3.1 Production of biosurfactant
3.1.1Availability of oil-soluble carbon sources
Utilizing waste motor oil or cooking oil as a single carbon source, the potential of strain GX7 to produce biosurfactants using oil-soluble carbon source were investigated (Fig. 1). Whether using waste oil or cooking oil as the carbon source, the surface tension of the fermentation liquid did not change significantly with the increase in carbon source concentration, which was similar to the blank control group. This could indicate that GX7 has a low utilization capacity for the oil-soluble carbon. The results were consistent with the previous experiments, which showed that GX7 had a low ability to utilize oleic acid in the carbon source optimization of the biosurfactant production [29]. Compared to water-soluble substrates like glucose and glycerol, these oils are rich in oleic acid and linoleic acid, which may be one of the reasons for low utilization[33]. Another reason is that waste motor oil and cooking oil contain more toxic compounds, including soluble monoaromatics like BTEX compounds, PAHs and heavy metals, which inhibits in the growth of GX7[34]. Studies have shown that used motor oil, even at lower concentrations, is highly toxic to microalgae[35].
3.1.2Availability of water-soluble carbon sources
When different concentrations of starch hydrolysate and wheat bran juice were added as carbon sources, the surface tension and emulsification index of the GX7 fermentation solution were obviously improved (Fig2). At a starch hydrolysate concentration of 1% in the medium, the surface tension of GX7 fermentation solution decreased to 27.35mN/m, and the emulsification index reached 39.87%, markedly higher than that of other groups (Fig2.a). As the concentration of starch hydrolysate increased, there were no significant changes observed in surface tension and emulsification index. Therefore, a concentration of 1% starch hydrolysate was determined to be optimal for the fermentation of strain GX7. The experiment with wheat bran juice had similar results (Fig.2.b). At a wheat bran juice concentration of 2% in the medium, the GX7 fermentation solution exhibited superior performance compared to other groups, with the surface tension decreasing to a minimum of 27.29 mN/m and the emulsification index reaching 40.52%. The main products of starch hydrolysis are oligosaccharides, including glucose syrup, syrup and dextrin, which serve as carbon sources for microbial synthesis of bacterial cellulose [36]. As a byproduct in the starch production process, wheat bran contains cellulose, hemicellulose, and lignin, serving as sufficient carbon sources during microbial fermentation processes[37]. The economical availability and broad distribution of these agricultural wastes reduce the cost of industrial fermentation production. Previous studies have also demonstrated the viability of agricultural waste utilization. Rivera has established a hydrocarbon-degrading consortium using papaya and mango as carbon sources, achieving promising results in diesel degradation[38]. Tatyana investigated the production of 17 pectinolytic enzymes using wheat bran as a substrate with Aspergillus niger 18FSDE16 via semi-solid-state fermentation (SSSF)[37]. Soumya investigated the production of EPS by a Lactobacilli using cassava starch hydrolysate as raw material, and indicated that starch hydrolysate could serve as a cost effective carbon source,potentially replacing the synthetic pure sugars[36].The results show that agricultural waste has great potential as a carbon source of petroleum degrading bacteria and has a positive effect on the production of biosurfactants from GX7.
3.1.3 Availability of corn steep liquor as a nitrogen source
The by-product of corn starch production via wet milling is corn steep liquor, which can provide essential nutrients such as amino acids, vitamins, and polypeptides for microbial fermentation as an excellent source of organic nitrogen[39]. Incorporating corn steep liquor as the sole nitrogen source in the medium showed significant experimental results (Fig.3). Compared to the blank control group, even a 0.1% concentration of corn steep liquor had a notable impact on the fermentation of strain GX7. The surface tension of the fermentation solution decreased to 28.04mN/m, indicating that Bacillus cereus GX7 can efficiently utilize corn steep liquor as a nitrogen source for fermentation, thereby producing surfactant. In Yang' s study, corn steep liquor enhanced the growth and production of B. subtilis, with lower concentrations boosting 2,3-butanedioland acetoin production, and higher concentrations improving microbial growth[40]. Therefore, corn steep liquor can serves as an economical and effective nitrogen source.
3.2 Application of GX7 and its surfactants in the remediation of oily sand
To evaluate the efficacy of the biosurfactant produced by GX7 in promoting crude oil degradation, an experimentation was designed to test the removal efficiency of crude oil using either the biosurfactant or chemical surfactants over a period of 3 days. As shown in Fig.4.a, significant crude oil removal efficiency was observed in the oily sand treated with the biosurfactant group within 24h, which remained stable until the third day. The crude oil removal efficiency ultimately reached 14.63%. Compared with the biosurfactant group, the removal efficiency of crude oil in sand is enhanced by chemical surfactants, with sodium dodecyl sulfonate and sodium dodecyl sulfate achieving superior removal rates, reaching approximately 25% (Fig.4.b). However, the critical micelle concentration of these two chemical surfactants are significantly higher than those of other surfactants. Although they exhibit better crude oil removal efficacy, their large required dosages increase application costs. Furthermore, studies have shown that all types of chemical surfactant exhibit acute toxicity to crustaceans[41]. Therefore, considering the environmental benefits, the eco-friendliness of biosurfactants is of great significance for crude oil remediation.
3.3 Biosurfactant-Enhanced Bioremediation in diesel polluted seawater
3.3.1 Diesel biodegradation
The ability of the biosurfactant produced by GX7 on promotion of the diesel oil degradation in seawater is shown in Fig. 5. The biodegradation rate of the treatments with added indigenous degrading bacteria (BD) or GX7 are 57.62% and 45.89% respectively. However, the biodegradation rate of the treatments with both BD+GX7 reached 70.36%, while the treatment with BD+FJ increased to 94.38%. It demonstrated that the biosurfactant produced by GX7 can effectively promote the degradation of petroleum hydrocarbons. Furthermore, the group with only BD had an exceeding oil degradation effect in seawater compared to only GX7. It may be that GX7 could not adapt to high salinity seawater, thus affecting its survival rate. The results of community structure analysis also confirmed that the GX7 had a low abundance in seawater. Although GX7 had a poor survival in seawater, its inoculation can cooperate with indigenous bacteria to enhanced the diesel degradation efficiency of contaminated seawater.
3.3.2 Analysis of degradation product
The composition of total petroleum hydrocarbon in treatments with both BD and GX7, as well as in the treatment with BD and fermentation supernatant of GX7, was further investigated by GC-MS. The changes of petroleum hydrocarbon components before and after treatment in the two groups, as well as the degradation rate of each alkane are illustrated in Fig. 6. The BD+GX7 and BD+fermentation supernatant groups showed 75.09% and 81.84% in total alkane degradation rate, respectively(Fig.6.b). When fermentation liquid and indigenous bacteria were added to the diesel polluted seawater, the content of short-chain alkanes (C11-C15) was completely degraded within 7 days. This is because short-chain alkanes are easily fragmented and degraded by microbes. Alkanes (C16-C20) were found to be residual, with the degradation rate exceeding 80% for all except C16, and the substance analysis showed that the content of dibutyl phthalate in the residual components increased(Fig.6.a). It is speculated that microorganisms first decompose easily available short-chain hydrocarbons and then break long-chain hydrocarbons into short chains for further utilization[42]. During the degradation process, the fragmentation of long chain substances leads to the formation of C16 hydrocarbons. The increase of dibutyl phthalate is consistent with earlier studies, which identified n-alkanes as intermediate degradation products with high carbon content, resulting in a slower degradation rate[37]. The degradation rate of each alkane in group BD+GX7 was not as high as group BD+fermentation supernatant, but showed s similar trend.
3.4 Microbial community analysis
To understand the changes of microbial community structure during the remediation of diesel polluted seawater, the microbial 16S rRNA genes at the genus levels were classified. The Circos plot reflects the abundance and composition of genera across different groups at the taxonomic level. As illustrated in Fig.7.b, after 7 days, the dominant genus in BD group was Enterobacter (97.58%), with Pseudomonas also existed at a low abundance. Enterobacter had several metabolic pathways and enzyme-encoding genes related to oil degradation by genome analysis[43]. In group BD+ GX7, the dominate genus were Pseudomonas(47.26%), Stenotrophomonas(33.78%) and Achromobacter(7.89%). The microbial community structure of the BD+fermentation supernatant group closely resembled that of BD+GX7. Pseudomonas (42.39%) and Stenotrophomonas (30.42%) were dominant, while Enterobacter (14.82%) and Achromobacter (5.93%) also showed a high abundance, with a minor presence of Brucella, Pseudomonas and Stenotrophomonas are genus that widely exist in soil and seawater. Pseudomonas had significant petroleum degradation potential in seawater under nutrient limited conditions assisted by biosurfactant with low CMC (15.0 g/L)[44]. Stenotrophomonas has shown promising potential in the degradation of polycyclic aromatic hydrocarbons (PAHs), possessing essential genes involved in the PAHs degradation[45]. Although Bacillus GX7 did not dominate in the treatment in the current study, Wang et al. proposed that despite the decrease in abundance of the target bacteria after inoculation, it could still contribute to enhancing composition of indigenous microbial communities[42]. The BD+GX7 group had lower abundance of Shannon index compared with BD +FJ group, especially the quantity of Bacillus, it may due to the concentration of NaCl in the medium limited its growth. Which also echoed the previous experiments in which the degradation effective of GX7 on diesel oil was lower than that of the fermentation broth(Fig.7.a). The results showed that the biosurfactant produced by GX7 can regulate the community structure in seawater and enhance the remediation effect of contaminated seawater.