The integration of probiotics in aquaponics systems is a strategy for mitigating environmental impacts and for promoting sustainable agriculture. In order to understand the role of probiotics, we investigated the effect of a commercial probiotic mixture of Bacillus subtilis and B. licheniformis on the growth of lettuce (Lactuca sativa L.) under deep-water culture integrated with Mozambique tilapia (Oreochromis mossambicus).
Aquaponics involves the cultivation of plants and fish in a recirculating system. In this system, plants use dissolved nutrients excreted by fish or generated from the microbial breakdown of their excretions for growth . Enhancing the productivity in an aquaponics system involves monitoring and managing environmental variables in order to provide optimal growth conditions for microbes, fish, and plants .
If the bacterial community diversity is not balanced, or if the environmental conditions are not suitable for plants, fish, and microbes, water quality may fluctuate in such a way that the environment becomes harmful to both fish and plants. As a result, operational parameters need to be managed to ensure optimal and stable growth conditions .
Microbial communities play important roles in nutrient recycling, degradation of organic matter, and control of plant pathogens in aquaponics systems . The need to increase resistance to diseases in aquatic species, optimize the growth of all farmed aquatic organisms, and improve feed conversion efficiency has motivated research designed to test the effect of probiotics in aquaculture practices. The term probiotics refer to microorganisms that are associated with beneficial effects for the host .
Currently, there are commercial probiotic products comprised of various bacterial species such as Bacillus sp., Lactobacillus sp., Enterococcus sp., Carnobacterium sp., and the yeast Saccharomyces cerevisiae . Research on the application of Bacillus as probiotics in recirculating aquaculture systems has focused mainly on enhancing feed utilization and health improvement supplements for aquatic animals [6,7], but there is a paucity of work on their use in aquaponic crop production.
In traditional agricultural systems, research has demonstrated that inoculating plants with plant-growth-promoting bacteria (PGPB) such as Bacillus spp. can be an effective strategy to stimulate crop growth ; however, research on the application of PGPB in aquaponics is still limited [9,10]. In soil-based systems, PGPB is an effective substitute for chemical inputs to meet both plant growth requirements and to reduce the impact of biotic stress .
The direct promotion by PGPB entails either providing the plant with growth-promoting substances that are synthesized by the bacteria or facilitating the uptake of certain plant nutrients from the environment. PGPB are capable of stimulating plant growth through a variety of mechanisms, including nitrogen fixation , stimulation of root growth , organic matter mineralization, suppression of disease-causing organisms , improvement of plant nutrition, and increasing the bioavailability of nutrients .
Bacteria beneficial to plants form stable biofilms on the roots . Various genera of bacteria, such as Pseudomonas, Enterobacter, Bacillus, Variovorax, Klebsiella, Burkholderia, Azospirillum, Serratia, and Azotobacter have been reported to exhibit plant growth-promoting characteristics [8,15]. The activity of these rhizobacteria has been attributed to a number of factors, such as their ability to produce antimicrobial compounds as well as competing for space and nutrients on the root system, thereby inducing resistance to plant-pathogenic organisms [8,12,15].
Regardless of the type of agricultural system, root health is essential to the growth of plants. In addition to advancing aquaponic crop production, research into the use of PGPB in soilless-based environments has the potential to advance our understanding of rhizosphere microorganism associations. Among Bacilli, strains of Bacillus subtilis are the most widely used PGPB because of their disease-reducing and antibiotic-producing capabilities when applied as seed treatments [9,16].
In this study, we investigated the effect of supplementation of a commercial product (Sanolife®PRO-W) containing a mixture of Bacillus subtilis and Bacillus licheniformis on the growth of ‘Locarno’ leaf lettuce cultivar (Lactuca sativa) under deep water culture integrated with Mozambique tilapia (Oreochromis mossambicus) in modular-coupled aquaponics systems, in comparison with a control treatment that did not receive the product. We tested whether integrating Bacillus spp. into an aquaponics system could influence plant growth, nutrient availability, and bacterial community diversity.
Water Quality Management in Fish Rearing Tanks
Water temperature, NH3, TAN, NO2−, pH, and dissolved oxygen were maintained at similar levels between Trial 1 and Trial 2. These parameters were within acceptable levels as reported for aquaponics [24,25,26]. The water temperature for the fish tanks in both the control and Bacillus-treated systems across the two trials ranged from 21.0 to 26.7 °C. NH3 and NO2− concentrations remained below 1 mg L−1 during the trials.
These low concentrations allow tilapia to be reared without negative effects on their health [24,26,27]. The pH values ranged between 6.63 and 7.8 in Growth Trial 1 and between 6.52 and 7.09 for Growth Trial 2. Fish tolerate a wide range of pH but do best at levels of 6.5–8.5 [24,28]. Even at the higher pH values, the concentrations of free ammonia (NH3) remained below harmful levels for tilapia. In addition, the dissolved oxygen concentrations were above 5 mg L−1, which is an acceptable level for aquaponics as recommended by Sallenave .
EC and TDS values increased in both systems across the two trials over time, indicating the presence of ions released from both the fish feed and from the mineralization of accumulated organic matter. However, levels of EC and TDS did not differ between treatments for each trial.
In our study, EC levels ranged from 0.55 to 0.84 mScm−1 for Trial 1, and from 0.46 to 0.78 mScm−1 for Trial 2. The TDS ranged from 350 to 584 mg L−1 and 330 to 471 mg L−1 for Trial 1 and Trial 2, respectively. Both EC and TDS were within the optimum range for culturing fish [26,29,30].
In general, lettuce growth was relatively low. After 30 days in Trial 1, the average final shoot weight was 24.84 and 20.07 g plant−1 for the Bacillus-treated and the control systems, respectively, compared to 33.08 and 25.57 g plant−1 in Trial 2. Hernandez et al.  reported a higher average final lettuce fresh weight in the range of 53.42 g and 62.7 g plant−1 with a crop cycle of 55 days.
Our study used the same cultivar as reported by Hernandez et al. ; however, there were variations in systems design. The differences in growth could be due to differences in growth duration, the scale and maturity of the systems, type of systems (hydroponics vs. aquaponics), and the light intensity. However, the growth trials demonstrated improved growth in the Bacillus-treated aquaponics systems.
The fresh weight of the harvested shoots and the shoot dry weight was significantly higher in the Bacillus-treated systems compared to the control systems in both crop cycles. Higher shoot weight could possibly be attributed to the higher nitrate and phosphate levels in the Bacillus treatments. A study by Cerozi et al.  showed enhanced lettuce growth and phosphorus accumulation in aquaponics systems treated with Bacillus.
In addition, Bacillus may influence factors that stimulate root growth, leading to greater nutrient uptake. Bacillus spp. are also known plant growth enhancers, with the ability to protect plants against pathogens through mechanisms associated with induced systemic resistance [13,32,33].
The Fv/Fm was significantly higher in the Bacillus-treated systems. According to Murchie and Lawson , the value of Fv/Fm for unstressed leaves is approximately 0.81–0.83. Similar values were observed in the Bacillus-treated systems in our study, suggesting that the lettuce plants were not stressed.
Nutrient Accumulation in Lettuce and Water
At the end of Trial 1, significantly higher levels of P, K, and Zn were observed in lettuce when Bacillus was added to the system. A gradual increase in phosphate concentration in deep water culture systems treated with Bacillus was reflected in phosphorus accumulation in leaves, implying that the uptake of phosphorus by plants was influenced by the phosphate concentration in the deep-water culture growth beds.
Plants were grown in Bacillus-treated systems accumulated approximately four times as much total phosphorus as the control. Bacillus spp. possess strong growth-promoting activities such as solubilization of minerals, nitrogen fixation, production of antibiotics, siderophore production, and production of secondary metabolites . Through oxidoreductase systems and proton extrusion, Bacillus strains can increase the bioavailability of Zn by producing chelating ligands and secreting organic acids , such as 2-ketogluconic acid and gluconic acid, which solubilize Zn.
Dissolved Nitrate and Phosphate in the Deep-Water Culture Growth Beds
In the two trials, the levels of nitrate in the deep-water nutrient solution were within tolerable limits (<150 mg L−1) for plants under aquaponics systems [17,37]. The average nitrate values in the Bacillus-treated systems were significantly higher than in the control systems. The increase in phosphate and nitrate concentrations in aquaponic systems treated with Bacillus enhanced lettuce growth.
Although aquaponics systems were inoculated with a commercial product FINCO as a source of nitrifying bacteria during biofilter establishment, the levels of nitrate and phosphate accumulated faster in the Bacillus-treated systems than in the control systems during the growth trials. It could be possible that Bacillus might have enhanced biological nitrification that resulted in significant changes in nitrate levels. The different nitrate and phosphate levels observed between the trials were because each was run independently and could be attributed to the total fish biomass at the beginning of each trial.
The phosphate levels in the control systems remained relatively low in both trials. Most plants need a phosphate concentration of 1.9–2.8 mg L−1 for adequate growth in culture solutions . The systems treated with Bacillus in contrast showed significant increases in the phosphate concentrations in the deep-water nutrient solution. Since Bacillus can mineralize different forms of phosphorus, it is possible that the addition of the probiotic increased organic phosphorus mineralization in the deep-water culture solutions.
It has been reported that species belonging to Bacillus are able to produce phytase enzymes for the mineralization of phytates [13,16,39], which could possibly have contributed to increased phosphate levels. Despite variations between trials in the changes in nitrate and phosphate levels over time, the addition of Bacillus product increased nitrate and phosphate levels in both trials, which further strengthens the conclusion that Bacillus can increase the levels of these plant nutrients. However, whether the increased levels of nutrients reported in our study are partly due to the inherent capacity of the commercial Bacillus product to supply nutrients requires further investigation.
Root Associated Core Microbiota
At the phylum level, our results suggest that there was a predominance of Proteobacteria and Bacteroidetes in both the control and Bacillus-treatments. Proteobacteria and Bacteroidetes are known to respond rapidly to carbon sources and are generally considered to be r-strategists and fast-growing bacteria [40,41]. The enrichment of Proteobacteria and Bacteroidetes in aquaponics was reported by Schmautz  and Eck et al. , but with different relative abundances to our study.
Our results confirmed that these organisms predominate in the roots. In reference to soil-based studies, Proteobacteria and Bacteroidetes were described as effective rhizosphere and root colonizers in several plants such as rice  and wheat  because of their ability to utilize root exudates .
Members of the bacterial phyla Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes, were more abundant in Bacillus-treated systems than in the control systems. These phyla are mostly involved in physiological functions such as carbon usage, nitrogen assimilation, metabolism of iron, antimicrobials, and abundance of transporters [47,48].
For example, Acidobacteria has a large proportion of genes encoding for transporters. The high number of different transport systems facilitates the acquisition of a broad range of substrate categories, including amino acids, peptides, siderophores, cations, or anions . The phylum Verrucomicrobia has been detected in different soil, freshwater, and marine environments . One of the most notable features of freshwater Verrucomicrobia is their role in polysaccharide degradation [49,50].
Firmicutes have been reported to utilize carbon sources and produce lactic acid, acetone, butanol, and ethanol, which contribute to nutrient turnover . The high abundance of Planctomycetes in our study may be an indication of nitrogen cycling in the aquaponics system. Members of the Planctomycetes are known to perform anaerobic ammonium oxidation (anammox). They oxidize ammonium with nitrite as the electron acceptor to yield nitrogen . Schmautz et al.  reported high numbers of unclassified planctomycetes in a lettuce-tilapia aquaponic system.
At the genus level, there were relatively high numbers of Arenimonas and Flavobacterium enriched in the lettuce roots in both the control and the Bacillus-treatments. The enrichment of Arenimonas could possibly be due to their versatile abilities to utilize root metabolites, degrade aromatic compounds, and produce anti-microbial substances. Li et al.  found Arenimonas to appear at early stages of succession, indicating that they may be copiotrophic and fast-growing bacteria, which are able to exploit a transient niche for nutrition at the early growth stage.
It has been reported that the abundance of the genus Flavobacterium is often associated with the capacity to degrade complex organic compounds . In nature, Flavobacterium is known to mineralize organic substrates (e.g., carbohydrates, amino acids, and proteins) and degrade organic matter and some organisms (bacteria, fungi, and insects) using a variety of enzymes .
Thermomonas was among the genus that was most influenced by Bacillus addition to the systems. The relative abundance of this group was 1.72% and 7.70% of the total genus-defined root-associated bacteria in control samples and Bacillus-treated samples, respectively. Thermomonas spp. are filamentous aerobic chemoorganotrophs that do not reduce nitrate to nitrite and do not usually utilize carbohydrates, suggesting the presence of several organic compounds in the aquaponics environment.
Wongkiew et al.  also reported dominant OTUs belonging to Thermomonas spp. in plant roots of aquaponic systems. Thermomonas has been previously demonstrated to play a role in denitrification, but not all denitrifiers can perform complete denitrification by reducing nitrate to molecular nitrogen. Some of them lack critical enzymes to reduce nitrate to nitrite, nitrite to nitric oxide, or nitrous oxide to nitrogen gas . This may explain the gradual increase of nitrate in Bacillus-treated systems. Further investigations are needed to determine if Thermomonas can be considered true denitrifiers.
In our study, a relatively high proportion of reads (2.9%) from the plant roots was assigned to the genus Bacillus and (0.08%) to Pseudomonas in samples from Bacillus-treated systems, indicating that lettuce plants could have selected a community that is able to perform inherent biocontrol on its roots. Schmautz et al.  reported similar findings with lettuce plants in aquaponics systems integrated with tilapia, although with different relative abundances.
However, the potential of plant root communities for the inherent biocontrol of plant pathogens in aquaponics is not yet known. A large group of Pseudomonas spp. and Bacillus spp. have been reported to produce antimicrobial compounds that can inhibit the growth of a wide range of pathogens . It has been reported that Bacillus spp. and Pseudomonas spp. possess large gene clusters involved in detoxification, as well as the production of antibiotics and siderophores [14,57].
Bacillus-treated systems were characterized by a significantly higher abundance of bacteria assigned to Chryseobacterium, Nitrospira, and Cloacibacterium. It has been demonstrated that siderophore production by Chryseobacterium spp. alleviates iron starvation in tomato plants . We identified Nitrospira as the dominant nitrite-oxidizing bacteria (NOB).
A similar observation was reported by Schmautz et al.  and Eck et al. , demonstrating Nitrospira as the most prevalent genus among the known nitrifiers in aquaponic systems. Nitrospira is generally considered K-strategists NOB, favoring oligotrophic environments . The abundance of Nitrospira in Bacillus-treated systems shows a versatile metabolic network driving nitrification in biofilters of aquaponics systems.
For instance, nitrite-oxidizing Nitrospira spp. possess a diverse array of metabolic pathways, such as complete ammonium oxidation , hydrolysis of urea, and cyanate to ammonia, thereby initiating nitrification [59,61]. This metabolic versatility enables Nitrospira to adapt to different environments characterized by low NH4+ and NO2− concentrations . Whether Nitrospira in aquaponic systems transforms complete nitrification through these alternate pathways requires further research.
The study has demonstrated the value of Bacillus supplementation in aquaponic systems. The addition of a commercial mixture of Bacillus spp. increased lettuce growth in two independent growth trials compared to control systems with no supplementation. The addition of the probiotic also increased the relative levels of phosphate and nitrate, which very likely contributed to the increased lettuce growth in the Bacillus-treated systems.
Bacillus supplementation resulted in significant changes in the composition of bacterial communities associated with lettuce roots. We found that several members of the bacterial phyla Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were more abundant in the Bacillus-treated systems than in the control systems.
Our study also found that Bacillus-treated systems were characterized by a significantly higher abundance of bacterial genera assigned to Thermomonas, Pseudomonas, Bacillus, Chryseobacterium, Nitrospira, and Cloacibacterium. From this study, we can conclude that the positive effects (lettuce growth, increased levels of phosphorus, and nitrate) observed in systems treated with the Bacillus mixture might be due to bacterial activity, although this will require further investigation.
Source: Kasozi, N., Kaiser, H., & Wilhelmi, B. (2021). Effect of Bacillus spp. on Lettuce Growth and Root Associated Bacterial Community in a Small-Scale Aquaponics System. Agronomy, 11(5), 947.