugar kelp, was a North Atlantic and North Sea natural seaweed. Numerous successful cases have demonstrated
S. latissima’s farming and economic potential for the food and cosmetic industry. Thus, there is a rising number of
S. latissima cultivation systems established in Europe [
22,
23,
24,
25,
26].
S. latissima can be found in some areas which can have problems with excessive nutrients (eutrophication), hydrocarbons and heavy metals, such as fishing ports, because seaweeds are natural bio-accumulators of various compounds which can be dangerous for direct consumption by humans. Thus, this seaweed, when found in or nearby polluted water, cannot be the best supply for the food industry. However, algal biomass that does not meet food-grade standards can be used in agriculture rather than as organic waste (as it is today in some eutrophicated places) [
27,
28,
29,
30,
31]. Regarding the regulations related with seaweed extract application in agriculture, Spanish regulation was considered an exemplar in Europe. Agricultural product chemical control and security, however, are crucial for assuring safe application. They usually refer to arsenic levels and heavy-metal-compound thresholds [
31].
Free-living bacteria can also be employed as an external nitrogen source for the crop, promoting plant growth [
32,
33]. For example,
Methylobacterium was a bacteria genus known for its eco-friendly ability to improve plant growth through atmospheric nitrogen fixation, phosphate solubilization, and the stimulation of plant growth promoters [
33,
34]. This bacterium contains an enzyme nitrogenase, which converts atmospheric nitrogen (N
2) into ammonia (NH
3). This bacterium colonizes the plant during its early growth stages, providing it with ammonia without any risk of volatilization or leaching [
35,
36]. Lettuce (
Lactuca sativa L.) is a leafy herbaceous vegetable and one of the most popular salad crops, in both fresh and ready-to-eat markets in the world, with 27 million tons of lettuce and chicory produced globally in 2020 [
37].
The purpose of this research was to better understand the biostimulant and fertilizer impact of the aqueous extracts of the brown seaweed Saccharina latissima, alone, and combined with a bacteria-based biofertilizer (Methylobacterium symbioticum), as a foliar spray on lettuce (Lactuca sativa L.) plant growth and its nutritional profile. During this work, it was important to monitory if a bacteria/seaweed-combined extract can show a synergistic potential to enhance lettuce growth and its biochemical affluence. On another hand, the study also aimed to understand if a more economic and simplistic approach applying one of the extracts (seaweed or bacteria), alone to the plant or a combination of both, can be effective as a good biofertilizer.
4. Discussion
The
S. latissima was collected from an eutrophicated area, although, there was a need to observe and search for information about other problematics in the regions where the seaweeds were collected from. Nevertheless, Lima river estuary was obviously a focus of great vigilance, and the environmental protection agency has a huge number of findings of analyses performed on this river; it was certified that there are no heavy metals or excess hydrocarbons present in the location [
65,
66,
67,
68].
The nutritional, macro-, and microelement analyses of an algal biomass can vary depending of the harvesting season and geographic region [
52,
53,
54]. Still, this biochemical characterization might differ considerably based on biotic and abiotic components, which can have direct or indirect impacts, such as seashore and the period of the year when the biomass is harvested. Thus, there is a need to perform biochemical characterization of the seaweed biomass for each assay batch. For example,
S. latissima harvested in April 2015, that was produced in a seaweed aquaculture in Northern France (Brittany), when compared with our results, presented increased concentrations of sodium (3.0483 g 100 g
−1), potassium (3.87 g 100 g
−1), calcium (0.92 g 100 g
−1), magnesium (0.61 g 100 g
−1), iron (0.19 g 100 g
−1), manganese (<0.01 g 100 g
−1), and zinc (0.00386 g 100 g
−1) [
53]. In contrast, lower concentrations of copper (<0.01 g 100 g
−1), total protein (0.01 g 100 g
−1) and lipids (0.01 g 100 g
−1) were registered [
53]. Meanwhile,
S. latissima wild harvested in the intertidal region of Fink Cove, (Nova Scotia, Canada) in April 2010, exhibited higher mineral content, representing 24.50 g 100 g
−1 of this seaweed dry biomass, but a lower amount of total protein (8.10 g 100 g
−1), fat (5.50 g 100 g
−1), carbohydrate (59.80 g 100 g
−1) and phenolic compounds (1.11 × 10
−4 g GAE 100 g
−1) [
54].
This heterogeneity in algal chemical characterization was caused by the fact that algae metabolic activity changes according to the season and geographical [
69]. Based on these findings and the literature reviewed, this brown seaweed appears to be a promising source of alginic acid (the majority of the carbohydrates present in this seaweed are alginic acid), which plays an important role in plant nutrition by lowering water surface tension, forming a polymeric (alginic acid, primarily) film on the plant’s surface, increasing contact area, and making it easier for water-soluble substances to enter the plant cell through the cell membrane [
70,
71]. The economic potential of this brown seaweed is highlighted by its alginic acid concentration, which may reach up to 20% of the algal dry weight and is now being investigated by a range of sectors, including agriculture [
55,
56]. In fact, alginic acid was recognized by the International Federation of Organic Agriculture Movements (IFOAM) as an approved additive [
72]. However, we compared dry seaweed biomass values, the study of Sangu [
73] and other preliminary small-scale studies demonstrated that the main difference between the seaweed biomass and the liquid extract was only the water content. Moreover, liquid extract analysis faces some technical–operational challenges and the dehydration of the extract can increase chemical oxidation, hindering further analysis.
The nutrition solution used as a foliar spray treatment in our study can have a significant impact on plant development and growth [
74,
75,
76], and for this reason, extracts’ physical–chemical characterization was crucial to attain good results. Thus, the electrical conductivity of the tested extracts was revealed to be suitable for lettuce growth, because previous research has found that using nutrient solutions with an electric conductivity higher than 1300 μS/cm in lettuce cultivation may cause nutrient imbalance, resulting in decreased leaf number, area, and plant weight [
77,
78]. Furthermore, high electrical conductivity values (>1700 μS/cm) in the nutrient solution can lead to early bolting, and chlorotic and necrotic spots on lettuce’s lower leaves [
40]. Another critical factor for plant development was pH; hence, for lettuce development, nutrient solutions with values around 6 are recommended [
75,
76,
79,
80,
81]. On the other hand, researchers found that at pH 5, the shoot and root weight was excellent, but with higher pH (above 7) these values were lower [
82]. Moreover, total dissolved solids should also be monitored because high levels (above 1000 mg/L) can impair plant growth [
83].
However, bacteria can be absorbed by the seaweed polymer and not be as efficient as expected. Observing the results, the nitrogen in the seaweed was mainly in organic form (amino acids and protein), which was not the preferable nitrogen source for nitrifying bacteria. This can explain the negative stimulant effect results of algal extracts on lettuce growth. The seaweed extract can also have an anti-microbial effect [
11,
12,
13,
14,
15,
16,
17], but in our case, the lettuce plants were grown in separated pots. There was no antimicrobial effect observed in the
S. latissima extract used in our experiment [
84].
The algal extract (E) alone and the combination of the algal extract and BlueN (EB) had a positive effect on lettuce leaf development and weight (
Figure 4), while the BlueN (B) alone was found to be ineffective for lettuce development (
Figure 4). The length and weight ratio between the root and the aerial parts showed that there was a dependent connection amongst these variables. Herein, it was possible to observe that the positive control (CP), the algal extract (E) and BlueN (B) treatments alone exhibited higher ratios (2.04, 1.95 and 2.07, respectively) than the remaining treatments, resulting in a more developed root, whereas the plant focused on the leaf growth development. In contrast, when compared with the other treatments, the one with BlueN (B) and the combination between BlueN and the algal extract (EB) led to a higher root vs. aerial part weight ratio (0.47 and 0.37, respectively), indicating that the plant spent more energy on root biomass looking for nutrients in the substrate, and thus not promoting foliar part development, which is the economic part of the lettuce. Thus, the seaweed extract demonstrated an enhancement of the lettuce aerial part development that results in less root development. This algal extract resulted in an improvement of the plant foliar metabolism and an enhancement of the lettuce root efficiency to absorb more nutrients to support better aerial part development.
As water and nutrients are not evenly distributed in the soil, the spatial organization of the root system was critical for regulating the most efficient use of the available resources [
85]. Herein, White [
86] showed that higher-crop-yielding cultivars are often grown at optimal nutrient concentrations, resulting in the selection of smaller and less plastic roots (less developed, lower specific root length, root demography, and biomass allocation within the patch zone) [
87,
88]. In fact, based on the prior literature, when root architecture contains a high number of nodal and lateral roots, the plant yields more and produces a higher biomass because a significant investment in lateral root growth results in the establishment of a shallow root system [
89]. This happens because roots often proliferate when they come upon a nutrient-rich zone, improving their physiological ion uptake capacity [
88]. According to the literature, a reduction in soil pH values could be caused by the use of a nitrogen-based fertilizer, which results in plant absorption of available nutrients, leaving others in the soil to be oxidized [
46]. Similarly, the electrical conductivity values could have also been increased due to the application of the fertilizers which added nutrients/minerals in the soil [
46]. The soil substrate exhibited an electrical conductivity lower than 0.50 mS/cm, except for the treatment with BlueN (B: 0.52 mS/cm), which was suitable for lettuce growth, because this species was sensitive to a high salt concentration [
46]. The root system’s plasticity or flexibility responses have been suggested as the main system by which plants handle with soil’s innately arising nutrient heterogeneity [
88]. So, if the plant root was longer and had more biomass than the aerial portion, it means that the plant was not efficient in its use of nutrients to develop the aerial part and to realize photosynthesis, which is very important for crops [
86].
When compared with other studies where the nutritional characterization of lettuces grown under greenhouse conditions was evaluated [
63,
64], it was possible to observe that, in general, the other values were lower when compared to the present research, mostly in calcium, potassium, zinc, iron, and manganese. This demonstrates that extract treatment promotes a better nutrient uptake for the plants. Mainly, there was an enhancement of manganese absorption in the lettuce treated with seaweed extract only.
Nonetheless, genetic differences and environmental factors had a direct influence on the phenotypic differences between treatments influencing the nutritional composition of the edible portion of the lettuce [
63].
When applied as a foliar spray alone or in conjunction with the biofertilizer BlueN,
S. latissima aqueous extract improved the bulk of lettuce leaves while also enhancing their nutrition, particularly in micronutrients such as zinc and manganese, which are important in the human diet. A part of 100 g can fulfil the zinc threshold value (10–11 mg, daily intake for an average adult) according to the European Parliament and Council of the UE’s daily recommended intakes of minerals and trace elements, while 35 g of lettuce per day is enough to fulfil an average adult’s manganese dietary requirements (2–2.3 mg). Furthermore, zinc and manganese are micronutrients that play important catalytic, structural, and regulatory functions in human metabolism, and are especially important for the brain and cardiovascular systems [
90,
91,
92]. Zinc is an essential trace element that controls and physically maintains cell membrane stability [
93]. Manganese is required for enzymes such as manganese superoxide dismutase, arginase, and pyruvate carboxylase [
94,
95]. Furthermore, this mineral participates in a variety of metabolic functions, including the metabolism of amino acids, cholesterol, glucose, and carbohydrates. Manganese is also involved in the scavenging of reactive oxygen species, bone formation, reproduction, and immunological response [
96,
97,
98].
Lettuce is a cool-season crop that grows best at daytime temperatures of 15–20 °C [
99]. Because of the plant’s shallow root structure, it may be cultivated in a variety of soils as long as they are fertile and moisture retaining [
99]. It prefers neutral soil (pH values between 6.5 and 7.2) and will not grow in acidic soil. Heat-resistant cultivars can be cultivated during the summer months; however, care should be taken to protect the leaves from direct sunlight by shading or covering the plants to prevent bolting [
29,
31].
There are currently no restrictions on the use of seaweeds in agriculture; nevertheless, due to the high-salt seaweed content, long-term or excessive use of row and not treated seaweeds may contribute to an increase in salt content in a soil [
100]. When analyzing the soil, only the BlueN treatment was higher than 0.5 mS/cm, which indicates a rise of 0.3 mS/cm. This can be a dangerous rise if using more cultivars without revolving or adding more healthy soil; this problem can be due a bigger root system which can shift the soil structure and available nutrients.
BlueN, a commercially accessible product, was composed by
Methylobacterium symbioticum, an endophyte bacterium that naturally provides nitrogen to plants [
101]. Several experiments with this product have shown that it was beneficial to crops, increasing yield and lowering the use of conventional nitrogen fertilization [
101]. For example, only one application of BlueN led to a yield increase of 56% in maize crop culture, 40% in grape, and 9% in raspberry [
101]. Furthermore, the use of BlueN reduced 40% of the application of conventional nitrogen fertilizer on wheat crop culture and resulted in a 60% reduction in chicory cultivation [
101]. Even though there have been no reports of this product (BlueN) being used on lettuces, earlier research has demonstrated that
Methylobacterium spp. can improve the growth and productivity of several important crop cultures, including sugarcane, wheat, corn, peanut, and tomato [
102,
103,
104].
A patent study showed that the foliar application of a bacteria from the genus
Methylobacterium (1 × 10
6 CFU/mL to 1 × 10
11 CFU/mL) can improve the rate of root and leaf lettuce growth, as well as overall biomass production [
105]. However, because lettuce was a fast-growing crop, BlueN may have a greater impact on annual crops, as it persists longer in soil.
Brown seaweeds, such as
Alaria esculenta,
Ascophyllum nodosum,
Fucus serratus,
F. spiralis,
F. vesiculosus,
Laminaria digitata and
Ecklonia maxima have been shown to enhance plant growth, and based on these findings, various authors state that a continuous application of seaweed-based fertilizers has more potential than plant-based or organic and synthetic fertilizers in agriculture and horticulture [
106,
107,
108,
109,
110]. For instance, the brown seaweed
Ecklonia maxima has shown a positive effect on lettuce growth, increasing the potassium (46%), magnesium (37%) and calcium (52%) concentration in plant leaves [
111]. Another example is the
A. nodosum extract, which has also been shown to improve lettuce seedling performance when exposed to high temperatures [
112]. Moreover, previous research using Profertil (in the same concentration) on lettuce presented slightly higher results, when compared with the results obtained in this study, achieving an average aerial-part diameter of 20 cm and an aerial-part weight of 80 g, as well as a higher root weight (24 g) [
113].
Nutrients are absorbed by plants, depending on where they are applied, through their roots or on the surface of their leaves. Thus, the biochemical components of seaweed extract can significantly affect the plant’s nutrient profile [
106]. Furthermore, seaweed extracts can modify the physical, biochemical, and biological aspects of soil, as well as the architecture of plant roots, resulting in increased nutrient absorption efficiency [
114]. In this context, extensive research regarding the chemical composition of several seaweed extracts indicated that the extracts’ nutrient content (usually macronutrients such as nitrogen, phosphorus, and potassium) can affect plant growth and yield [
115,
116,
117]. Furthermore, temperature, humidity, light intensity and leaf age all influence the stomatal opening and the permeability of the cuticle and cell wall, influencing nutrition absorption from the leaf surface [
106,
115,
118].
In addition, brown seaweeds contain polysaccharides including alginates and fucoidans, which are beneficial to crop plants, promoting their growth [
6]. Alginic acid, for example, has soil-conditioning capabilities as well as the capacity to bind metal ions and produce high-molecular-weight polymers [
114,
119]. In this setting, the presence of a substantially cross-linked polymeric network improves soil water retention and root development [
120,
121,
122]. From another standpoint, alginate can compete with plants for cation uptake, limiting the growth-promoting effect [
120]. In this experiment, the brown seaweed A. nodosum had nearly twice the alginate concentration of Laminaria species, which could explain why the positive control had lower development in terms of leaf weight, when compared with the aqueous algal extract of
S. latissima (previously known as
Laminaria saccharina). Moreover, phenolic compounds have chelating properties, which may explain why seaweed extracts can release soil components that are otherwise unavailable [
108,
123,
124].
However, not all seaweeds, and hence not all seaweed extracts, are the same; even the same raw material extracted using different procedures yields extracts of varying quality [
107]. Thus,
S. latissima ever-changing biomolecular profile was one of the major bottlenecks that hinders this species large-scale seaweed-based biostimulant production [
125].
Furthermore, the concentration of seaweed extract is a crucial issue for good plant growth that deserves additional investigation, as was the timing and frequency of its application to achieve the desired results [
106,
107]. Moreover, the interaction of the brown algal extract with the bacteria present in the BlueN was investigated to determine whether or not both products used had synergistic effects that stimulated plant development. Previous research has demonstrated that the application of a product composed by plant-growth-boosting bacteria (
Bacillus licheniformis,
Bacillus megatherium,
Azotobacter sp.,
Azospirillum sp., and
Herbaspirillum sp.) and by the green microalgae
Chlorella vulgaris substantially affected the plant weight of romaine (18.9% at spring) and leaf lettuce (22.7% at summer) [
126].
Murugan et al. [
33] investigated the effect of bacterial–algal interaction on plant growth. In this context, the bacteria
Methylobacterium oryzae and a methanolic extract of the brown seaweed
Sargassum wightii collected on the Palladam coast (India) were utilized in the investigation. As a result, the extract with the best yield in both crop cultures (tomato/
Lycopersicon esculentum L. and red pepper/
Capsicum annum L.) had a seaweed:methanol ratio of 40:2500, and it outperformed the algal extract and the bacteria alone.
Still, there is little information regarding the interaction of Methylobacterium sp. with seaweed-based liquid fertilizers on plant growth; therefore, more research is need.