Probiotic Bacillus spp. modulate biofilm microbial assembly in Litopenaeus vannamei culture water

Probióticos Bacillus spp. modulam a montagem microbiana do biofilme na água de cultivo de Litopenaeus vannamei

Abstract

Shrimp aquaculture faces persistent challenges arising from microbial imbalances and pathogenic dominance, often linked to biofilm-forming bacterial communities. Probiotics, particularly Bacillus spp., offer a sustainable alternative to antibiotic and chemical management; however, their structural and ecological roles in biofilm modulation remain insufficiently understood. This study applied an integrative approach combining Scanning Electron Microscopy (SEM), 16S rRNA amplicon sequencing, and nutrient monitoring to evaluate the influence of Bacillus-based probiotic supplementation on biofilm communities in Litopenaeus vannamei culture water. Amplicon-based analysis revealed a distinct reconfiguration of bacterial assemblages: untreated biofilms were dominated by Vibrio spp., whereas probiotic supplementation reduced their relative abundance and transiently increased marine-associated genera such as Marivita, Pseudoalteromonas, and Marinobacter during early succession. Bacillus exhibited increased relative abundance under probiotic treatment, coinciding with a shift toward a more compositionally balanced community structure. SEM observations corroborated these findings, showing that probiotic application was associated with less compact and more heterogeneous biofilm architecture. Nutrient analyses demonstrated moderately lower concentrations of ammonia, nitrite, and nitrate in probiotic-treated systems, whereas phosphate concentrations remained consistently low across treatments. Although Bacillus abundance was negatively associated with inorganic nitrogen parameters, functional mechanisms were not directly measured. These findings therefore represent ecological associations rather than mechanistic confirmation of enhanced nutrient turnover. Overall, the results highlight the potential of Bacillus-based probiotics as environmentally compatible biofilm modulators in shrimp aquaculture systems.

Keywords:
Bacillus spp.; biofilm ecology; probiotics; Litopenaeus vannamei; aquaculture sustainability

Resumo

A carcinicultura enfrenta desafios persistentes decorrentes de desequilíbrios microbianos e da dominância de patógenos, frequentemente associados a comunidades bacterianas formadoras de biofilme. Os probióticos, sobretudo os do gênero Bacillus spp., oferecem uma alternativa sustentável ao manejo com antibióticos e produtos químicos; no entanto, seus papéis estruturais e ecológicos na modulação do biofilme ainda não são totalmente compreendidos. Este estudo aplicou uma abordagem integrativa combinando Microscopia Eletrônica de Varredura (MEV), sequenciamento de amplicons de rRNA 16S e monitoramento de nutrientes para avaliar a influência da suplementação com probióticos à base de Bacillus nas comunidades de biofilme na água de cultivo de Litopenaeus vannamei. A análise baseada em amplicons revelou uma reconfiguração distinta dos conjuntos bacterianos: os biofilmes não tratados eram dominados por Vibrio spp., enquanto a suplementação com probióticos reduziu sua abundância relativa e aumentou transitoriamente gêneros associados ao ambiente marinho, como Marivita, Pseudoalteromonas e Marinobacter, durante a sucessão inicial. Bacillus apresentou aumento na abundância relativa sob tratamento com probióticos, coincidindo com uma mudança para uma estrutura comunitária mais equilibrada em termos de composição. Observações por microscopia eletrônica de varredura (MEV) corroboraram esses achados, mostrando que a aplicação de probióticos estava associada a uma arquitetura de biofilme menos compacta e mais heterogênea. Análises de nutrientes demonstraram concentrações moderadamente menores de amônia, nitrito e nitrato em sistemas tratados com probióticos, enquanto as concentrações de fosfato permaneceram consistentemente baixas em todos os tratamentos. Embora a abundância de Bacillus estivesse negativamente associada aos parâmetros de nitrogênio inorgânico, os mecanismos funcionais não foram medidos diretamente. Portanto, esses achados representam associações ecológicas, em vez de confirmação mecanística de uma maior renovação de nutrientes. No geral, os resultados destacam o potencial dos probióticos à base de Bacillus como moduladores de biofilme ambientalmente compatíveis em sistemas de cultivo de camarão.

Palavras-chave:
Bacillus spp.; ecologia de biofilme; probióticos; Litopenaeus vannamei; sustentabilidade na aquicultura

1. Introduction

Shrimp aquaculture has become one of the fastest-growing sectors of global food production and plays a pivotal role in food security and rural economic development. Among cultivated species, the whiteleg shrimp (Litopenaeus vannamei) has emerged as the industry’s cornerstone due to its rapid growth, high reproductive capacity, and tolerance to diverse environmental conditions. These biological attributes have positioned L. vannamei as a key driver of export-oriented economies in Asia and Latin America while contributing to broader sustainability agendas (Eissa et al., 2022; Tran et al., 2022). However, the intensification of farming practices has introduced substantial ecological and managerial challenges. Overstocking, limited water exchange, and high feeding inputs accelerate the accumulation of organic matter and nitrogenous wastes, destabilizing microbial communities and promoting the proliferation of opportunistic pathogens such as Vibrio spp. (Tian et al., 2017; Gunathilake et al., 2022). These microbial imbalances not only compromise shrimp survival but also generate nutrient-rich effluents that exacerbate eutrophication and ecosystem degradation in receiving waters (Conley et al., 2009; Pramudia et al., 2022; Lieke et al., 2019).

Within these processes, biofilms represent a critical yet often underexplored ecological component of aquaculture systems. Biofilms are surface-attached microbial assemblages embedded in extracellular polymeric substances (EPS) that regulate nutrient cycling, influence water quality, and shape microbial succession. In intensive shrimp systems, biofilms can become dominated by opportunistic taxa such as Vibrio spp., forming structured microbial reservoirs that enhance persistence under environmental stress and complicate disease control (Liu et al., 2019). Consequently, understanding how biofilm communities assemble, consolidate, and respond to probiotic intervention is central to developing ecologically grounded management strategies.

In the present study, the system boundary is explicitly defined as a controlled, tank-scale culture-water biofilm system using water collected from L. vannamei ponds. The investigation focuses on surface-attached biofilms formed on inert high-density polyethylene (HDPE) substrates, rather than bulk-water microbial communities, sediment biofilms, or host-associated microbiota. Probiotic effects were evaluated over an early-stage succession window (0–168 h), representing the short-term time scale in which Bacillus-based probiotics are expected to initiate microbial community restructuring during initial biofilm development. Accordingly, conclusions are restricted to culture-water surface biofilms under controlled tank conditions and do not directly represent long-term pond-scale dynamics.

Historically, pathogen control in shrimp ponds has relied heavily on antibiotics and chemical treatments. While temporarily effective, these approaches accelerate antimicrobial resistance and disrupt ecological balance (Cabello et al., 2016; Hoseinifar et al., 2017). As a sustainable alternative, probiotics, particularly Bacillus spp., have gained attention due to their spore-forming capacity, environmental resilience, and antagonistic activity against pathogenic bacteria (Huerta-Rábago et al., 2019; Hui et al., 2019). Beyond pathogen suppression, Bacillus species are reported to participate in organic matter degradation and nutrient transformation processes and may influence microbial communication pathways such as quorum sensing (Tarnecki et al., 2019).

Ecologically, Bacillus supplementation may influence biofilm assembly through several non-mutually exclusive mechanisms. First, competitive exclusion may occur through rapid colonization of available surfaces and nutrient niches, thereby limiting space and resources for opportunistic taxa (Moehammad et al., 2025). Second, extracellular enzyme production may modify or partially disrupt EPS matrices, potentially affecting biofilm consolidation and structural integrity (Karygianni et al.,2020). Third, quorum quenching activity reported in certain Bacillus strains may interfere with cell–cell signaling pathways that regulate biofilm maturation and virulence expression. In addition, aerobic metabolism and organic matter degradation by Bacillus may alter local redox microenvironments within biofilms, influencing microbial niche partitioning (Markowska et al., 2024). Finally, shifts in nitrogen transformation pathways including processes related to nitrification or denitrification may indirectly modify inorganic nutrient availability, thereby favoring taxa adapted to altered nutrient regimes.

However, most previous studies have primarily emphasized the reduction of pathogenic bacteria or shifts in bulk water microbial communities. Considerably less attention has been given to whether Bacillus supplementation actively promotes the enrichment of beneficial marine-associated taxa within surface-attached culture-water biofilms. Genera such as Marivita, Pseudoalteromonas, and Marinobacter are widely recognized for their roles in nutrient turnover, antimicrobial metabolite production, and ecological stabilization in marine environments. Yet, explicit evidence demonstrating probiotic-driven positive selection of these taxa within biofilm assemblages remains limited. Furthermore, structural confirmation of probiotic-associated biofilm modification has rarely been integrated with high-resolution taxonomic profiling.

Therefore, this study addresses a critical knowledge gap by investigating whether Bacillus supplementation restructures culture-water biofilms not only through reduction of opportunistic dominance but also through ecological enrichment of beneficial bacterial groups. We employed an integrative approach combining Scanning Electron Microscopy (SEM), 16S rRNA amplicon sequencing, and nutrient profiling to evaluate structural, taxonomic, and chemical responses within biofilm systems. While 16S rRNA amplicon sequencing resolves community composition at high taxonomic resolution, SEM provides complementary structural insight into biofilm architecture, including matrix compactness, cell aggregation patterns, and surface colonization structure. By linking microbial succession with biofilm morphology and nutrient dynamics, this study aims to clarify how Bacillus probiotics modulate early-stage biofilm assembly toward more ecologically favorable configurations in shrimp aquaculture systems.

2. Materials and Methods

2.1. Experimental design

A controlled, tank-scale comparative experiment was conducted to evaluate the short-term effects of a Bacillus-based probiotic on surface-attached biofilm microbial communities and nutrient dynamics in Litopenaeus vannamei culture water. The system boundary was restricted to culture-water biofilms developing on inert substrates under controlled laboratory conditions.

Two experimental treatments were established: Biofilm Control (BC) and Biofilm Treatment (BT). Each treatment consisted of three independent biological replicates (n = 3 tanks per treatment). Twelve-liter tanks were filled with 10 L of shrimp pond-derived culture water and randomly assigned to BC or BT treatments. All tanks were maintained under identical abiotic conditions (temperature 28–30 °C, continuous aeration, and a natural photoperiod) and handled with an identical sampling regime to minimize experimental bias (Boyd, 2020).

To facilitate biofilm development, sterile high-density polyethylene (HDPE) sheets (10 × 10 cm) were vertically suspended in each tank as attachment substrata. HDPE is commonly used in aquatic biofilm studies due to its chemical inertness and strong colonization potential (Battin et al., 2016; Dang and Lovell, 2016). HDPE sheets within each tank were treated as subsamples of the same experimental unit (tank) rather than independent replicates(Figure 1).

Figure 1
Experimental design showing the setup of probiotic-treated (T) and control (C) biofilm systems using culture water from Litopenaeus vannamei ponds.

A commercial Bacillus spp. probiotic suspension was applied once to BT tanks at time zero (0 h) at a final concentration of 1 mg L⁻¹ (1 ppm), consistent with dosages used in shrimp aquaculture water management (Hui et al., 2019). Control tanks received an equivalent volume of sterile carrier solution. Biofilm and water samples were collected at 0, 24, 72, and 168 h (7 days) to capture early-stage biofilm succession and short-term probiotic effects.

2.2. Bacillus‑based probiotic and cultural conditions

A commercial Bacillus-based probiotic formulation was used as the probiotic inoculum (commercial product (QuickPro-Direct)). The product was activated by dissolving the recommended amount in sterile seawater and incubating under gentle aeration at 28–30 °C for 6 h. The activated suspension was serially diluted (10-fold) and spread-plated on appropriate agar media to determine viable cell concentration (CFU·mL⁻¹). The suspension was adjusted to an operational concentration of 1 × 10⁷ CFU·mL⁻¹. A single application was performed at time zero to achieve a final concentration of 1 mg·L⁻¹ (1 ppm) of the commercial product in the experimental system.

2.3. Quantification of microbial density in biofilms

Biofilm samples were aseptically scraped from HDPE sheets (10 × 10 cm) using sterile cell scrapers at each sampling point (0, 24, 72, and 168 h). Each sample was suspended in 10 mL of sterile 0.85% saline solution and vortexed for 3 min to dislodge cells from the substrate surface. The resulting suspension was serially diluted (10⁻¹–10⁻⁶) and spread-plated onto nutrient agar plates in triplicate. Plates were incubated at 30 °C for 24 h, and colony-forming units (CFU·cm⁻²) were enumerated to estimate total heterotrophic bacterial density.

2.4. DNA extraction and high‑throughput sequencing

Biofilm samples were collected from HDPE sheets, suspended in sterile buffer, and filtered through a 0.2-μm pore-size polycarbonate membrane prior to DNA extraction. Total genomic DNA was extracted from samples using the DNeasy PowerSoil Kit (Qiagen) with mechanical disruption (bead‑beating) and chemical lysis per the manufacturer’s protocol, quantified with a Qubit dsDNA HS fluorometer (Thermo Fisher Scientific) and assessed for integrity by agarose gel electrophoresis. The V3–V4 hypervariable region of the 16S rRNA gene (~464 bp) was PCR‑amplified with universal primers 341F (5′‑CCTACGGGNGGCWGCAG‑3′) and 805R (5′‑GACTACHVGGGTATCTAATCC‑3′) using an initial denaturation at 95 °C for 3 min, followed by 25–30 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 5 min. Amplicons were purified with AMPure XP magnetic beads (Beckman Coulter), subjected to dual‑indexing using Nextera XT adapters in a second PCR according to established protocols, and libraries were quantified by qPCR (KAPA Library Quantification Kit), normalized to 4 nM, pooled and sequenced on an Illumina MiSeq platform (v3 chemistry) to obtain 2 × 250 bp paired‑end reads. Raw reads were trimmed to remove residual adapters and primers using Cutadapt, then processed with the DADA2 pipeline in R for quality filtering, error modelling, dereplication and chimera removal to infer amplicon sequence variants (ASVs) at single‑nucleotide resolution, and ASVs were taxonomically assigned using the SILVA v138.1 (silva_nr99) reference database with a naïve‑Bayes classifier.

2.5. Determination of nutrient concentrations

Nutrient parameters in biofilm suspensions and culture water were analyzed to evaluate the influence of probiotic application on nutrient cycling. Samples were collected at 0, 24, 72, and 168 h from both control (BC) and probiotic-treated (BT) tanks. Ammonium (NH₄⁺-N) was quantified using the indophenol blue method, nitrate (NO₃⁻-N) by the cadmium reduction method, and nitrite (NO₂⁻-N) by the sulfanilamide–NED reaction, following Standard Methods for the Examination of Water and Wastewater (APHA, 2017). These colorimetric techniques are widely applied in aquaculture water-quality monitoring due to their sensitivity and reliability for nitrogenous compounds (Grasshoff et al., 2009; Boyd, 2020). Absorbance readings were measured using a UV–Vis spectrophotometer (HITACHI U-1900) at wavelengths of 640 nm for NH₄⁺-N, 543 nm for NO₃⁻-N, and 543 nm for NO₂⁻-N, respectively.

Soluble reactive phosphate (PO₄³⁻-P) concentrations were determined using the ascorbic acid–molybdenum blue method at 880 nm with the same UV–Vis spectrophotometer (HITACHI U-1900), following APHA protocols (APHA, 2017). This method is recognized as the standard approach for assessing bioavailable phosphorus in aquatic systems and has been extensively applied in aquaculture and eutrophication studies (Murphy and Riley, 1962; Conley et al., 2009).

2.6. Scanning Electron Microscopy (SEM) of biofilm structure

Biofilm samples for SEM analysis were collected from HDPE sheets at 24 h and 168 h in both control (BC) and probiotic-treated (BT) systems. HDPE sections bearing intact biofilms were gently rinsed with sterile seawater to remove loosely attached particles without disturbing the biofilm architecture.

Samples were fixed in 2.5% (v/v) glutaraldehyde prepared in phosphate-buffered saline (PBS, pH 7.2) for 2–4 h at 4 °C. Following fixation, specimens were rinsed three times with PBS and dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%). The dehydrated samples were subsequently dried by critical point drying (or air-drying, according to facility protocol). Dried specimens were mounted on aluminum stubs and sputter-coated with a thin layer of gold or gold–palladium to improve electrical conductivity.

Biofilm morphology was examined using a field emission scanning electron microscope (FESEM; Quanta 650 FEG, FEI Company, Hillsboro, OR, USA) operated at an accelerating voltage of X kV. Images were obtained at multiple magnifications to assess biofilm matrix compactness, cellular aggregation patterns, and surface coverage. All micrographs include scale bars.

2.7. Data analysis

All nutrient concentrations were expressed in mg·L⁻¹ and analyzed in triplicate, with results presented as mean ± standard deviation (SD). Differences in nutrient concentrations (ammonia, nitrite, nitrate, and phosphate) and microbial density (CFU) between probiotic and control treatments over time were analyzed using linear mixed-effects models (LMMs), with treatment and time as fixed effects and sampling unit as a random effect. Pearson’s correlation analysis was used to assess relationships among microbial density, nutrient parameters, and Bacillus abundance, and results were visualized as a heatmap. Microbial community structure differences were evaluated using PERMANOVA based on Bray–Curtis dissimilarity (999 permutations) after confirming homogeneity of multivariate dispersion. Principal Component Analysis (PCA) was performed to explore multivariate associations and identify variables contributing most to treatment-related variance. All statistical analyses were conducted in R (v4.3.0).

3. Results and Discussion

3.1. Microbial density in biofilm

Culturable heterotrophic bacterial counts (CFU·cm⁻²) exhibited distinct temporal patterns between control (BC) and probiotic-treated (BT) biofilms (Figure 2). In the control group, CFU values gradually declined from baseline to 168 h, indicating a reduction in the cultivable fraction of the biofilm community under static incubation, a pattern previously observed in closed aquaculture systems without microbial intervention (Kurniawan et al., 2012; Al Zamzami et al., 2023; Huerta-Rábago et al., 2019; Sánchez-Ortiz et al., 2016). In contrast, probiotic-treated biofilms showed a pronounced increase in CFU at 72 h, followed by a moderate decrease by 168 h.

Figure 2
Culturable heterotrophic bacterial density (CFU·cm⁻²) in control (BC) and probiotic-treated (BT) biofilms over time.

Importantly, CFU-based enumeration reflects only the cultivable and fast-growing heterotrophic fraction of the biofilm microbiota and does not represent total biofilm biomass, total cell abundance, or community-wide metabolic activity. Therefore, the observed increase in CFU after probiotic supplementation should be interpreted as an expansion of cultivable populations potentially including Bacillus spp. rather than a direct proxy for overall biofilm growth.

Taken together, these results indicate that Bacillus supplementation was associated with a transient enrichment of cultivable heterotrophic bacteria during early biofilm development, followed by a subsequent decline consistent with community adjustment over time.

3.2. Microbial community in biofilm

This study employed an integrative approach combining 16S rRNA amplicon sequencing and structural analysis to characterize the compositional and architectural dynamics of shrimp culture-water biofilms with and without Bacillus spp. probiotic supplementation. The sequencing results revealed clear differences in bacterial community composition among the control biofilm (BC1), 24 h after probiotic application (BT1), and 168 h after probiotic application (BT2) (Figure 3).

Figure 3
Relative abundance of dominant bacterial genera in control (BC1) and probiotic-treated biofilms (BT1 and BT2).

In the control biofilm (BC1), Vibrio spp. predominated, accounting for the largest proportion of the community. This dominance highlights the susceptibility of untreated culture-water biofilms to opportunistic taxa, reinforcing the importance of microbiome-targeted management strategies in shrimp aquaculture systems.

Following probiotic supplementation, the relative abundance of Vibrio decreased at 24 h (BT1), accompanied by increased representation of several marine-associated genera, including Marivita, Pseudoalteromonas, and Marinobacter. These genera are commonly associated with nutrient cycling, antimicrobial metabolite production, and quorum-quenching activity, contributing to microbial community resilience in marine environments (Tarnecki et al., 2019; Kuebutornye et al., 2020; Gram et al., 2010). However, the enrichment of Marivita and Pseudoalteromonas observed at 24 h was not sustained; by 168 h (BT2), their relative abundance declined and they no longer represented dominant community members, indicating a transient enrichment during early biofilm succession.

By 168 h (BT2), Bacillus exhibited a marked increase in relative abundance compared with the control and represented one of the most prevalent identified genera in the treated biofilm. Nevertheless, Bacillus did not completely replace other taxa, suggesting a shift toward a more evenly distributed community structure rather than absolute taxonomic dominance. Although the relative abundance of Vibrio remained substantially lower than in the control, it was still detectable at 168 h, indicating that probiotic supplementation reduced but did not eliminate this opportunistic genus.

Similar successional patterns following probiotic supplementation have been reported in shrimp and fish aquaculture systems (Huerta-Rábago et al., 2019; Pramudia et al., 2022). Overall, these findings indicate that probiotic application was associated with a restructuring of the biofilm assemblage, characterized by reduced pathogen dominance and the development of a more compositionally balanced microbial community.

3.3. Nutrient concentrations in biofilm

Analysis of nutrient concentrations (Figure 4) revealed treatment-related differences primarily in inorganic nitrogen compounds. Throughout the experimental period, probiotic-treated (BT) tanks generally exhibited lower concentrations of ammonia (NH₄⁺–N), nitrite (NO₂⁻–N), and nitrate (NO₃⁻–N) relative to controls. In contrast, phosphate (PO₄³⁻–P) concentrations remained consistently low across all treatments and time points, with no pronounced treatment-related divergence. These concentration-based observations therefore suggest that probiotic supplementation was associated mainly with shifts in inorganic nitrogen dynamics under the experimental conditions.

Figure 4
Temporal variation in dissolved inorganic nutrient concentrations: (A) Ammonia (NH₄⁺–N), (B) Nitrite (NO₂⁻–N), (C) Nitrate (NO₃⁻–N), and (D) Phosphate (PO₄³⁻–P) in control (BC) and probiotic-treated (BT) systems.

Ammonia concentration decreased to approximately 4 mg·L⁻¹ in the probiotic-treated system. Although this reduction was moderate rather than drastic, it remained consistently lower than in the control treatment. However, as this study relied on nutrient concentration measurements and 16S rRNA amplicon profiling, it does not directly resolve the specific biochemical processes responsible for these changes. Functional pathways such as nitrification, denitrification, or phosphorus assimilation were not quantified, and no process-rate measurements were performed. Accordingly, the observed nitrogen reductions should be interpreted as ecological associations coinciding with Bacillus supplementation rather than direct evidence of enhanced nutrient turnover or specific transformation mechanisms.

Nevertheless, similar patterns of reduced inorganic nitrogen concentrations following Bacillus-based probiotic application have been reported in shrimp aquaculture systems (Hui et al., 2019; Kuebutornye et al., 2020; Huerta-Rábago et al., 2019). Future studies incorporating functional gene profiling, targeted enzymatic assays, or direct process-rate measurements will be necessary to elucidate the precise biogeochemical pathways underlying these nutrient dynamics.

3.4. Principal Component Analysis (PCA)

Principal Component Analysis (Figure 5) summarized the multivariate relationships among microbial and nutrient variables across treatments. The first two principal components (PC1 and PC2) explained 93.6% of the total variance, with PC1 (77.4%) primarily structured by inorganic nitrogen parameters and PC2 (16.2%) associated mainly with microbial variables. The high proportion of variance explained by these axes indicates that nutrient gradients constituted the dominant source of variation among samples, whereas microbial metrics contributed secondary structuring.

Figure 5
Principal Component Analysis (PCA) showing multivariate relationships among microbial and nutrient variables in control (BC) and probiotic-treated (BT) systems.

Two Bacillus-related variables were included in the PCA: Bacillus relative abundance derived from 16S rRNA amplicon sequencing and Bacillus density estimated from CFU-based enumeration. These metrics represent different ecological dimensions. Relative abundance reflects proportional representation within the total microbial community, whereas density represents the cultivable fraction expressed as colony-forming units. In the biplot, Bacillus relative abundance was oriented opposite to inorganic nitrogen variables, indicating negative co-variation consistent with the Pearson correlation results (Section 3.5). In contrast, the Bacillus density vector showed weaker alignment with nutrient gradients. The longer vector length of Bacillus density reflects greater overall variance captured by CFU measurements across samples rather than a stronger ecological effect on nutrient conditions.

Samples from probiotic-treated systems clustered distinctly from controls along PC1, suggesting that probiotic supplementation was associated with coordinated shifts in inorganic nitrogen profiles and microbial composition. Comparable treatment-driven separation in ordination space has been reported in aquaculture microbiome studies where probiotic application coincided with altered community structure and changes in water-quality indicators (Pramudia et al., 2022; Tarnecki et al., 2019). However, PCA provides evidence of multivariate co-variation rather than mechanistic confirmation of functional coupling or enhanced nutrient transformation processes (Xiong et al., 2015; Boyd, 2020; Dang and Lovell, 2016; Flemming et al., 2016).

3.5. Correlation analysis between microbial and nutrient parameters

Pearson correlation analysis (Figure 6) revealed significant associations between microbial and chemical variables within the biofilm systems. Bacillus relative abundance exhibited strong negative correlations with ammonia (r = −0.70), nitrite (r = −0.68), and nitrate (r = −0.83), indicating that higher proportional representation of Bacillus co-occurred with lower inorganic nitrogen concentrations. Although a negative correlation was also observed with phosphate (r = −0.73), phosphate concentrations remained consistently low across treatments, and therefore this association should be interpreted cautiously. Similar inverse relationships between probiotic-associated taxa and nitrogenous waste levels have been reported in shrimp aquaculture systems (Zokaeifar et al., 2012; Kuebutornye et al., 2020).

Figure 6
Pearson correlation heatmap showing relationships between microbial variables and nutrient concentrations in control (BC) and probiotic-treated (BT) biofilm systems.

When considering CFU-based measurements, Bacillus density showed weaker or non-significant associations with nutrient parameters compared with relative abundance. This distinction reflects methodological differences: relative abundance captures proportional community restructuring inferred from sequencing data, whereas density represents the cultivable fraction of the microbial assemblage. These findings are consistent with the PCA results (Figure 5), where Bacillus relative abundance aligned more closely with nitrogen gradients than did Bacillus density.

Conversely, total culturable heterotrophic bacterial counts exhibited moderate positive correlations with nutrient concentrations. This pattern suggests that elevated nutrient levels may coincide with increased abundance of fast-growing heterotrophic populations, a phenomenon commonly observed in nutrient-enriched aquaculture environments (Boyd, 2020; Liu et al., 2019). Importantly, correlation analysis does not establish causation; thus, these relationships should be interpreted as statistical co-variation rather than direct evidence of nutrient transformation processes mediated by Bacillus spp.

Although Bacillus species have been widely documented to participate in nitrogen and phosphorus cycling in aquaculture systems (Hoseinifar et al., 2017; Zokaeifar et al., 2012; Huerta-Rábago et al., 2019; Kuebutornye et al., 2020), the present study did not directly quantify functional genes or metabolic rates. Accordingly, the observed correlations are best understood as ecological associations consistent with prior reports rather than confirmation of specific biogeochemical mechanisms or regulatory roles within the biofilm system.

3.6. SEM (Scanning Electron Microscopy)

In control biofilms (BC1 and BC2; Figure 7), bacterial cells formed dense and compact aggregates at both 24 h and 168 h, with extensive matrix-like material surrounding the cells. The overall structure appeared more cohesive at 168 h, consistent with progressive biofilm development over time. Dense microcolony-like arrangements are commonly associated with mature biofilms and have been reported in Vibrio-dominated biofilm systems in aquaculture contexts (Liu et al., 2019).

Figure 7
Scanning Electron Microscopy analysis of the biofilm without treatment.

In contrast, probiotic-treated biofilms (BT1 and BT2; Figure 8) displayed a less compact and more heterogeneous surface structure, characterized by visible void spaces and fewer densely packed aggregates. Cells appeared more dispersed and less embedded within the surrounding matrix-like material. By 168 h, the treated biofilm remained relatively thin and irregular compared with controls, suggesting that probiotic supplementation was associated with altered biofilm consolidation under the experimental conditions.

Figure 8
Scanning Electron Microscopy analysis of the biofilm with treatment.

These morphological patterns are consistent with the 16S rRNA amplicon results indicating a shift from Vibrio-dominated assemblages toward increased Bacillus relative abundance. However, SEM provides structural observations only; it does not directly resolve biochemical mechanisms such as EPS degradation, quorum interference, or antimicrobial activity. Therefore, mechanistic interpretations should be regarded as hypothesis-generating and interpreted in the context of prior literature describing Bacillus-associated interference with biofilm maturation and stability (Kuebutornye et al., 2020; Sánchez-Ortiz et al., 2016).

Overall, SEM observations support the conclusion that probiotic supplementation coincided with measurable differences in biofilm architecture, complementing the taxonomic and nutrient patterns observed in this study.

3.7. Integrative interpretation of biofilm responses

The combined taxonomic (16S rRNA amplicon), structural (SEM), and chemical datasets reveal coordinated ecological patterns in biofilms exposed to Bacillus-based probiotic supplementation. Probiotic-treated systems were associated with a reduced relative dominance of Vibrio spp., shifts in community composition toward Bacillus and other marine-associated taxa, and a less compact biofilm architecture. Concurrently, lower concentrations of inorganic nitrogen compounds were observed in treated systems.

These observations suggest that Bacillus spp. supplementation may influence biofilm community structure and nutrient dynamics in culture water. However, the present dataset is limited to taxonomic composition and structural characterization; functional genes, metabolic pathways, and specific biogeochemical processes were not directly measured. Therefore, the proposed ecological interpretations should be regarded as correlative and hypothesis-generating rather than mechanistic confirmation.

Future studies employing shotgun metagenomic sequencing, targeted functional gene assays, or metabolic activity measurements are necessary to validate the specific biochemical pathways underlying the observed community shifts.

4. Conclusion

This study integrated SEM, 16S rRNA amplicon profiling, and nutrient monitoring to evaluate biofilm responses to Bacillus-based probiotic supplementation in Litopenaeus vannamei culture water. Probiotic treatment was associated with a shift away from Vibrio-dominated assemblages toward a more compositionally balanced microbial community, accompanied by a less compact biofilm architecture and moderately lower inorganic nitrogen concentrations, while phosphate levels remained consistently low across treatments. Although Bacillus relative abundance was negatively associated with nitrogen parameters, functional genes and process rates were not directly measured; therefore, these findings should be interpreted as ecological associations rather than mechanistic confirmation of enhanced nutrient turnover. Further studies incorporating functional gene profiling and activity-based analyses are required to clarify the specific biogeochemical mechanisms underlying these community-level changes.

Acknowledgements

This research received funding from the Directorate of Research and Community Service, Ministry of Education, Culture, Research and Technology, Republic of Indonesia (Kemendiktisaintek) through Fundamental Research Grant (Pendidikan Magister menuju Doktor untuk Sarjana Unggul) under master contract number: 064/C3/DT.05.00/PL/2025 (dated 28 Mei 2025), and derivative contract number: 690/UN10.A0501/B/PT.01.03.2/2025 (dated 2 Juni 2025) with the Directorate Research and Community Service, Brawijaya University (DRPM, UB). The authors thank the Coastal and Marine Research Center of the University of Brawijaya for the assistance and facilities provided during this research. The authors thank the Integrated Research Laboratory - Brawijaya University (LRT-UB).

Data Availability Statement

The datasets generated during this study are available from the corresponding author upon reasonable request. All relevant data supporting the findings, including nutrient measurements, microbial density (CFU), and processed sequencing outputs, are included within the article and its supplementary materials. Raw sequencing data can be made available upon request.

References

• AL ZAMZAMI, I.M., KILAWATI, Y., IBDIAH, R. and KURNIAWAN, A., 2023. Analysis of Protein Profile and Functional Groups in Biofilms in the Hypersaline Environment. Jurnal Penelitian Pendidikan IPA, vol. 9, no. 12, pp. 11351-11358. https://doi.org/10.29303/jppipa.v9i12.4464
  » https://doi.org/10.29303/jppipa.v9i12.4464
• AMERICAN PUBLIC HEALTH ASSOCIATION – APHA. (2017). Standard methods for the examination of water and wastewater 23rd ed. Washington, DC: APHA, AWWA, & WEF.
• BATTIN, T.J., BESEMER, K., BENGTSSON, M.M., ROMANI, A.M. and PACKMANN, A.I., 2016. The ecology and biogeochemistry of stream biofilms. Nature Reviews. Microbiology, vol. 14, no. 4, pp. 251-263. https://doi.org/10.1038/nrmicro.2016.15 PMid:26972916.
  » https://doi.org/10.1038/nrmicro.2016.15
• BOYD, C.E., 2020. Water quality: an introduction 3rd ed. Cham: Springer. https://doi.org/10.1007/978-3-030-23335-8
  » https://doi.org/10.1007/978-3-030-23335-8
• CABELLO, F.C., GODFREY, H.P., BUSCHMANN, A.H. and DÖLZ, H.J., 2016. Aquaculture as yet another environmental gateway to the development and globalisation of antimicrobial resistance. The Lancet. Infectious Diseases, vol. 16, no. 7, pp. e127-e133. https://doi.org/10.1016/S1473-3099(16)00100-6 PMid:27083976.
  » https://doi.org/10.1016/S1473-3099(16)00100-6
• CONLEY, D.J., PAERL, H.W., HOWARTH, R.W., BOESCH, D.F., SEITZINGER, S.P., HAVENS, K.E., LANCELOT, C. and LIKENS, G.E., 2009. Controlling eutrophication: nitrogen and phosphorus. Science, vol. 323, no. 5917, pp. 1014-1015. https://doi.org/10.1126/science.1167755 PMid:19229022.
  » https://doi.org/10.1126/science.1167755
• DANG, H. and LOVELL, C.R., 2016. Microbial surface colonization and biofilm development in marine environments. Microbiology and Molecular Biology Reviews : MMBR, vol. 80, no. 1, pp. 91-138. https://doi.org/10.1128/MMBR.00037-15 PMid:26700108.
  » https://doi.org/10.1128/MMBR.00037-15
• EISSA, A., CHEN, P., BROWN, P.B. and HUANG, J.Y., 2022. Effects of feed formula and farming system on the environmental performance of shrimp production chain from a life cycle perspective. Journal of Industrial Ecology, vol. 26, no. 6, pp. 2006-2019. https://doi.org/10.1111/jiec.13370
  » https://doi.org/10.1111/jiec.13370
• FLEMMING, H.C., WINGENDER, J., SZEWZYK, U., STEINBERG, P., RICE, S.A. and KJELLEBERG, S., 2016. Biofilms: an emergent form of bacterial life. Nature Reviews. Microbiology, vol. 14, no. 9, pp. 563-575. https://doi.org/10.1038/nrmicro.2016.94 PMid:27510863.
  » https://doi.org/10.1038/nrmicro.2016.94
• GRAM, L., MELCHIORSEN, J. and BRUHN, J.B., 2010. Antibacterial activity of marine culturable bacteria collected from a global sampling of ocean surface waters and surface swabs of marine organisms. Marine Biotechnology, vol. 12, no. 4, pp. 439-451. https://doi.org/10.1007/s10126-009-9233-y PMid:19823914.
  » https://doi.org/10.1007/s10126-009-9233-y
• GRASSHOFF, K., KREMLING, K. and EHRHARDT, M., 2009. Methods of seawater analysis. 3rd ed. Weinheim: Wiley-VCH. https://doi.org/10.1002/9783527613984
  » https://doi.org/10.1002/9783527613984
• GUNATHILAKE, T., AKANBI, T.O., SULERIA, H.A., NALDER, T.D., FRANCIS, D.S. and BARROW, C.J., 2022. Seaweed phenolics as natural antioxidants, aquafeed additives, veterinary treatments and cross-linkers for microencapsulation. Marine Drugs, vol. 20, no. 7, pp. 445. https://doi.org/10.3390/md20070445 PMid:35877738.
  » https://doi.org/10.3390/md20070445
• HOSEINIFAR, S.H., SUN, Y.Z., WANG, A. and ZHOU, Z., 2017. Probiotics as means of diseases control in aquaculture, a review of current knowledge and future perspectives. Frontiers in Microbiology, vol. 9, pp. 2429. https://doi.org/10.3389/fmicb.2018.02429 PMid:30369918.
  » https://doi.org/10.3389/fmicb.2018.02429
• HUERTA-RÁBAGO, J.A., MARTÍNEZ-PORCHAS, M., MIRANDA-BAEZA, A., NIEVES-SOTO, M., RIVAS-VEGA, M.E. and MARTÍNEZ-CÓRDOVA, L.R., 2019. Addition of commercial probiotic in a biofloc shrimp farm of Litopenaeus vannamei during the nursery phase: effect on bacterial diversity using massive sequencing 16S rRNA. Aquaculture (Amsterdam, Netherlands), vol. 502, pp. 391-399. https://doi.org/10.1016/j.aquaculture.2018.12.055
  » https://doi.org/10.1016/j.aquaculture.2018.12.055
• HUI, Y., ZHANG, X., YANG, K. and CHEN, L., 2019. Antagonistic activity of Bacillus spp. and their effects on nitrogen transformation in shrimp aquaculture systems. Journal of Applied Microbiology, vol. 127, no. 2, pp. 522-534.
• KARYGIANNI, L., REN, Z., KOO, H. and THURNHEER, T., 2020. Biofilm matrixome: extracellular components in structured microbial communities. Trends in Microbiology, vol. 28, no. 8, pp. 668-681. https://doi.org/10.1016/j.tim.2020.03.016 PMid:32663461.
  » https://doi.org/10.1016/j.tim.2020.03.016
• KUEBUTORNYE, F.K.A., ABARIKE, E.D. and LU, Y., 2020. A review on the application of Bacillus as probiotics in aquaculture. Fish & Shellfish Immunology, vol. 87, pp. 820-828. https://doi.org/10.1016/j.fsi.2019.02.010 PMid:30779995.
  » https://doi.org/10.1016/j.fsi.2019.02.010
• KURNIAWAN, A., YAMAMOTO, T., TSUCHIYA, Y. and MORISAKI, H., 2012. Analysis of the ion adsorption–desorption characteristics of biofilm matrices. Microbes and Environments, vol. 27, no. 4, pp. 399-406. https://doi.org/10.1264/jsme2.ME11339 PMid:22673305.
  » https://doi.org/10.1264/jsme2.ME11339
• LIEKE, T., MEINERS, M., HÖRNIG, M. and HASSENRÜCK, C., 2019. Impacts of shrimp aquaculture effluents on coastal microbial communities. Marine Pollution Bulletin, vol. 140, pp. 334-343.
• LIU, H., WANG, Q., LIU, Q., CAO, X., SHI, C. and ZHANG, Y., 2019. Role of Vibrio biofilms in disease outbreaks in aquaculture systems. Aquaculture (Amsterdam, Netherlands), vol. 507, pp. 1-8.
• MARKOWSKA, K., SZYMANEK-MAJCHRZAK, K., PITUCH, H. and MAJEWSKA, A., 2024. Understanding quorum-sensing and biofilm forming in anaerobic bacterial communities. International Journal of Molecular Sciences, vol. 25, no. 23, pp. 12808. https://doi.org/10.3390/ijms252312808 PMid:39684519.
  » https://doi.org/10.3390/ijms252312808
• MOEHAMMAD, K.S., KURNIAWAN, A. and FAQIH, A.R., 2025. Application of probiotic bacteria Bacillus Spp. on biofilm formation and water quality: an experimental study on vaname shrimp culture media. Journal of Fisheries and Marine Research, vol. 9, no. 1, pp. 10-19. https://doi.org/10.21776/ub.jfmr.2025.009.01.2
  » https://doi.org/10.21776/ub.jfmr.2025.009.01.2
• MURPHY, J. and RILEY, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta, vol. 27, pp. 31-36. https://doi.org/10.1016/S0003-2670(00)88444-5
  » https://doi.org/10.1016/S0003-2670(00)88444-5
• PRAMUDIA, Z., FAQIH, A.R. and KURNIAWAN, A., 2022. Analysis of growth and water quality dynamics in white shrimp (Litopenaeus vannamei) cultivation using the millennial shrimp farming system in Indonesia. Journal Ecology, Environment & Conservation, vol. 28, no. 2, pp. 664-671.
• SÁNCHEZ-ORTIZ, A.C., ANGULO, C., LUNA-GONZÁLEZ, A., ÁLVAREZ-RUIZ, P., MAZÓN-SUÁSTEGUI, J.M. and CAMPA-CÓRDOVA, Á.I., 2016. Effect of mixed-Bacillus spp isolated from pustulose ark Anadara tuberculosa on growth, survival, viral prevalence and immune-related gene expression in shrimp Litopenaeus vannamei. Fish & Shellfish Immunology, vol. 59, pp. 95-102. https://doi.org/10.1016/j.fsi.2016.10.022 PMid:27744059.
  » https://doi.org/10.1016/j.fsi.2016.10.022
• TARNECKI, A.M., WAFAPOOR, M., PHILLIPS, R.N. and RHODY, N.R., 2019. Benefits of a Bacillus probiotic to larval fish survival and transport stress resistance. Scientific Reports, vol. 9, no. 1, pp. 4892. https://doi.org/10.1038/s41598-019-39316-w PMid:30894554.
  » https://doi.org/10.1038/s41598-019-39316-w
• TIAN, T., WANG, Z. and ZHANG, J., 2017. Pathomechanisms of oxidative stress in inflammatory bowel disease and potential antioxidant therapies. Oxidative Medicine and Cellular Longevity, vol. 2017, no. 1, pp. 4535194. https://doi.org/10.1155/2017/4535194 PMid:28744337.
  » https://doi.org/10.1155/2017/4535194
• TRAN, L., NHUT, T.C., ALFREY, K.B., BARROWS, F.T., KUHN, D. and MCLEAN, E., 2022. Performance of pacific white leg shrimp fed an open-source marine resource-free diet under commercial conditions. International Journal of Fisheries and Aquatic Studies, vol. 10, no. 4, pp. 33-41. https://doi.org/10.22271/fish.2022.v10.i4a.2702
  » https://doi.org/10.22271/fish.2022.v10.i4a.2702
• XIONG, J., DAI, W., LI, C., ZHANG, J. and QIU, Q., 2015. Response of host–bacterial colonization in shrimp to developmental stage, environment and disease. Molecular Ecology, vol. 27, no. 18, pp. 3686-3699. https://doi.org/10.1111/mec.14822 PMid:30070062.
  » https://doi.org/10.1111/mec.14822
• ZOKAEIFAR, H., BALCÁZAR, J.L., SAAD, C.R., KAMARUDIN, M.S., SIJAM, K., ARSHAD, A. and NEJAT, N., 2012. Effects of Bacillus subtilis on growth performance, digestive enzymes, immune response, and disease resistance of white shrimp (Litopenaeus vannamei). Fish & Shellfish Immunology, vol. 33, no. 4, pp. 683-689. https://doi.org/10.1016/j.fsi.2012.05.027 PMid:22659618.
  » https://doi.org/10.1016/j.fsi.2012.05.027

Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  15 June 2026
• Date of issue
  2026

History

• Received
  16 Dec 2025
• Accepted
  23 Mar 2026