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Article

Could Hydroinfiltrators Made with Biochar Modify the Soil Microbiome? A Strategy of Soil Nature-Based Solution for Smart Agriculture

by
Azahara Navarro
1,
Ana del Moral
2,*,
Gabriel Delgado
1,
Jesús Párraga
1,
José Ángel Rufián
3,
Raúl Rojano
1 and
Juan Manuel Martín-García
1,4
1
Department of Soil Science, University of Granada, 18071 Granada, Spain
2
Department of Microbiology, University of Granada, 18071 Granada, Spain
3
Department of Nutrition and Bromatology, University of Granada, 18071 Granada, Spain
4
Institute of Research in Olive Groves and Olive Oil, University of Jaen, 23071 Jaen, Spain
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(15), 8503; https://doi.org/10.3390/app15158503 (registering DOI)
Submission received: 4 July 2025 / Revised: 22 July 2025 / Accepted: 28 July 2025 / Published: 31 July 2025
(This article belongs to the Special Issue The Soil Microbiome: Diversity, Genomics and Potential Applications in Climate Smart Agriculture)
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Abstract

Featured Application

The use of a biochar hydroinfiltrator could enhance water infiltration and, therefore, the availability for olive tree growth—as demonstrated in previous studies—but the influence on soil microbiota has never been previously described and will be proven in this study.

Abstract

Climate change negatively affects agriculture, causing desertification, salinisation, and drought. The biochar hydroinfiltrator (ES Patent No.: ES2793448 B2) is a device that increases the capture of rainwater or irrigation water for crops by increasing infiltration rates. Biochar, produced via biomass pyrolysis, has emerged as a promising agricultural amendment, as it helps to optimise moisture retention and improve soil structure, key aspects for boosting crop yields. There is growing interest in microorganisms’ plant-growth-promoting activity (PGP) by carrying out different activities considered growth promoters. The aim of the present study is to evaluate the use of a biochar hydroinfiltrator as a promoter of microbial activity when it is used in soil. Metagenomic analysis of soils with and without the device reveals that genera Bacillus and Sphingomonas became particularly enriched in soils with hydroinfiltrators. Also, in order to understand the interaction between the uses of biochar together with bacteria PGP, an in vitro test was carried out. Two microorganisms, previously selected for their characteristics as plant growth promoters, were inoculated in soils with and without biochar and they grew better after 15 to 30 days of inoculation, showing major CFU counts. This combined strategy—biochar hydroinfiltrator and PGP bacteria—offers an innovative, eco-friendly approach to sustainable agriculture, particularly under drought stress.

1. Introduction

Soil constitutes a complex ecosystem whose functionality is largely dependent on its own microbiota. This microbial community can be influenced by environmental factors such as climate, moisture levels and soil physicochemical properties.
In the Mediterranean region, climate change is exacerbating drought conditions, reducing annual precipitation and raising temperatures, thereby intensifying water scarcity. This climatic situation not only threatens agricultural productivity but also accelerates some processes involving desertification, soil salinisation and prolonged drought periods. In this context, sustainable agricultural practices, enhanced irrigation efficiency and technological innovation are essential tools [1].
Under the current climate scenario, there is a pressing need to develop sustainable strategies to increase crop productivity and restore degraded soils, which are environmentally friendly strategies. To achieve this goal, one feasible strategy might be the installation of hydroinfiltrators in agricultural soils, a cutting-edge and nature-based device composed of biochar [2].
Biochar is a carbon-rich material produced by heating biomass at temperatures above 250 °C under limited or no oxygen supply through a thermochemical process known as pyrolysis or charring, which is also used in the production of charcoal [3]. However, unlike charcoal, it is specifically produced for use as soil amendment or in broader environmental management. A key feature of biochar is its high content of stable organic carbon, largely in the form of fused aromatic ring structures, formed during pyrolysis and crucial in the mineralisation and adsorption properties. Consequently, biochar typically contains high levels of carbon, and it is often enriched in phosphorus (P), as well as metals like calcium (Ca), magnesium (Mg), and occasionally nitrogen (N). Biochar may offer several benefits improving soil structure, enhancing microbial activity and contributing to the reduction in greenhouse gas emissions [4].
The influence of biochar on soil microorganisms is diverse and complex. According to several studies [5,6], biochar can affect microorganisms both directly through potential toxicity and the release of volatile organic compounds (VOCs), as well as indirectly, by modifying soil physicochemical properties, altering nutrient availability and influencing enzymatic activity [4,7].
Plant-growth-promoting bacteria (PGPB) comprise a wide variety of microorganisms with the ability to colonise plants, promoting their growth through both direct and indirect mechanisms. Direct mechanisms include the supply of essential nutrients (N, P, Fe…) and the synthesis of phytohormones (auxins, cytokinins, gibberellins, ethylene…) [8]. On the other hand, indirect mechanisms may enhance plant protection against pathogens through the secretion of diverse substances (hydrolytic enzymes, siderophores, antibiotics, volatile compounds…) and the induction of systemic resistance, among other processes [9,10].
Under saline conditions, halotolerant PGP bacteria exhibit a selective advantage: they can maintain their active metabolism, produce beneficial compounds, and promote plant adaptation by accumulating osmoprotectants, antioxidants, and protective enzymes [1,11]. Studies involving Bacillus, Pseudomonas, Azospirillum, and Halomonas strains have reported salt tolerance enhancement in crops such as wheat, rice, and tomato [8]. Additionally, these studies have also shown other advantages such as improved root development, water retention, higher chlorophyll content, and larger biomass production [1,12].
Another approach involves the use of biochar as an organic amendment in combination with the application of plant-growth-promoting bacteria, representing an innovative and effective alternative. Both strategies, when combined, can act synergistically to mitigate the effects of salt stress, improve soil physicochemical properties and reactivate its microbiota [13].
The aim of the present study is to evaluate the use of hydroinfiltrators and biochar as promoters of microbial activity when used as water-harvesting systems in soils. To achieve this objective, we propose two different assays. The first is an in vivo experiment focused on the metagenomics analysis of the microbial diversity in two soils, with and without a biochar hydroinfiltrator, at two different depths (20 and 40 cm) from the same rainfed olive grove. The second is an in vitro assay that examines the direct effect of biochar on two PGP microorganisms inoculated in samples from the same agricultural soil amended with different proportions of biochar.
The interest of this study lies in the fact that most Andalusian olive grove soils are in an advanced state of degradation, with low biological activity due to the effects of climate change and increasing drought conditions. The use of a biochar hydroinfiltrator could enhance water infiltration and, therefore, the availability for olive tree growth—as demonstrated in previous studies—and promote microbial species with benefits for the soil–plant system, as will be proven in this study.

2. Materials and Methods

2.1. Sampling Site and Soil

The experimental plot for dryland olive grove is located in Baena (Córdoba, southern Spain) in “La Agusadera” area (37°26′42.07″; 4°20′57.33″) (Figure 1).
The soil is characterised by a clay–loam texture, low organic carbon content (1.26%), basic pH (8.3), non-saline conditions (0.46 dS/m) and a cation exchange capacity of 44.8 cmol+ kg−1. It is classified as Haplic Calcisol [14].
The hydroinfiltrator is a cylindrical mesh-like shell structure primarily filled with biochar—obtained from oak wood and olive pruning waste—which was semi-buried, in 2020, next to the tree (or shrub) to capture and retain rainwater or irrigation water in the root zone, thereby enhancing infiltration and reducing losses due to evaporation or runoff [2,15]. Furthermore, it can be used in conjunction with fertilisers or advanced irrigation systems, increasing water use efficiency and olive grove production. This system also contributes to soil improvement and carbon sequestration, representing a sustainable tool in the context of climate change.
The characteristics of the studied plot, soil properties and olive grove management practices have been previously described by Rojano-Cruz et al. (2023) [2].

2.2. Metagenomic Analysis of Soil at Different Depths near Olive Trees with and Without Biochar Hydroinfiltrators

Four soil samples were collected 20 cm away from the hydroinfiltrator (soil material surrounding the hydroinfiltrator) or equivalent position in control trees without the hydroinfiltrator, which was installed 0.5 m upstream from the base of each olive tree. Samples were taken in October 2022: SC20 (control soil at 20 cm depth), SC40 (control soil at 40 cm depth), SH20 (soil with hydroinfiltrator at 20 cm depth) and SH40 (soil with hydroinfiltrator at 40 cm depth).

2.2.1. DNA Extraction

The microbial DNA of each sample was obtained from 25 g of soil (Table A1 in Appendix A). The soil was added to a Lysis Matrix E tube with 1 mL of CTAB solution and shaken twice in a FastPrep homogeniser at 5.5 m/s for 30 s. After that, 40 µL of proteinase K was added and incubated for 10 min at 70 °C. The sample was centrifuged for 5 min at 12,000 rpm and 300 µL of supernatant was recovered. The DNA was then purified using the PureFood GMO extraction and Authentication Kit on a Maxwell RSC robot (Promega; Fisabio, Valencia, Spain).

2.2.2. Sequencing of 16S rRNA Gene Amplicons

To obtain amplicons from the V3-V4 region of the 16S rRNA gene and prepare sequencing libraries, the Illumina protocol (16S Metagenomic Sequencing Library Preparation, Cod 15044223 RevA; Fisabio, Valencia, Spain) was followed, with slight modifications due to the low initial DNA quantity (15 μL of amplicons and 12 amplification cycles were used in the index PCR, which is performed to label the amplicons with dual adapters and indexes that allow for multiplex sequencing). Sequencing of the indexed amplicons was carried out on an Illumina MiSeq sequencer using the MiSeqReagent v3 kit in a 2 × 300-cycle run (Fisabio, Valencia, Spain).
Meta-taxonomic analysis of the sequences was performed with qiime2 [16]. Noise reduction, paired-end joining, and chimera removal were performed using DADA2 [17]. The Silva138 database was used for taxonomic assignment [18].

2.2.3. Indexes of Bacterial Diversity

Venn diagrams were obtained by using Venny v.2.1 tool [19]. Rarefaction curves were employed to check the depth level and the quality of the metagenomics analysis [20]. Alpha diversity comprised the Shannon (H′) and the complementary Simpson (1-D) indexes. Beta diversity included the Jaccard (IJ) and the Whittaker indexes (IW). Both alpha and betta diversity indexes were calculated with PAST v.4.16c software [21].

2.3. Microbiological Study Under In Vitro Conditions

2.3.1. Bacterial Strains

A total of 8 halotolerant plant-growth-promoting bacteria, from the microbial collection of the BIO 188 research group (https://www.bio188.es/ accessed on 23 May 2025), were used. These microorganisms had been isolated from different origins and locations (Table A1).

2.3.2. Culture Media and Conditions

TSA and LB media were used for the growth, maintenance and determination of the possible enzymatic activities of the microorganisms (Table S1).

2.3.3. Tolerance Tests and Determination of Enzymatic Activities

To determine salt tolerance, bacteria were grown in LB medium with different concentrations of NaCl (1.0; 1.5; 2.0; 3.5; 6.0; 7.5; 10.0; 15.0; and 20.0%). After that, a volume of 10 µL from each inoculum was poured on a plate and incubated at 28 °C for 24–48 h.
Bacterial tolerance to water stress was evaluated by using polyethylene glycol (PEG) 8000, a stress-inducing agent. For doing so, bacteria were inoculated into TSB 10% (w/v) and supplemented with PEG at different concentrations (0; 5.0; 10.0; and 15.0% (w/v)). Inoculates were incubated at 28 °C, under shaking conditions (120 rpm) for 72 h. Bacterial growth was then determined by optical density measurements (OD600).
Different culture media were used to evaluate the following enzyme activities (Table S1): Nase in N-free Burk’s medium, basic Burk’s medium with 1-aminocyclopropane acid (ACC) deaminase, acid phosphatase, alkaline phosphatase, phytase, caseinase, and amylase.

2.3.4. Experiments with Substrates of Soil and Biochar

Test tubes were prepared with different proportions of soil (fine earth fraction) and biochar: 100% soil (S), soil and biochar in weight/weight ratios of 90/10% (SB10) and 80/20% (SB20), and 100% biochar (B) (Figure 2). The soil material used was the arable layer (first 20 cm) of control soil. The biochar used was a commercial charcoal sieved to a size minor than 0.5 mm. A gram of soil, biochar, or soil/biochar mixture was added to each tube, being subsequently sterilised in an autoclave (112 °C, 0.5 atm, 20 min). The sterilisation process was repeated three times, with a 24 h incubation period between each one, to remove resisting spore-forming bacteria.
The moisture content of each treatment was determined at field capacity using Richard’s membrane and gravimetric weight before and after incubation at 105 °C for 8 h (Table 1). These moisture values were used to determine the amount of water (inoculum) to be added to soil, biochar, and soil/biochar mixtures to maintain the best moisture conditions (field capacity).
Strains 11C and 15C, with the best PGP activity under water stress and salinity conditions, were chosen for this test. Both bacteria were grown in LB and incubated at 28 °C for 24 h, under shaking conditions. After that, test tubes containing sterilised substrates of soil, biochar, and soil/biochar mixtures were inoculated with each strain. The inoculum quantity was calculated according to field moisture capacity values (Table 1). The same procedure was followed for control tubes, using sterile distilled water instead of the inoculum. Each experiment was performed twice.
After inoculation, test tubes containing different substrates were incubated in a climate chamber at 28 °C and 50% humidity.
The effect of biochar on bacterial growth was evaluated by microbial counting in the inoculum (time 0) and after different time periods (15, 30, and 60 days).
Microorganisms were recovered from the test tubes by adding 2 mL of saline solution, shaking vigorously, and gently stirring for one hour. The test tubes were allowed to stand for 15 min and the microorganisms suspended in the saline solution were subsequently recovered from the supernatant (Figure 2).

3. Results and Discussion

3.1. Metagenomic Analysis of Soil Samples with and Without Hydroinfiltrators

A total of 39 phyla and 790 genera were detected (Table S2). The rarefaction curves obtained from these data reached an asymptote, indicating that the sequencing depth was sufficient and adequate for all samples (Figure 3). Thus, the saturation achieved suggests that the representation of microbial diversity is reliable. Sample SH40 exhibited the highest number of taxa (S) and sequences (specimens). Samples SH20 and SC40 showed intermediate values: the number of taxa (S) was similar in both samples, while the number of sequences was higher in SC40. The lowest values were observed in sample SC20.
The Venn diagram shows that the four samples shared 107 genera (Figure 4). A comparative analysis reveals that the number of exclusive genera shared between hydroinfiltrator soil samples (SH20-SH40 = 132) is considerably higher than in the control soil samples (SC20-SC40 = 7). The highest number of specific genera was found in sample SH40 (268), followed by sample SC40 (66); both samples were taken at a depth of 40 cm. In contrast, the number of unique genera was much lower in samples SC20 (10) and SH20 (2), both collected at a depth of 20 cm.
The alpha diversity indices reveal a high genus-level diversity across all samples (Figure A1). According to the Shannon index (H′), the highest value was observed in sample SH40 (5.084) and the lowest in sample SC20 (4.517), corroborating the results obtained from the rarefaction curves (Figure 3). The complementary Simpson index (1-D) showed the highest value in sample SC40 (0.9851), although this result is not very significant because the rest of samples had similar values (≈0.98).
The beta diversity indices reflect the heterogeneity of the bacterial community among the samples (Table 2). The Jaccard index (IJ) indicates that similarity between samples ranges from moderate (≈0.50) to low (≈0.25). The highest value was found for the SH20-SH40 pair (0.465) and the lowest for the SC20-SH40 pair (0.253). Similarly, the Whittaker index (IW) shows that the turnover rate or dissimilarity between samples ranges from moderate (0.25–0.5) to moderately high (0.5–0.75). In this case, the highest value was also observed in the SC20-SH40 pair (0.596) and the lowest in the SH20-SH40 pair (0.366), supporting the values obtained with the Jaccard index.
The most abundant phyla were Pseudomonadota (29%), Actinomycetota (18%), Bacillota (15%), Acidobacteriota (9%), and Bacteroidota (6%), according to the average relative abundance across samples (Figure 5). These results are consistent with previous studies on soil microbial communities [22].
The comparative analysis of control soils and soils with a hydroinfiltrator revealed notable differences (Figure 5). The phyla Acidobacteriota Cyanobacteriota 4, Pseudomonadota 36, and Gemmatimonadota 3.7 were more abundant in SC20 soils (control), while Bacillota (23.8%), Planctomycetota (9.2%) and Chloroflexota (4.8%) predominated in SH20 soils (hydroinfiltrator). Additionally, the phyla Actinomycetota (26%), Bacillota (23%) and Acidobacteriota (11%) were enriched in SC40 soils (control), whereas others such as Pseudomonadota (33%) and Bacteroidota (5.7%) were more abundant in SH40 soils (hydroinfiltrator).
The bacterial community also exhibits differences according to soil depth (Figure 5). In control soils, the phylum Pseudomonadota (36.6%) was more abundant in SC20 (20 cm), while other phyla such as Actynomycetota (26.2%) and Bacillota (23.8%) were more prevalent in SC40 (40 cm). In soils with hydroinfiltrators, the phylum Bacillota (16%) was more abundant in SH40, while other phyla such as Planctomycetota (9.2%) were enriched in SH20.
In general, soil microorganisms belong mainly to the phyla Acidobacteriota, Actinomycetota, Bacteroidota, Bacillota, and Pseudomonadota [22]. The use of biochar as a soil amendment induces phylum-level shifts in soil microbial communities. Kolthon et al. (2011) [23], in a study on cucumbers grown with biochar in greenhouse conditions, observed that the dominant phyla were Pseudomonadota, Bacteroidota, Actinomycetota, and Bacillota, both in control soil and soil/biochar samples. However, these authors reported that biochar application increased the relative abundance of certain phyla, such as Bacteroidota and Actinomycetota, while negatively affecting others, such as Pseudomonadota. Similarly, Sun et al. (2013) [24], in a soybean study, observed enrichment of Acidobacteriota, Gemmatimonadota, Pseudomonadota and Actinomycetota after biochar application. In turn, Hu et al. (2014) [25], in a study of forest soils in China, reported that Pseudomonadota, Actinomycetota and Acidobacteriota were the predominant phyla in soils amended with biochar.
In general, soil depth is a key factor in the structure of bacterial communities [26]. However, in our study, at the genus level, soil with the hydroinfiltrators did not show substantial shifts in populations across different depths (Figure 6 and Table S2), as corroborated by the beta diversity indices (Table 2). This finding will be discussed further below.
At the genus level, the most abundant were Bacillus (6.0%) and Sphingomonas (4.7% based on the average relative abundance (Figure 6). Among the major genera, others such as Blastococcus, (1.8%) Microvirga (1.7%) and Skermanella (1.6%) were also found. Additionally, the presence of uncultured genera and others corresponding to unclassified environmental taxa was also noteworthy.
The composition of the bacterial community at the genus level was clearly influenced by the use of hydrofiltrators in the soil (Figure 6). According to our results, Bacillus (6.0%) and Sphingomonas (4.7%) showed the greatest proportions in soils with hydrofiltrators. Other genera such as Blastococcus (1.8%), Microvirga (1.7%), and Skermanella (1.6%) were also positively affected, although to a lesser extent. Other studies also reported a positive effect on bacterial populations after biochar application, including Bacillus [27,28], Sphingomonas [29,30], Blastococcus [31,32] and Microvirga [33,34,35].
As previously stated, Bacillus and Sphingomonas were the dominant genera [33,34] in soils with hydroinfiltrators (SH20 and SH40) (Figure 6), showing substantial differences in their abundance when compared to the control soils. Both genera were considered beneficial for plant growth, as will be discussed henceforth.
Bacillus is one of the most abundant genera in soil biota and plays multiple relevant functions in ecosystems [22,36]. Bacillus spp. actively participated in biogeochemical cycles and enhanced nutrient availability. Certain species possess the ability to fix atmospheric nitrogen (diazotrophs), producing NH3 available for plants [37,38,39]. Others can solubilise P [40] and several nutrients such as potassium and zinc [41,42,43]. In addition, many species contribute to the production of plant phytohormones and act as plant-growth-promoting rhizobacteria (PGPR) [44,45]. Bacillus spp. also play a key role as a biocontrol agents and in the bioremediation of soils contaminated with heavy metals through the production of siderophores and other metabolites [46,47].
The proportion of Sphingomonas was also predominant in soils with hydroinfiltrators (Figure 6), although differences were observed according to soil depth. Its proportion is somewhat lower in soils with hydroinfiltrators (SH20) (5.5%) compared to the control soils at the same depth (SC20) (6.5%). However, the relative abundance increased considerably in soils with hydroinfiltrators (SH40) (6.0 5) compared to the control soils, both collected at 40 cm (SC40) (0.9%). The genus Sphingomonas actively participates in N and P biogeochemical cycles [48]. Some species facilitate P solubilisation [49], while others contribute to nitrogen fixation [50]. Additionally, some cases involving the bioremediation of contaminated soils and the degradation of polycyclic aromatic hydrocarbons (PAH) have also been described [51,52]. Furthermore, certain strains are considered extremophiles due to their tolerance to drought, high salinity, and elevated heavy metal concentrations [53,54].
Steroidobacter and Blastococcus also showed the highest relative abundance in soil with hydroinfiltrators (Figure 6). The presence of Steroidobacter was larger in soils with hydroinfiltrators compared to control soils, with higher abundance at 20 cm depth (SH20) (2.4%) than at 40 cm (SH40) (1.5%). Similarly, Blastococcus showed a greater abundance in soil with hydroinfiltrators only at 40 cm depth (SH40 vs. SC40) (1.4% vs. 0.13%), although its presence slightly decreased to 20 cm (SH20 vs. SC20) (2.3% vs. 3.2%).
Microorganisms belonging to Blastoccocus perform various beneficial functions for soil and plants. This genus can promote the bioremediation of contaminated soils [55]. Some species are involved in the degradation of organic substrates [56], while others exhibit PGP properties [57]. Additionally, certain strains are resistant to extreme conditions of drought, salinity [58], and alkalinity [59], and demonstrate broad tolerance to heavy metals [60]. Similarly, the genus Steroidobacter is also effective in the bioremediation of contaminated soils [61]. Some species play a key role in the degradation of PAH, while others are efficient in the uptake of heavy metals [62].
The use of hydroinfiltrators, on the other hand, has a negative impact on the development of some soil bacterial populations. The relative abundances of Cutibacterium (1.3%), Ramlibacter (1.2%), Blautia (1.0%) and Weissella (1.4%) decreased in soil with the hydroinfiltrators (Figure 6). Specifically, for Ramlibacter, its proportion was considerably lower in SH20 soils (0.1%) compared to SC20 4.5 soils (4.5%). However, at 40 cm depth, similar proportions were observed between SH40 (0.19) and SC40 (0.1%). A negative effect was also noted on some genera of non-culturable bacteria and others corresponding to unassigned environmental taxa. Nevertheless, these findings differ from other studies involving the application of biochar to soil, reporting beneficial effects for several genera such as Cutibacterium [63] and Ramlibacter [33].
The genus Ramlibacter is involved in P solubilisation, and precipitation processes and exhibits PGP properties [64,65]. Some species promote the degradation of organic compounds (e.g., PAHs) and participate in the bioremediation of contaminated soils [33,66]. Additionally, certain strains are tolerant to stress conditions such as high temperature, aridity, and nutrient scarcity [67].
As previously mentioned, soil depth is a key factor influencing the structure of bacterial communities, although this effect is less pronounced in soil with the hydroinfiltrators (Figure 6). However, the genera Skermanella and Microvirga are notably more abundant in SH20 soils compared to SH40 soils (Figure 6). These genera are actively involved in the nitrogen cycle and are capable of degrading organic compounds [68,69,70,71]. In addition, Skermanella also participates in the P cycle through the production of alkaline phosphatase [72,73].
The results from control soils indicate more pronounced shifts in bacterial community structure with depth (Figure 6). The genera Sphingomonas (6.6%), Ramlibacter (4.5%) and Blastococcus (3.3%) were enriched in SC20 soils, taken at a depth of 20 cm. Similarly, a moderate increase in the abundance of other genera such as Microvirga (3.2%) and Skermanella (2.8%) was also observed.
Although not displayed in Figure 6 due to its low average relative abundance (<1%), Aquicella was found in an appropriate proportion of around 2% in SH40 soils (Table S2). This genus is typically associated with aquatic environments but has occasionally been detected in humid soils or in relation to irrigation water [74]. Although not the primary focus of this study, Aquicella is highlighted here as its presence supports that hydroinfiltrators increases soil water content at depth, a remarkable aspect for olive groves.

3.2. In Vitro Microbiological Study

3.2.1. Strain Selection Based on Tolerance Tests and Enzymatic Activities

The range of salts tested and the NaCl tolerance of the eight bacterial strains studied are shown in Table A2. Strains P6 and B38 were classified as moderately halophilic microorganisms, whereas the remaining strains were halotolerant.
Considering water stress tolerance (Table A3), strains N3, B23, and B38 exhibited the highest growth at low PEG concentrations (0–15%). In contrast, strains 8C, 11C, 14C, and 15C demonstrated a broader tolerance range, as they were able to grow—albeit weakly—even at PEG concentrations up to 25%.
The enzymatic activities related to PGP properties are shown in Table A4. Strains B23, 8C, 11C, 14C and 15C showed positive results for five out of seven biochemical tests, suggesting the highest PGP properties. These strains belong to the genera Kushneria (B23), Bacillus (8C, 11C and 14C) and Pseudomonas (15C). Previous studies have reported PGP activity in the genera Bacillus and Pseudomonas, supporting our results [75,76].
Of the five aforementioned strains, Gram-positive (11C, Bacillus cabrialesii) and Gram-negative bacteria (15C, Pseudomonas neuropathica) were selected to perform growth tests with soil/biochar substrates (described in Section 2.3.4.). According to de Santos-Villalobos et al. (2023) [77], Bacillus cabrialesii is involved in phosphate solubilisation in soil, increases chlorophyll content in plants, and can grow under saline and water stress conditions. Additionally, these authors highlight its Nase and ACC deaminase activities, as well as the production of hydrolytic enzymes such as amylase and caseinase. Although no previous studies have demonstrated PGP activity in Pseudomonas neuropathica, it was selected for this assay due to its broad tolerance to salinity and water stress, as well as its enzymatic activity (Table A2, Table A3 and Table A4 in Appendix A)

3.2.2. Experiments with Substrates of Soil and Biochar

The results of growth assays for the two selected strains (11C and 15C) on different substrates with varying soil/biochar ratios will be discussed below. The proportions used are those indicated in Table 1 (Section 2.3.4).
For strain 11C at 15 days, the count in SB10 was 3.92 × 104 CFU and in SB20 was 2.42 × 104 CFU (Figure 7). At the same time, substrates S and B yielded very similar counts, around 1.02–1.04 × 104 CFU. After 30 days of incubation, bacterial growth was also observed, but with differences depending on the substrate: SB10 showed the highest count at 1.4 × 104 CFU, followed by B with 1.11 × 104 CFU. After 60 days, a significant decline in microbial count was observed across all substrates, likely due to nutrient depletion. Although the behaviour of Bacillus cabrialesii at 60 days has not been specifically described in the literature, some Bacillus strains have been reported to exhibit population decreases in soil under low-nutrient conditions [78].
For strain 15C, the highest counts were also recorded after 15 days of incubation across all substrates (Figure 7). The highest value (1.787 × 104 CFU) corresponded to substrate B. Elevated counts (8.2 × 103 CFU) were also observed in substrate SB20. After 30 days, the counts were reduced by approximately by half in all substrates compared to those observed after 15 days. In this case, substrate B again yielded the best results, with 8.56 × 103 CFU. Over the same period, SB substrates also supported bacterial growth, though to a lesser extent. In contrast, in soil without biochar (substrate S), no bacterial growth was detected after 30 days. After 60 days, as observed with strain 11C, bacterial growth was negligible. This decline might be attributed again to nutrient depletion.
Bacterial counts for the two strains (Bacillus cabrialesii and Pseudomonas neuropathica) demonstrated their ability to colonise substrates of soil and biochar, with biochar exerting a positive influence on their growth. However, a notable difference was observed: strain 15C growth was considerably higher in substrates with pure biochar (B) at 15 days (805 CFU vs. 17.875 CFU, Figure 7), despite the high toxicity commonly reported for fresh biochar [72,79].
In recent studies, the combined application of biochar inoculated with Bacillus subtilis, nanomaterials, and proline was proven to enhance wheat’s tolerance to salinity [80]. This approach might be promising, as biochar improves soil structure and water retention capacity, while PGP bacteria facilitate nutrient uptake and modulate plant hormonal responses (e.g., ethylene). The use of this biochar/PGP bacteria amendment can modulate the hormonal signalling pathways, thereby improving plants’ ability to withstand water and salt stress related to drought and salinity [81].
However, our growth trial was conducted under in vitro conditions, with specific moisture conditions and in the absence of other biotic and abiotic factors typically encountered in agricultural soils (in vivo). Consequently, further research focused on field and plant experiments is required.

3.3. Influence of Biochar Hydroinfiltrators on Microbial Communities

Our results confirm that the use of hydroinfiltrators combined with biochar produces significant effects on the structure and diversity of soil microbial communities (Figure A1 and Table 2). This is consistent with previous research pointing out the multifactorial impact of biochar on soil ecosystems. Biochar is composed of recalcitrant carbon, exhibits a low mineralisation rate and contains essential plant nutrients such as P, K, S, Mg, Ca, and micronutrients [82,83]. The surface of biochar contains multiple negatively charged sites (OH-, COOH-, phenolic compounds, etc.) that enhance its adsorption capacity at neutral pH, increase electrical conductivity and cation exchange capacity [84]. Its porous structure, high specific surface area, and nutrient retention capacity promote the survival and activity of beneficial soil bacteria, which can lead to improvements in soil fertility and crop productivity [85,86]. The use of biochar in combination with bacterial inoculation should be considered as a climate change adaptation strategy in agriculture [87].
Metagenomic analysis revealed an increase in diversity and functional shifts associated with the presence of the hydroinfiltrator (Figure 6 and Figure 7), particularly within the charosphere zone, where multiple biogeochemical processes occur [88,89,90]. Alterations in key parameters such as pH, porosity and nutrient availability in this zone [91] could explain the expansion of ecological niches for new bacterial genera, as also reported in previous works [92,93].
The observed bacterial colonisation aligns with the mechanisms described by Gorotsov et al. (2020) [7], in which the quality and dose of biochar, along with the soil type, determine the rate and selectivity of microbial settlement. Our results indicate selective colonisation by genera such as Sphingomonas and Bacillus (Figure 6), both with physiological abilities including active motility, exopolysaccharide (EPS) production, and biofilm formation [53,94,95]. In particular, the increase in Sphingomonas in SH40 soil compared to SC40 soil could be related to its motility and adhesion capabilities mediated by structures such as flagella and EPS, as described by Halverson (2005) [96] and Rummel et al. (2017) [97].
In contrast, the decrease in Weissella in SH40, despite also being an EPS-producing genus [98], suggests that it may have been displaced by other genera with better functional adaptability, such as Sphingomonas (Figure 6). This type of competition has been described as a key process in microbial structuring under biochar-modified conditions [96].
Changes in aerobic and anaerobic conditions were also noticeable. Our results revealed a reduction in anaerobic genera (Cutibacterium, Blautia) and facultative anaerobes (Weissella) in soils with a hydroinfiltrator (Figure 6). The anaerobic and facultative anaerobic nature of the three genera mentioned has been described by some authors [98,99,100]. The effect of the hydroinfiltrator aligns with the findings of Lehman & Joseph (2015) [4], who point out that biochar improves soil aeration and, therefore, favours the development of aerobic microbial communities. Consequently, the soil environment becomes less favourable for strictly anaerobic microorganisms, being consistent with the results obtained.
Regarding the toxicity of biochar, some authors have reported that its alkalinity may reduce or inhibit soil microbial activity in the short term due to its toxic effects [79]. According to our results, biochar does not exert an inhibitory effect across all bacterial populations, but instead, has a select effect for resistant taxa such as Bacillus, Sphingomonas, Blastococcus, and Steroidobacter (Figure 6), as documented in previous studies [54,55,61,62]. Other adaptive advantages would be accounted for the tolerance to heavy metals and the ability to degrade toxic compounds, including polycyclic aromatic hydrocarbons (PAHs), for these microorganisms [52,62].
On the other hand, the increase in Bacillus and Sphingomonas in soils with hydroinfiltrators could also be associated with the use of resources released from the death of microorganisms more sensitive to the initial toxicity of fresh biochar, as proposed in other studies [7,71]. Additionally, biofilm formation is also promoted under these conditions, facilitating microbial cooperation via quorum sensing and improving environmental resistance [95,100,101].
Under environmental stress conditions, the use of hydroinfiltrators with biochar showed positive effects on microbial diversity, presumably due to improved water retention and nutrient availability, as previously pointed out by some authors [5,76,102]. However, a decrease in the abundance of Ramlibacter (Figure 6), common in arid environments [66], was also observed. This finding would suggest that increased moisture conditions may be unfavourable for certain xerotolerant populations. Nevertheless, other genera, such as Sphingomonas, are able to thrive due to their multiple physiological functions [53,66].
An in vitro assay with halotolerant bacteria highlighted the efficacy of Bacillus cabrialesii compared to Pseudomonas neuropathica (Figure 7), likely due to the high ecological adaptability of Bacillus in saline environments and its ability to maintain key metabolic functions under adverse conditions [21,35]. Studies with strains of Bacillus, Pseudomonas, Azospirillum and Halomonas have also reported improvements in salt tolerance in crops such as wheat, rice and tomato, including enhanced root development, water retention, chlorophyll content and higher biomass production [1,11]. This finding reinforces the use of biochar as a vector for microbial inoculation in saline environments, as previously proposed by several authors [22,27,103]. Under the current climate change scenario, where an increase in soils with aridity and salinity problems is expected, this approach could be a recommended strategy for adaptation.
Finally, the increase in genera such as Microvirga, Skermanella, Bacillus, Sphingomonas, and Blastococcus (Figure 6) indicates that the use of hydroinfiltrators promotes microbial communities functionally involved in N, P, and carbon biogeochemical cycles [29,32,35,70,104,105,106,107]. This pattern aligns with previous studies indicating that biochar can improve ammonium retention, reduce denitrification, and increase P solubilisation [108,109,110,111,112,113].

4. Conclusions

Metagenomic analysis has confirmed the presence of typical phyla commonly found in soil and rhizosphere environments (mainly Pseudomonadota, Actynomycetota, and Bacillota). Notably, the genera Bacillus and Sphingomonas became particularly enriched in soils with hydroinfiltrators. Bacillus is classically recognised as a relevant agent in terrestrial ecosystems, but the prominent occurrence of Sphingomonas, with plant-growth-promoting (PGP) properties, constitutes a novel finding. Differences in genus-level diversity and abundance were observed across soil depth, with effects being more pronounced at 40 cm compared to 20 cm.
An in vitro study with soil and biochar mixtures exhibited an increase in bacterial growth, particularly at 15 days after inoculation, demonstrating a positive influence of biochar. Having confirmed these beneficial effects, we propose the use of biochar hydroinfiltrators, in combination with the inoculation of PGP bacteria into soils, as an innovative and environmentally friendly strategy to mitigate the effects of drought caused by climate change.
To apply these findings in agricultural soils, further research is needed both under laboratory conditions and in the field, where the introduced bacteria would coexist with native microbiota. In that case, the success of the system would depend not only on the type of microorganism but also on soil characteristics, nutrient availability, and environmental conditions. Therefore, it is also essential to continue exploring various combinations of soil, biochar, and bacteria, with the aim of developing new sustainable strategies to improve the health and productivity of soils.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15158503/s1.

Author Contributions

J.P., A.d.M., J.M.M.-G. and G.D. conceived and contributed to experimental design. A.N., contributed to bacterial strains selection. R.R. and A.N. collected soil samples. J.Á.R. contributed to metagenomic and bioinformatic analyses. J.M.M.-G. collaborated in the assays in vitro. A.d.M., A.N., G.D. and J.P. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by PID2023-150154OB-I00 funded by MICIU/AEI/10.13039/501100011033 and by ERDF/EU.

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

Data are available through this paper.

Acknowledgments

The authors gratefully acknowledge Inmaculada Llamas and Inmaculada Sampedro for the loan of the strains studied and for their guidance in the experimental design of the work. During the preparation of this manuscript/study, the authors used ChatGPT’s free version for the purposes of translating the text and improving the style. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Description of the bacterial strains used in this study.
Table A1. Description of the bacterial strains used in this study.
StrainTaxonomic IdentificationLocationOrigin
P6Pseudomonas segetisRhizosphere of Salicornia europeaSaladar El Margen (Granada, Spain)
B38Psychrobacter sp.Endophyte (aerial part) of Salicornia hispanicaSaladar El Margen (Granada, Spain)
B23Kushneria endophyticaEndophyte (aerial part) of ArthrocaulonSaladar El Margen (Granada, Spain)
N3Peribacillus castrilensisOtter faecesCastril (Granada)
8CBacillus siamensisEndophyte (aerial part) of Salicornia europeaSalobral de Ocaña (Toledo, Spain)
11CBacillus cabrialesiiEndophyte (root) of Caroxylon vermiculatumSalobral de Ocaña (Toledo, Spain)
14CBacillus siamensisSaline soilSalobral de Ocaña (Toledo, Spain)
15CPseudomonas neuropathicaSaline soilSalobral de Ocaña (Toledo, Spain)
Table A2. Salt spectrum of the bacterial strains studied *.
Table A2. Salt spectrum of the bacterial strains studied *.
Strains% NaCl
11.523.567.5101520
P6
B38
B23
N3
8C
11C
14C
15C
* The major intensity of grey indicates the highest tolerance to NaCl concentration.
Table A3. Optical density at different PEG concentrations.
Table A3. Optical density at different PEG concentrations.
Strain% Polyethylene Glycol (PEG)
0510152025
P60.7020.6640.5300.65300
N31.1511.2571.2931.06500.035
B231.3431.4551.4181.0800.1050.050
B381.6861.8091.4921.2540.1140.093
8CND1.0750.7360.5710.2220.220
11C1.0990.9480.8690.6650.2140.217
14C0.7650.7220.9770.9750.2430.160
15C0.9130.6970.4630.5450.1590.111
ND: Not detected.
Table A4. Enzymatic activities of the bacterial strains studied.
Table A4. Enzymatic activities of the bacterial strains studied.
StrainNaseACC
Desaminase		Alkaline
Phosphatase		Acid
Phosphatase		FitaseCaseinaseAmilase
P6+−+++−−
B23+−+−+++
B38−−−++−+
N3+−−−+−−
8C++−−+++
11C+−+−+++
14C++−−+++
15C++−++++
Applsci 15 08503 g0a1
Figure A1. Indexes of alpha diversity of the bacterial community of soil samples.
Figure A1. Indexes of alpha diversity of the bacterial community of soil samples.
Applsci 15 08503 g0a1

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Figure 1. Location of the experimental plot in La Agusadera area, near Baena town (Córdoba, Spain).
Figure 1. Location of the experimental plot in La Agusadera area, near Baena town (Córdoba, Spain).
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Figure 2. Experimental design to evaluate bacterial growth in samples with different proportions of soil and biochar. Created in BioRender. M.Lepe.2025.
Figure 2. Experimental design to evaluate bacterial growth in samples with different proportions of soil and biochar. Created in BioRender. M.Lepe.2025.
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Figure 3. Rarefaction curves. Number of taxa (taxa S) vs. number of sequences (specimens) belonging to the bacterial community of soil samples.
Figure 3. Rarefaction curves. Number of taxa (taxa S) vs. number of sequences (specimens) belonging to the bacterial community of soil samples.
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Figure 4. Venn diagram. Number of shared and specific genera in the bacterial community of soil samples.
Figure 4. Venn diagram. Number of shared and specific genera in the bacterial community of soil samples.
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Figure 5. Study of the bacterial community in soil samples. Phyla with an average relative abundance > 1%.
Figure 5. Study of the bacterial community in soil samples. Phyla with an average relative abundance > 1%.
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Figure 6. Study of the bacterial community in soil samples. Genera with an average relative abunance > 1%.
Figure 6. Study of the bacterial community in soil samples. Genera with an average relative abunance > 1%.
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Figure 7. Microbial counts in substrates soil/biochar (CFU) vs. incubation time (days). Legend: S (soil 100%), SB10 (soil/biochar 90/10%), SB20 (soil/biochar 80/20%) and B (biochar 100%). (A) Strain 11C (Bacillus cabrialesii) and (B) strain 15C (Pseudomonas neuropathica).
Figure 7. Microbial counts in substrates soil/biochar (CFU) vs. incubation time (days). Legend: S (soil 100%), SB10 (soil/biochar 90/10%), SB20 (soil/biochar 80/20%) and B (biochar 100%). (A) Strain 11C (Bacillus cabrialesii) and (B) strain 15C (Pseudomonas neuropathica).
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Table 1. Substrates and moisture (%) at field capacity.
Table 1. Substrates and moisture (%) at field capacity.
SampleSubstrateMoisture (%)
SSoil 100%25.5
SB10Soil/biochar 90/10%57.4
SB20Soil/biochar 80/20%53.8
BBiochar 100%60.9
Table 2. Indexes of beta diversity of the bacterial community of soil samples.
Table 2. Indexes of beta diversity of the bacterial community of soil samples.
(a) Jaccard Index (IJ)
SC20SH20SC40SH40
SC201.0000.3320.3900.253
SH20 1.0000.3510.465
SC40 1.0000.338
SH40 1.000
(b) Whittaker Index (IW)
SC20SH20SC40SH40
SC200.0000.5020.4390.596
SH20 0.0000.4810.366
SC40 0.0000.495
SH40 0.000
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Navarro, A.; del Moral, A.; Delgado, G.; Párraga, J.; Rufián, J.Á.; Rojano, R.; Martín-García, J.M. Could Hydroinfiltrators Made with Biochar Modify the Soil Microbiome? A Strategy of Soil Nature-Based Solution for Smart Agriculture. Appl. Sci. 2025, 15, 8503. https://doi.org/10.3390/app15158503

AMA Style

Navarro A, del Moral A, Delgado G, Párraga J, Rufián JÁ, Rojano R, Martín-García JM. Could Hydroinfiltrators Made with Biochar Modify the Soil Microbiome? A Strategy of Soil Nature-Based Solution for Smart Agriculture. Applied Sciences. 2025; 15(15):8503. https://doi.org/10.3390/app15158503

Chicago/Turabian Style

Navarro, Azahara, Ana del Moral, Gabriel Delgado, Jesús Párraga, José Ángel Rufián, Raúl Rojano, and Juan Manuel Martín-García. 2025. "Could Hydroinfiltrators Made with Biochar Modify the Soil Microbiome? A Strategy of Soil Nature-Based Solution for Smart Agriculture" Applied Sciences 15, no. 15: 8503. https://doi.org/10.3390/app15158503

APA Style

Navarro, A., del Moral, A., Delgado, G., Párraga, J., Rufián, J. Á., Rojano, R., & Martín-García, J. M. (2025). Could Hydroinfiltrators Made with Biochar Modify the Soil Microbiome? A Strategy of Soil Nature-Based Solution for Smart Agriculture. Applied Sciences, 15(15), 8503. https://doi.org/10.3390/app15158503

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