Objectives High concentrations of heavy metals can cause severe stress damage to microorganisms, thereby limiting their large-scale application in the remediation of heavy metal pollution. This article aims to investigate the protective effect of biomass-based collagen peptide (BCP) on Bacillus licheniformis against chromium stress, and to reveal the intrinsic correlation between the amino acid composition of BCP and its protective efficacy, thereby providing new ideas for the protection of microorganisms against heavy metal stress.

Methods Taking BCP as the research object and B. licheniformis as the experimental strain, a systematic study was conducted on the chromium stress protection performance of BCP on B. licheniformis. BCP was introduced into the chromium-containing medium (PS_Cr) to explore the effect of BCP on the growth behavior of B. licheniformis under chromium stress, in which the viable cell concentration was quantified by colony forming units count method and the cell morphology was observed using scanning electron microscopy (SEM). Additionally, different concentrations of BCP were added into the PS_Cr media to evaluate the effect of BCP concentration on the cell growth. Moreover, the chromium stress protection performance of bovine liver peptide, corn peptide, soybean peptide and BCP were compared, and the contents of glycine (Gly), proline (Pro) and hydroxyproline (Hyp) in peptides were determined. The influence of free amino acids including Gly, Pro and Hyp on the chromium stress tolerance of B. licheniformis was investigated. The elemental distribution on the surface of B. licheniformis was analyzed using energy dispersive spectroscopy to further elucidate the intrinsic correlation between the amino acid composition of BCP and its protective efficacy.

Results and Discussion High concentrations of chromium caused severe stress damage to B. licheniformis, but its viable concentration increased by 4-fold after 6 h with the introduction of BCP (Fig. 1). Numerous shrunken and concave cells were observed after B. licheniformis was cultured in the PS_Cr medium for 120 h. However, the cells exhibited a predominantly plump morphology in the presence of BCP, and the proportion of damaged cells was obviously lower than that in the PS_Cr. The biomass of B. licheniformis was significantly increased to 124% when the added BCP concentration was 2 mmol/L (Fig. 2). Moreover, OD₆₀₀ further increased with increasing BCP concentration, demonstrating that the chromium stress protection of B. licheniformis by BCP is concentration-dependent. BCP showed the highest contents of Gly, Pro and Hyp, coupled with greater stress-protective efficacy, compared with bovine liver, corn, and soybean peptides (Fig. 3). Supplementation of the chromium stress medium with Gly, Pro and Hyp resulted in 9.80%, 15.08% and 35.97% increase in OD₆₀₀ at 120 h, respectively (Fig. 4). Meanwhile, cell integrity and stability were greatly maintained under the protection of Gly, Pro and Hyp, with a substantial distribution of chromium on the cells surface (Fig. 5). These results indicate that the chromium stress protection of BCP on Bacillus licheniformis is critically linked to its unique amino acid composition.

Conclusion BCP can markedly enhance the survival of B. licheniformis under chromium stress. High concentrations of chromium cause severe stress damage to the cell morphology of B. licheniformis, while BCP can effectively maintain the cell morphology to resist chromium stress. The chromium stress protection of B. licheniformis by BCP is concentration-dependent. BCP exhibited more robust stress protection performance, compared with other peptides. Gly, Pro and Hyp can effectively enhance the tolerance of B. licheniformis against chromium stress, suggesting that the protection of BCP is closely related to its specific amino acid composition. Therefore, BCP possesses significant potential for application in the protection of microbial heavy metal stress.