Fermentation is one of humanity’s oldest food preservation technologies, and in West Africa it remains a cornerstone of daily nutrition. Maize is processed into staples through successive soaking in water, while African locust bean seeds are boiled and fermented to produce pungent, protein-rich condiments that flavor dishes across Nigeria, Benin, and beyond. Bacillus species are central players in these transformations. Their enzymes, including alpha-amylase, cellulases, and proteases, break down complex polysaccharides and proteins into sugars, amino acids, and peptides, generating the characteristic textures and flavors of these products. But the same biochemical versatility that makes Bacillus invaluable to food producers also means these bacteria thrive in the waste streams that fermentation generates, and some members of the genus carry a darker capability: the production of enterotoxins.

The research team, led by Oluwafemi Bamidele Daramola, focused on ten commercial fermentation sites in Ile-Ife, in Osun State, Nigeria, five dedicated to maize processing and five to African locust bean. Between March and June 2022, they aseptically collected top-horizon soil cores from depths of five to thirty centimeters at two spots within each site, targeting areas where liquid and solid effluents had been dumped. Back in the laboratory, the samples were serially diluted, plated on nutrient agar, and incubated, after which colonies displaying typical Bacillus morphology were isolated and identified through biochemical testing based on Bergey’s Manual of Determinative Bacteriology. In total, 62 Bacillus isolates were recovered, spanning sixteen species, including Bacillus firmus and Bacillus cereus at 16 percent each, Bacillus licheniformis at 15 percent, and Bacillus megaterium at 10 percent, alongside rarer species such as B. decolorationis, B. nealsonii, and B. schlegellii.

The critical question was not simply which Bacillus species were present, but what genetic weaponry they carried. Using polymerase chain reaction, the researchers screened every isolate for genes encoding three major enterotoxin systems associated with the Bacillus cereus group: the non-hemolytic enterotoxin complex, with subunit genes nheA, nheB, and nheC; the hemolysin BL complex, detected through the hblC and hblD subunit genes; and the Bacillus cereus enterotoxin T gene, bceT. DNA was extracted by boiling bacterial cells and centrifuging away the debris, then amplified using specific oligonucleotide primers in single and multiplex PCR reactions. Amplified products were separated on ethidium bromide-stained agarose gels and visualized under ultraviolet light, allowing the team to determine precisely which toxin genes each isolate possessed.

The results were striking. The bceT gene appeared in 32.26 percent of the isolates, with a 95 percent confidence interval of 23.1 to 41.4 percent. The hemolysin BL subunit genes were detected in 40.32 percent of isolates, and the non-hemolytic enterotoxin genes in 38.71 percent. Breaking the nhe complex down further, nheA was present in 38.70 percent of isolates, nheB in 35.48 percent, and nheC in 37.09 percent. Most alarming was the finding that 32.26 percent of the recovered Bacillus species carried all three categories of enterotoxin genes simultaneously, meaning nearly one in three environmental isolates had the complete genetic apparatus for producing the full spectrum of diarrheal toxins assayed. Only 59.68 percent of the isolates lacked any of the targeted genes altogether.

The architecture of these toxin systems proved as interesting as their prevalence. In classical descriptions, the three subunit genes of either the hbl or nhe complexes occur together within a single operon, and structural studies have shown that the tripartite toxin components assemble sequentially on target cells and are not interchangeable between toxin types. Yet in this study, the researchers found isolates deficient in one or two subunit genes, with 23 Bacillus encoding both hblC and hblD while two carried only hblD, and a single isolate encoding only nheA. This suggests that genes within an operon can be present and potentially function independently, a deviation from the canonical model that echoes findings from other research groups. The team also detected bceT, a gene traditionally associated with B. cereus, in other Bacillus species, consistent with earlier reports of this gene in B. coagulans, B. thuringiensis, B. alvei, and B. brevis, a distribution that has been attributed to horizontal gene transfer, the process by which bacteria exchange genetic material across species boundaries.

The study builds on and extends earlier work by some of the same researchers, who had screened 74 Bacillus isolates from cassava processing environments in Nigeria and found that 50 percent carried bceT, 47.3 percent carried hblD, and 37.8 percent carried nheA. Although the current study recovered fewer isolates, it documented a higher proportion encoding the nheB and nheC subunits. Together, the two investigations paint a consistent picture: enterotoxigenic Bacillus are not rare curiosities in Nigerian food processing environments but common residents of the waste streams those environments generate. Comparable surveys in other settings, from Tunisian foodstuffs to dairy products, vegetables, meat, and fermented millet, have likewise documented enterotoxin genes among Bacillus isolates, indicating that the phenomenon is global rather than local.

Why does this matter for public health? The route of transmission for pathogenic bacteria typically runs through contaminated soil, water, and plants, particularly vegetables. Effluents from these fermentation sites are discarded indiscriminately into open channels, uncompleted structures, undeveloped land, and along streets, where runoff can carry bacteria into surface and groundwater, onto crops, and into contact with people. Previous research has documented that fermentation effluents pollute soil and water, breed mosquitoes and other parasitic insects in stagnant channels, and contribute to the burden of insect-borne disease in Nigeria. The new findings add a microbial dimension to this environmental hazard: if toxin-gene-carrying Bacillus colonize processing equipment, raw materials, or finished products, they could trigger foodborne illness through point-source or non-point-source contamination, turning a traditional food practice into a vehicle for disease.

There is an important caveat to the study’s conclusions. The Bacillus species were identified presumptively using morphological and biochemical characterization rather than molecular methods, which means the species assignments carry some uncertainty. The researchers themselves acknowledge this limitation and call for molecular characterization of the enterotoxigenic isolates in future work to clarify their identities and potential. Gene presence also does not automatically equal toxin production; the PCR approach detects genetic potential rather than expressed protein. Nevertheless, the detection of enterotoxin genes in such a high proportion of environmental isolates is a legitimate warning sign, particularly because hbl toxins are known to produce extensive hemolytic and cytotoxic effects, and nhe toxins, while somewhat less deleterious, act through a similar pore-forming mechanism and have been shown to activate the NLRP3 inflammasome in host cells.

The authors argue that the solution lies in sanitation and regulation rather than in abandoning fermentation itself. They call for awareness programs aimed at artisanal fermenters, highlighting the dangers of indiscriminate effluent disposal and teaching appropriate disposal methods, alongside national and local regulatory guidelines with strict compliance requirements for the responsible handling of maize and African locust bean waste. In a country where fermentation centers serve a rapidly growing population using both traditional and mechanical techniques, the stakes are considerable. Fermented foods offer genuine nutritional and health benefits, and Bacillus species will remain indispensable to their production. The challenge, this study makes clear, is to manage the waste those processes generate before the environment transforms a beneficial microbiology into a public health liability.

Subject of Research: Enterotoxigenic Bacillus species in fermentation effluent-contaminated soils at maize and African locust bean processing sites in Nigeria

Article Title: Potential health and safety risk of enterotoxin-producing Bacillus species isolated from fermentation effluents discharged into the environment

Article References: Daramola, O. B., Torimiro, N., Omole, R. K., Okugbesan, T. O., Olubamise, O. J., Akinsola, I. T., & Akinfolarin, O. O. (2025). Potential health and safety risk of enterotoxin-producing Bacillus species isolated from fermentation effluents discharged into the environment. Discover Toxicology, 2(1), Article 3. https://doi.org/10.1007/s44339-025-00020-2

Image Credits: AI Generated

DOI: 10.1007/s44339-025-00020-2

Keywords: Bacillus, enterotoxins, fermentation effluents, food safety, foodborne illness, Nigeria, PCR, hemolysin BL, non-hemolytic enterotoxin, bceT, environmental contamination, fermented foods