Combined effects of brewer’s spent grain biostimulation and Bacillus bioaugmentation on crude oil biodegradation in contaminated soil

Abstract

Petroleum hydrocarbon contamination is a major environmental challenge because of its persistence and adverse effects on soil quality and ecosystem functioning. This study evaluated the combined effects of brewer’s spent grain (BSG) biostimulation and Bacillus bioaugmentation on crude oil biodegradation in contaminated soil. The study was performed using standard microbiological and physicochemical procedures to assess the composition of BSG, soil properties, microbial populations, and petroleum hydrocarbon degradation during remediation. Soil microcosms were amended with 1%, 10%, and 20% BSG and bioaugmented with Bacillus subtilis. Heterotrophic bacterial abundance (HBA), oil-degrading bacteria (ODB), Nitrosomonas, and Nitrobacter populations were monitored during the remediation period, while residual petroleum hydrocarbons were determined at the end of the experiment. The experimental data were subjected to analysis of variance (ANOVA) to determine differences among treatments. The composition of BSG included organic carbon of 76.0 ± 0.84%, protein of 27.5 ± 0.36%, nitrogen of 2.33 ± 0.05%, and a calorific value of 399.4 ± 0.35 kcal, indicating its suitability as a nutrient-rich amendment for stimulating microbial activity. The soil was acidic, with a pH of 5.05 ± 0.18, 7.15 ± 0.25 mg/kg total petroleum hydrocarbons, and 0.57 ± 0.21 mg/kg polycyclic aromatic hydrocarbons. The heterotrophic bacterial abundance of 7.30 ± 0.32 log₁₀ CFU/g and hydrocarbonoclastic bacterial abundance of 2.60 ± 0.61 log₁₀ CFU/g included genera such as Bacillus, Pseudomonas, Alcaligenes, and Micrococcus sp., indicating organisms with intrinsic biodegrading potential. HBA and ODB populations in Controls 1 and 2 differed significantly from those in the amended and treated soils (ANOVA, p < 0.05), indicating enhanced microbial proliferation following nutrient supplementation and bioaugmentation. Biostimulation with 1%, 10%, and 20% BSG amendments improved soil physicochemical conditions, with pH values ranging from 7.26 ± 0.03 to 8.21 ± 0.03, redox potential from 54.0 ± 0.2 to 90.0 ± 0.1 mV, and moisture content from 14.97 ± 0.06 to 16.52 ± 0.43% compared with the untreated controls. Combined treatment stimulated microbial proliferation, with the 10% BSG treatment recording the highest HBA of 8.82 ± 0.88 log₁₀ CFU/g, while oil-degrading bacteria were enriched during remediation. Bioaugmentation with Bacillus subtilis alone resulted in 49.93% hydrocarbon degradation, whereas combined bioaugmentation and BSG biostimulation improved degradation efficiencies to 88.58%, 76.85%, and 68.61% in the 1%, 10%, and 20% BSG treatments, respectively. Preferential degradation of low- and mid-molecular-weight hydrocarbons (C10–C22) and substantial reduction of biodegradation indicator compounds such as pristane and phytane indicated enhanced microbial activity. Although 10% BSG supported heterotrophic bacterial abundance, the 1% BSG treatment achieved the highest hydrocarbon degradation efficiency, suggesting that moderate nutrient supplementation through BSG amendment, rather than excessive organic loading, favored effective biodegradation. These findings indicate that synergy between nutrient-driven microbial stimulation, functional hydrocarbon degraders, and optimized nutrient amendment strategies enhanced bioremediation efficiency and support the sustainable use of agro-industrial wastes as effective biostimulants for environmental remediation.