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Article

Baccilus amyloliquefacins Strains Isolated in a Wastewater Treatment Plant: Molecular Identification and Amylase/Protease Production Capacity

by
Jean Jules Nana Ndangang
1,*,
Alain-Martial Sontsa-Donhoung
2,
Elvire Hortense Biye
3,
Dumitra Raducanu
4,
Narcis Barsan
5,*,
Anne Ayo
6,
Guy Valerie Djumyom Wafo
7,
Emilian Mosnegutu
5,
Valentin Nedeff
5,
Christelle Kebassa Nkwefuth
1,
Florin-Marian Nedeff
5,
Mirela Panainte-Lehadus
5,
Dana Chițimus
5 and
Ives Magloire Kengne Noumsi
1
1
African Wastewater Research Unit, Department of Plant Biology, Faculty of Science, University of Yaoundé I, Yaounde P.O. Box 812, Cameroon
2
Laboratory of Regional Biological Control and Applied Microbiology, Institute of Agricultural Research for Development (IRAD), Yaounde P.O. Box 2123, Cameroon
3
Laboratory of Ecology, Departement of Plant Biology, University of Yaounde I, Yaounde P.O. Box 812, Cameroon
4
Faculty of Science, “Vasile Alecsandri” University of Bacau, Calea Marasesti 156, 600115 Bacau, Romania
5
Faculty of Engineering, “Vasile Alecsandri” University of Bacau, Calea Marasesti 156, 600115 Bacau, Romania
6
Laboratory of Agroecology and Agronomic Science, ISABEE-Belabo Department of Plant Production, University of Bertoua, Belabo P.O. Box 60, Cameroon
7
Research Unit on Applied Botany, Department of Plant Biology, University of Dschang, Dschang P.O. Box 96, Cameroon
*
Authors to whom correspondence should be addressed.
Environments 2025, 12(11), 412; https://doi.org/10.3390/environments12110412
Submission received: 24 September 2025 / Revised: 30 October 2025 / Accepted: 31 October 2025 / Published: 2 November 2025
(This article belongs to the Special Issue Advances in Wastewater Bio-Management: Microbial Community Relationships, Monitoring and Assessment)
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Abstract

This study centred on isolating and characterizing Bacillus amyloliquefaciens strains derived from wastewater sludge to assess their potential for sludge treatment. Samples were collected from the Etoa wastewater sludge treatment plant in Yaounde, Cameroon. The isolates were obtained on nutrient agar medium and were identified through morphological and biochemical characterization, followed by 16S rRNA gene sequencing analysis. The sequences showed 99–100% similarity with Bacillus amyloliquefaciens strains in the NCBI database. The isolates exhibited significant in vitro enzymatic activities, including catalase, amylase, and protease production, indicating their ability to degrade hydrogen peroxide starch and proteins, respectively. The results confirmed the in vitro potential of Bacillus amyloliquefaciens as a promising microbial agent for organic matter degradation in wastewater sludge. Although the findings were limited to laboratory conditions, they provided a foundation for future pilot-scale or in situ studies aimed at validating their practical efficiency. This research contributes to the development of microbial-based and eco-efficient strategies for sustainable sludge management.

1. Introduction

Treating fecal sludge is a major challenge for public health and environmental protection, particularly in urbanizing regions. Conventional physicochemical treatment methods are often costly, energy-intensive, and inefficient in removing complex organic matter and pathogens [1]. However, recent studies suggest that the use of microorganisms could be a promising and effective solution [2,3]. Boruszko and Butarewicz [4] demonstrated that bacterial microorganisms could effectively reduce organic load in wastewater sludge, while Wardhani et al. [5] highlighted the biodegradation efficiency of Priestia aryabhattai in septage treatment. Treating fecal sludge is crucial to preventing the spread of disease and protecting the environment. Fecal sludge is a mixture of fecal matter, wastewater, urine and other organic waste collected in wastewater systems not connected to the public sewer system [6]. They contain toxic substances such as heavy metals, chemicals, and pathogens which may lead to environmental degradation through soil and groundwater pollution, atmospheric emissions, and dissemination of infectious diseases [7,8]. In Yaounde, Cameroon, accelerated urbanization and population growth have intensified the generation of liquid waste, posing serious environmental, health and economic challenges [9].
Members of the Bacillus genus offer a promising biotechnological solution due to their spore-forming ability, metabolic versatility, and resilience in harsh environmental conditions [10]. Bacillus spp. is recognized for producing extracellular enzymes that hydrolyze complex organic matter, reduce biochemical oxygen demand (BOD) and chemical oxygen demand (COD), and decrease volatile solids, enhancing sludge biodegradation [11,12]. Among them, Bacillus amyloliquefaciens is widely found in soil, water, and plant-associated environments, with applications in agriculture, medicine, and environmental remediation [13,14].
Recent studies demonstrated that Bacillus amyloliquefaciens produces antimicrobial lipopeptides (surfactin, fengycin, and iturin) and exhibits heavy metal and organic pollutant bioremediation capacity [15,16]. For example, strain BAB-807 removed up to 96% of Cr and over 90% of Pb, Ni, and Cu from contaminated effluents under optimized laboratory conditions [17]. However, most studies to date have been performed under controlled conditions, rather than full-scale sludge treatment systems. Thus, additional research is needed to evaluate the enzymatic potential of Bacillus amyloliquefaciens strains specifically isolated from wastewater sludge. Thorough morphological, biochemical, and genomic analyses of Bacillus amyloliquefaciens strains represent an essential step in validating their utility for wastewater sludge bioremediation.
Compared with other Bacillus species such as Bacillus subtilis and Bacillus licheniformis, Bacillus amyloliquefaciens exhibits higher extracellular enzyme productivity and better adaptability to environmental stress, making it a suitable candidate for sludge treatment. Other bacterial genera, including Trichococcus, can dominate sludge digestion processes, particularly when supplemented with substrates such as citric acid, highlighting the need to identify and characterize key microbial contributors to optimize wastewater treatment efficiency [3].
Bacillus amyloliquefaciens is a widely distributed Gram-positive microorganism characterized by its remarkable metabolic adaptability and ability to secrete a broad spectrum of extracellular enzymes and bioactive secondary metabolites. These attributes confer substantial industrial, medical, and agricultural relevance, and highlight its emerging role as a sustainable and versatile chassis organism for synthetic biology, metabolic engineering, and heterologous protein production [18]. Also, Bacillus amyloliquefaciens has been shown to effectively utilize starch-based industrial wastewater as a fermentation substrate for amylase biosynthesis, underscoring its relevance as a cost-efficient microbial platform for sustainable enzyme production within circular bioeconomy frameworks [19].
Therefore, the present study focuses on the isolation and in vitro characterization of Bacillus amyloliquefaciens strains obtained from the Etoa wastewater sludge treatment plant in Yaounde, Cameroon. Specifically, it seeks to identify the isolated strains using morphological, biochemical, and molecular approaches, and assess their enzymatic activities relevant to the biodegradation of organic pollutants present in wastewater, notably improving sanitary quality.

2. Materials and Methods

2.1. Sample Collection

The Etoa wastewater sludge treatment plant is in the Yaounde 3 subdivision, as shown in Figure 1. The sampling points were defined based on the depth and total thickness of 20 cm of the dehydrated sludge, which had been left to dry for two weeks. Samples were collected once at nine random points on 14 March 2024, in the drying bed, at a depth of approximately 5 to 10 cm, using a shovel to obtain representative samples of the top layer of sludge, where the microorganisms are mostly active. The samples were collected in previously labelled sterile jars and then placed in a cool box to maintain an adequate temperature during transport to the laboratory, where they were maintained at 4 °C, according to Koné et al. [20].

2.2. Microbial Isolation and Characterization of Bacillus amyloliquefaciens Strains

The selection method described by Travers et al. [21] and Daniel et al. [22] was used to isolate the bacteria belonging to the Bacillus genus from wastewater sludge samples. First, 1 g of fecal sludge was taken using a sterilized spatula and diluted in 9 mL of 0.85% sterile saline to obtain a 10−1 dilution. Successive decimal dilutions were then made up to 10−5. The different isolates were grown on nutrient agar (NA) consisting of 10 g peptone casein, 5 g yeast extract, 5 g sodium chloride, and 20 g bacteriological agar in distilled water. The prepared medium underwent sterilization via autoclaving (121 °C, 15 min), followed by cooling to approximately 50 °C prior to solidification. Next, 0.1 mL of each bacterial suspension was streaked onto NA plates containing 30 mg/L chloramphenicol. For optimal Bacillus spp. growth, plates were incubated at 35 °C for 24–48 h. Serial dilutions were performed in triplicate to ensure reproducibility. Distinct colonies with morphological features consistent with Bacillus spp. (including shape, colour, and texture) were selected. Each isolate was purified through successive subcultures until a stable colony morphology was achieved. Pure cultures were stored at 4 °C for short-term use. Isolates were morphologically and biochemically characterized; thus, they were identified using appropriate molecular methods.

2.3. Morphological Characterization of Isolate

All macroscopic and microscopic analyses were performed in triplicate to ensure observation consistency, and representative isolates were photographed for reference.
Macroscopic and microscopic observations of the bacterial colonies obtained on NA were carried out by noting the shape, relief, contour, surface, colour, texture, and diameter of the colonies, following the method described by Vos et al. [23]. Gram staining of the bacterial isolates was performed to observe their microscopic morphology under a light microscope to visualize the morphology and the cellular organization. In addition, bacterial spore formation was examined based on endospore staining, as described by Oktari et al. [24].
The Schaeffer–Fulton method was employed to stain the endospores. To induce sporulation, bacterial cultures were exposed to thermal stress (44 °C in an oven or 8 °C refrigeration) for 24–48 h. Sporulated cells were then suspended, thinly smeared on glass slides, and heat-fixed. Slides were flooded with 5% malachite green solution and gently heated over a Bunsen burner until vapour emission, followed by a 6–10 min staining period. The filter paper was taken off, and the slide was washed with water. Finally, the slide was treated with 0.5% safranin solution for 30 s to visualize the spores in green and the bacilli in red as previously described by Oktari et al. [24].

2.4. Enzymatic Production Capacity of Isolates

Identification of the bacterial isolates was accomplished by evaluating their metabolic characteristics through biochemical analysis. Each test was performed in triplicate, and appropriate negative controls were used.

2.4.1. Catalase Activity

Catalase activity was evaluated following the method described by MacFaddin [25]. Fresh bacterial colonies (24 h) were transferred onto a clean glass slide using a sterile loop, and one drop of 3% hydrogen peroxide (H2O2) solution was added. The immediate formation of oxygen bubbles indicated a positive catalase reaction.

2.4.2. Amylase Activity

The starch hydrolysis assay was performed according to Hankin and Anagnostakis [26]. Bacterial isolates were cultured on starch-supplemented nutrient agar (0.2% w/v). Following incubation, plates were flooded with Lugol’s iodine solution, revealing amylolytic activity as distinct clearance zones surrounding positive colonies.

2.4.3. Protease Activity

Bacterial isolates were spot-inoculated on Skim Milk Agar plates and incubated for 2–5 days. Proteolytic activity was identified by the formation of transparent hydrolysis zones surrounding colonies, following established methodologies by Chaiharn et al. [27] and Ullah et al. [28].

2.4.4. Arginine and Ornithine Decarboxylase Activities

Arginine and ornithine decarboxylase activities were demonstrated on solid media (g bactopeptone, 5 g meat extract, 0.5 g glucose, 5 mg pyridoxine, 20 mg phenol red, and 18 g/L agar for 1 litre of distilled water, final pH of 6.0) containing 0.2% arginine L hydrochloride or 0.2% ornithine L hydrochloride, respectively [29]. Enzyme activity was assessed after one day’s incubation due to the change in colour of the medium from yellow to pink.

2.4.5. Gelatinase Activity

Bacterial isolates were tested for gelatinolytic activity using nutrient gelatin medium, which included appropriate positive and negative controls. Following incubation, tubes were refrigerated in an ice bath for 30 min to determine liquefaction persistence, with temperature-stable fluidification indicating gelatinase synthesis, according to [29].

2.5. Molecular Analysis of Bacterial Strains

Molecular analysis was performed for representative isolates showing distinct morphological and biochemical profiles.

2.5.1. Extraction of Bacterial Genomic DNA

Genomic DNA was extracted from bacterial cultures cultivated for 24 h in nutrient broth. To increase DNA yield, mechanical cell disruption was performed using a Mini Bead Beater-8 homogenizer (BioSpec, Bartlesville, OK, USA) for 1 min. The DNA was then purified using the High Pure PCR Template Preparation Kit (Roche, Germany). The extracted DNA was tested for concentration and purity using a SpectraMax® QuickDrop™ Micro-Volume Spectrophotometer (Molecular Devices, Carlson Circle Tampa, FL, USA).

2.5.2. Bacterial Strain Identification by Sequencing

Species-level identification of bacterial strains was achieved by targeting the highly conserved 16S rDNA region. Amplification of the 16S rRNA gene was carried out using universal bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACTT-3′) [30]. The PCR reaction (25 µL total volume) contained 20 ng of template DNA, along with 1X Buffer, 2 mM MgCl2, 0.2 mM dNTPs (Thermo Scientific, Vilnius, Lithuania), 0.5 µM of each primer, and 0.25 U of MangoTaq DNA Polymerase (BioLine, UK), prepared in MilliQ water. The annealing temperature was optimized to 55.5 °C. Following amplification, PCR products were visualized via agarose gel electrophoresis, then purified and sequenced using the Sanger dideoxy method (CeMIA, Larissa, Greece). The resulting forward and reverse primer sequences were aligned using BioEdit Sequence Alignment Editor (v7.2.5). For taxonomic classification, the assembled sequences were analyzed using NCBI’s NBLAST (Nucleotide BLAST + 2.17.0) tool, comparing them against the NCBI microbial database for homology-based identification. Phylogenetic analysis was conducted in MEGAX according to Kumar et al. [31]. Sequence alignment was performed with ClustalW ClustalW 2.0, and the phylogenetic tree was constructed via UPGMA clustering according to [30].

3. Results

3.1. Morphological and Microscopic Characteristics of Isolates

3.1.1. Morphological Characteristics of Isolates

The following three bacterial isolates based on colony morphology and colours were obtained from wastewater sludge samples. These isolates are registered under the designations BEM1, BEM2, and BEM3 (Table 1). All isolates exhibited a round shape and were smooth. BEM 1 and 3 display a slightly higher relief, while BEM 2 shows a moderate relief, indicating some variability in colony structure.
The isolates varied slightly in size, ranging from 1 to 3 mm for isolates 1 and 3, and from 1 to 2 mm for BEM 2. All isolates exhibited a rough surface and opacity, which are typical characteristics of bacterial colonies. In terms of colour, all the isolates BEM 1, BEM 2 and BM3 were yellowish, highlighting a colorimetric similarity (Figure 2).

3.1.2. Gram and Endospore Staining

The Gram staining results showed that all three isolates were Gram-positive (Figure 3). Bacteria with a thick peptidoglycan cell wall are characterized by their vivid violet colouration and well-defined rod-like appearance. Concerning endospore staining, the results revealed the presence of oval-shaped endospores frequently observed inside bacterial cells in all isolates (Figure 3). The endospores under the microscope are presented in Figure 4.

3.2. Molecular Identification of Isolates

BEM 1 was identified as Bacillus amyloliquefaciens based on 16S rDNA sequencing. Comparative analysis revealed 100% sequence identity and 99.86% query coverage with 60 reference strains in the NCBI database (including accession CP054415.1). The genomic sequence of BEM 1 has been deposited in GenBank under accession number PV355678. Similarly, BEM 2 was confirmed as Bacillus amyloliquefaciens, sharing 100% identity and 99.86% coverage with 59 database strains (including CP054415.1). Its sequence is publicly available under accession PV355830. The BEM 3 isolate also exhibited 100% 16S rDNA identity and full query coverage with 64 Bacillus amyloliquefaciens strains (including CP054415.1). Its sequence is archived in GenBank (PV355845) (Table 2).
The 16S rRNA gene sequences of the isolates were aligned with reference sequences from NCBI using ClustalW in MEGA 11 software. Phylogenetic relationships were inferred via the Neighbour-Joining algorithm with 1000 bootstrap replications for branch support. The resulting tree revealed distinct phylogenetic clustering of the studied isolates, as illustrated in Figure 5.
Multiple sequence alignment was performed using ClustalW, followed by phylogenetic tree construction via the Neighbour-Joining algorithm. A bootstrap consensus tree (1000 replicates) was generated to assess topological robustness, with poorly supported branches (<50% bootstrap value) collapsed. Branch labels indicate the frequency (%) of cluster formation across bootstrap iterations. Evolutionary distances were calculated using the Maximum Composite Likelihood approach, expressed as nucleotide substitutions per site.

3.3. Enzymatic Production Capacity of the Strains

The enzymatic activities of the isolates are summarized in Table 3. Catalase activity (Figure 6A) demonstrates the ability of all isolates to decompose hydrogen peroxide (H2O2) into water and oxygen, as evidenced by release of oxygen bubbles upon contact with H2O2. Protease activity was confirmed by the formation of clear halos around colonies (Figure 6C), while amylolytic activity was also detected (Figure 6B). All enzymatic assays were qualitative, except for ornithine decarboxylase and gelatinase, for which halo diameters were measured to estimate relative enzyme activity levels. All isolate isolates exhibited ornithine decarboxylase activity, with BEM 3 displaying the highest activity (+16 mm). None of the isolates (BEM 1 and BEM 3) produced arginine decarboxylase activity, indicating a limited capacity to metabolize this amino acid. Notably, BEM 3 exhibited the strongest gelatinase production (31 mm), suggesting a high proteolytic potential.

4. Discussion

The round shape and smoothness of the colonies may indicate healthy and homogeneous growth of the isolates, suggesting adaptation to the culture conditions. However, minor variations among isolates were observed in relief (slightly higher vs. moderate), opacity and elevation. These subtle morphological differences could reflect inherent strain variability. However, without quantitative morphometric or genetic data, such interpretations remain speculative. For example, the similar size of isolates BEM 1 and BEM 3 may suggest adaptation to similar ecological niches within the sludge, where resource availability and physical conditions are comparable, whereas the slightly smaller size of isolate BEM 2 could hint at differences in nutrient uptake or metabolic efficiency. Future microscopic and image analysis studies would be needed to confirm these hypotheses.
The Gram-positive rod morphology observed supports classification within Bacillus or related genera characterized by their rod morphology and staining and consistent with the standard description as in Bergey’s Manual [23]. Lee et al. [32] and Li et al. [33] reported predominance of Gram-positive rods in Bacillus amyloliquefaciens isolates, reinforcing this result.
One key adaptive characteristic of Bacillus amyloliquefaciens isolates is catalase activity, which decomposes hydrogen peroxide (H2O2)) into water and molecular oxygen. This enzymatic capacity is likely advantageous in sludge environments where oxidative stress or residual peroxides may occur. In our study, all isolates exhibited positive catalase reactions, which aligns with literature reports on Bacillus survival under fluctuating redox conditions [33]. However, we did not measure catalase kinetics or quantitative activity levels, which constrains our ability to compare strains.
The isolates also demonstrated protease and amylase activities. These enzymes are central to sludge biodegradation, as they hydrolyze proteins, polysaccharides, and starches into simpler compounds, thereby facilitating the breakdown of organic matter [14]. Proteases catalyze the hydrolysis of peptide bonds in proteins, leading to the formation of peptides and free amino acids. This activity facilitates the degradation of proteinaceous materials commonly found in sludge, thereby contributing to the reduction in organic nitrogen content. Liberated amino acids can be assimilated by other microorganisms as nutrient sources, enhancing microbial activity and accelerating the overall biodegradation process during sludge treatment [34]. Hou et al. [35] isolated endogenous proteases from sludge environments and showed their effect on sludge solubilization. Our results are consistent with the expected enzymatic profile of Bacillus amyloliquefaciens [36,37]. Amylase plays a key role in the degradation of polysaccharides present in fecal sludge, particularly starch, by catalyzing the hydrolysis of glycosidic bonds that convert complex polysaccharides into simple sugars (starch → dextrins → maltose → glucose). This enzymatic activity enhances the breakdown of organic matter and contributes to the removal of carbohydrate pollutants. The resulting simple sugars provide readily available carbon sources that can stimulate the growth and metabolic activity of other microorganisms, thereby promoting further biodegradation and improving the overall efficiency of sludge treatment [38]. The positive amylolylic and proteolytic responses are comparable to previous findings [36,37]. However, the exact contribution of these enzymes in sludge treatment was not assessed here. This link must be validated at pilot scale before claiming operational efficacy.
Interestingly, gelatinase activity was detected in BEM1 and BEM2, indicating their ability to degrade protein substrates such as gelatin [39]. Fugaban et al. [40] have previously reported gelatinase production in B. amyloliquefaciens. Additionally, positive ornithine and arginine decarboxylase was detected in all the isolates. Several studies [41,42] also highlighted the capacity of B. amyloliquefaciens to produce these enzymes. They may hint at amino acid metabolism capabilities under nitrogen-limited conditions in sludge.
Beyond wastewater applications, enzymatic versatility suggests potential exploitation for high-value bioproducts, such as biosurfactants and bioflocculants. For example, molecular engineering of B. amyloliquefaciens has been explored to enhance protease synthesis [43]. Moreover, bioflocculation as a pollutant removal strategy has been validated in recent studies [44]. While intriguing, applying these bioproducts at industrial scale requires further optimization of yields, stability, and integration into existing sludge treatment systems.
In summary, although the observed morphological traits and enzymatic activities of B. amyloliquefaciens isolates are promising, their practical utility in sludge treatment remains to be demonstrated under realistic operational conditions.

5. Conclusions

In this study, Bacillus amyloliquefaciens strains were successfully isolated from wastewater sludge and characterized for their in vitro enzymatic activities, including the production of catalase, amylase and protease, gelatinase, arginine and ornithine decarboxylase. These results demonstrate their potential as promising microbial agents for sustainable sludge treatment and confirm their biotechnological relevance for the biodegradation of organic waste.
Although this work demonstrates in vitro enzymatic performance, further validation under real or pilot-scale conditions is needed to assess their practical efficiency in sludge treatment. Future studies should include in situ sludge digestion trials, as well as life cycle and carbon footprint assessments to quantify their environmental benefits. Such efforts will support the development of microbial-based and eco-efficient strategies for wastewater sludge treatment within a circular economy framework.

Author Contributions

Conceptualization, J.J.N.N.; validation, A.-M.S.-D. and N.B.; methodology, D.R. and A.A.; software, G.V.D.W. and E.M.; supervision, V.N.; investigation, J.J.N.N., A.-M.S.-D. and C.K.N.; writing—review and editing, J.J.N.N., A.-M.S.-D. and E.H.B.; visualization, F.-M.N. and M.P.-L.; formal analysis, D.C. and I.M.K.N. I.M.K.N. passed away prior to the publication of this manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research work is funded by the Conservation Action Research Network (CARN) and the “Eugen Ionescu” doctoral and postdoctoral research scholarships organized by the “Agence Universitaire de la Francophonie (AUF). The research performed by Dumitra Raducanu, Narcis Barsan, Emilian Mosnegutu, Valentin Nedeff, Florin-Marian Nedeff, Mirela Panainte-Lehadus and Dana Chițimus was funded by the Ministry of Education, through the National Council for the Financing of Higher Education, Romania, grant number CNFIS-FDI-2025-F-0483: Improving the quality of RDI activities at “Vasile Alecsandri” University of Bacău through national and international cooperation (acronym: Connect-UBc).

Data Availability Statement

Data is contained within the article. The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors would like to express their thanks to the “Vasile Alecsandri” University of Bacau for providing access to the laboratory.

Conflicts of Interest

The authors declare no conflicts of interest.

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Environments 12 00412 g001
Figure 1. Schematic representation of the wastewater treatment facility located in Etoa, Yaounde: 1: Access road. 2: Entry/exit gate. 3: Guard post. 4: Truck maneuvering area. 5: Unloading points. 6: Thickening basins. 7: Anaerobic lagoon. 8: Optional lagoon. 9: Treated effluent discharge point. 10: Sludge pumping station to drying beds. 11: Drying beds for sludge from thickening basins. 12: Drying beds for sludge from thickening basins (repeated). 13: Drying beds for sludge from anaerobic and optional lagoons. 14: Solid fraction storage and recovery building. 15: Technical room. 16: Fence. 17: Sampling points:
Figure 1. Schematic representation of the wastewater treatment facility located in Etoa, Yaounde: 1: Access road. 2: Entry/exit gate. 3: Guard post. 4: Truck maneuvering area. 5: Unloading points. 6: Thickening basins. 7: Anaerobic lagoon. 8: Optional lagoon. 9: Treated effluent discharge point. 10: Sludge pumping station to drying beds. 11: Drying beds for sludge from thickening basins. 12: Drying beds for sludge from thickening basins (repeated). 13: Drying beds for sludge from anaerobic and optional lagoons. 14: Solid fraction storage and recovery building. 15: Technical room. 16: Fence. 17: Sampling points:
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Figure 2. Colonies of bacterial isolates on culture medium nutrient agar.
Figure 2. Colonies of bacterial isolates on culture medium nutrient agar.
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Figure 3. Microscopic observation of Gram-positive BEM1, BEM2 and BM3 in the form of rods.
Figure 3. Microscopic observation of Gram-positive BEM1, BEM2 and BM3 in the form of rods.
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Figure 4. Microscopic observation of endospores.
Figure 4. Microscopic observation of endospores.
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Figure 5. Phylogenetic tree constructed with Mega 11 based on the 16 rRNA gene.
Figure 5. Phylogenetic tree constructed with Mega 11 based on the 16 rRNA gene.
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Figure 6. Biochemical characterization of the isolates: (A) test of catalase, control (1), BEM 1 activity (2), BEM 2 activity (3), BEM 3 activity (4); (B) amylase production of selected bacteria; (C) protease production of selected bacteria.
Figure 6. Biochemical characterization of the isolates: (A) test of catalase, control (1), BEM 1 activity (2), BEM 2 activity (3), BEM 3 activity (4); (B) amylase production of selected bacteria; (C) protease production of selected bacteria.
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Table 1. Morphological characteristics of isolates.
Table 1. Morphological characteristics of isolates.
CriteriaBEM 1BEM 2BEM 3
ShapeRoundRoundRound
ReliefSlightly highModerateSlightly high
ContourSmooth SmoothSmooth
Size1 to 3 mm1 to 2 mm1 to 3 mm
SurfaceRoughRoughRough
OpacityOpaqueOpaqueOpaque
ColourYellowishYellowishYellowish
Table 2. Molecular characterization of bacterial strains isolated from fecal sludge through 16S ribosomal DNA sequencing.
Table 2. Molecular characterization of bacterial strains isolated from fecal sludge through 16S ribosomal DNA sequencing.
IsolatesNCBI Accession NumberSequence Analysis
Closest NCBI Database Match with
Accession Number		Query CoverPercentage of Identity
BEM 1PV355678Bacillus amyloliquefaciens CP054415.199.86%100
BEM 2PV355830Bacillus amyloliquefaciens CP054415.199.86%100
BEM 3PV355845Bacillus amyloliquefaciens CP054415.1100%100
Table 3. Extracted secondary metabolite data.
Table 3. Extracted secondary metabolite data.
IsolatesCatalaseAmylaseProteaseArginine DecarboxylaseOrnithine Decarboxylase (mm)Gelatinase (mm)
BEM 1++++++−1526
BEM 2++++++150
BEM 3++++−1631
(−) negative production; (+) low production; (++) medium production.
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MDPI and ACS Style

Ndangang, J.J.N.; Sontsa-Donhoung, A.-M.; Biye, E.H.; Raducanu, D.; Barsan, N.; Ayo, A.; Wafo, G.V.D.; Mosnegutu, E.; Nedeff, V.; Kebassa Nkwefuth, C.; et al. Baccilus amyloliquefacins Strains Isolated in a Wastewater Treatment Plant: Molecular Identification and Amylase/Protease Production Capacity. Environments 2025, 12, 412. https://doi.org/10.3390/environments12110412

AMA Style

Ndangang JJN, Sontsa-Donhoung A-M, Biye EH, Raducanu D, Barsan N, Ayo A, Wafo GVD, Mosnegutu E, Nedeff V, Kebassa Nkwefuth C, et al. Baccilus amyloliquefacins Strains Isolated in a Wastewater Treatment Plant: Molecular Identification and Amylase/Protease Production Capacity. Environments. 2025; 12(11):412. https://doi.org/10.3390/environments12110412

Chicago/Turabian Style

Ndangang, Jean Jules Nana, Alain-Martial Sontsa-Donhoung, Elvire Hortense Biye, Dumitra Raducanu, Narcis Barsan, Anne Ayo, Guy Valerie Djumyom Wafo, Emilian Mosnegutu, Valentin Nedeff, Christelle Kebassa Nkwefuth, and et al. 2025. "Baccilus amyloliquefacins Strains Isolated in a Wastewater Treatment Plant: Molecular Identification and Amylase/Protease Production Capacity" Environments 12, no. 11: 412. https://doi.org/10.3390/environments12110412

APA Style

Ndangang, J. J. N., Sontsa-Donhoung, A.-M., Biye, E. H., Raducanu, D., Barsan, N., Ayo, A., Wafo, G. V. D., Mosnegutu, E., Nedeff, V., Kebassa Nkwefuth, C., Nedeff, F.-M., Panainte-Lehadus, M., Chițimus, D., & Magloire Kengne Noumsi, I. (2025). Baccilus amyloliquefacins Strains Isolated in a Wastewater Treatment Plant: Molecular Identification and Amylase/Protease Production Capacity. Environments, 12(11), 412. https://doi.org/10.3390/environments12110412

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