Biosurfactant production by Bacillus cereus GX7 utilizing organic waste and its application in the remediation of hydrocarbon-contaminated environments

Biosurfactant production by Bacillus cereus GX7 utilizing organic waste and its application in the remediation of hydrocarbon-contaminated environments | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biosurfactant production by Bacillus cereus GX7 utilizing organic waste and its application in the remediation of hydrocarbon-contaminated environments Yunyun Zhang, Jin Gao, Qintong Li, Jingjing Yang, Yu Gao, Jianliang Xue, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4721567/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Oct, 2024 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 17 You are reading this latest preprint version Abstract The use of biosurfactants represents a promising technology for remediating hydrocarbon pollution in the environment. This study evaluated a highly effective biosurfactant strain- Bacillus cereus GX7’s ability to produce biosurfactants from industrial and agriculture organic wastes. GX7 showed poor utilization capacity for oil soluble organic waste but effectively utilized of water soluble organic wastes such as starch hydrolysate and wheat bran juice as carbon sources to enhance biosurfactant production. This led to significant improvements in surface tension and emulsification index. Corn steep liquor was also effective as a nitrogen source for GX7 in biosurfactant production. The biosurfactants produced by strain GX7 demonstrated a remediation effect on oily beach sand, but are slightly inferior to chemical surfactants. Inoculation with GX7 (70.36%) or its fermentation solution (94.38%) can effectively enhanced the degradation efficiency of diesel oil in polluted seawater, surpassing that of indigenous degrading bacteria treatments (57.62%). Moreover, Inoculation with GX7’s fermentation solution notably improved the community structure by increasing the abundance of functional bacteria such as Pseudomonas and Stenotrophomonas in seawater. These findings suggest that the GX7 as a promising candidate for bioremediation of petroleum hydrocarbons. Biosurfactants Bacillus cereus GX7 Organic waste application Petroleum hydrocarbons pollution Bioremediation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Bacillus cereus GX7 effectively utilize water-soluble organic wastes as the sole carbon and nitrogen sources to produce surfactant . GX7 and its biosurfactant efficiently degrade petroleum hydrocarbons in polluted seawater collaborated with indigenous bacteria. GX7 and its biosurfactant optimized the microbial community structure for remediating diesel-contaminated seawater. 1. Introduction Globally, petroleum production and utilization serve as pivotal elements in the energy landscape, underpinning various sectors including transportation, manufacturing, and energy generation. In 2023, the world's petroleum production was estimated at approximately 100 million barrels per day, catering to a vast array of energy demands across the globe[1]. Despite its critical role in economic development, the extensive use of petroleum has precipitated substantial environmental and health challenges[2]. Petroleum hydrocarbon pollution primarily arises from oil spills, oily wastewater discharge, pipeline leaks, and extraction activities, significantly contaminating soil and water[3-4]. Aromatic hydrocarbons in petroleum, particularly polycyclic aromatic hydrocarbons (PAHs) such as bicyclic and tricyclic hydrocarbons, are highly toxic to humans and animals. PAHs can enter the body through inhalation, skin contact, and dietary intake, adversely affecting the normal functioning of vital organs like the liver and kidneys, and may even lead to cancer[5]. Therefore, the development of effective remediation techniques of petroleum pollution is urgently needed[6]. Compared to conventional physio-chemical treatment methods which incur high maintenance cost, consume significant and generate toxic intermediate products during the remediation process[7-8], bioremediation techniques are environmentally friendlyand cost-effective[9]. Therefore, bioremediation is considered as the most promising technology in petroleum contamination remediation. Bioremediation techniques include biostimulation, which enhances the activity of indigenous microorganisms, and bioaugmentation, which involves adding specific degrading microbes in environments[6]. In essence, it is the decomposition of petroleum hydrocarbons by petroleum degrading microorganisms during their growth and metabolism. However, petroleum bioavailability and microbial activities are usually limited due to the low solubility and hydrophobicity of petroleum hydrocarbons[10]. The sticky nature of oil, combined with its ability to be easily absorbed by rocks and sand, makes it particularly challenging to remove once discharged into the environment[11]. Therefore, improving the solubility of petroleum hydrocarbons is crucial for enhancing their biodegradation[12].Some bacteria possess specific enzymatic abilities that enable them to produce surfactants, which facilitate the emulsification of hydrocarbon[13]. The utilization of biosurfactants has been effective addressing the issue of bioavailability and is often recommended to enhance remediation efforts[6]. This can be achieved by introducing biosurfactant-producing microorganisms or applying microbial biosurfactants directly to contaminated sites[3]. This process significantly accelerates the degradation rate of petroleum hydrocarbons by increasing their bioavailability to microbial action[14]. Biosurfactants were first discovered in the late 1960s as extracellular amphiphilic compounds produced during hydrocarbon fermentation[15]. These molecules are amphipathic, containing both hydrophilic and hydrophobic groups, which enable them to significantly reduce the surface and interfacial tensions. By attaching to various hydrophobic compounds, biosurfactants increase the solubility of petroleum, facilitating its degradation in environmental remediation processes[16-17]. In addition, biosurfactants possess distinct advantages over chemical surfactants, which have increasingly been recognized for their poor environmental stability and the potential to contribute to chemical pollution. In contrast, biosurfactants are biodegradable and generally exhibit lower toxicity, making them more environmentally friendly alternatives[18]. In the remediation of petroleum contaminated soil, the use of biosurfactants significantly enhance biological functions within the soil, such as soil conductivity and core enzyme activities involved in the degradation of petroleum hydrocarbon. Furthermore, they facilitate quorum sensing among different species[19]. This suggests that biosurfactants have a less negative impact on the microbial community in the soil compared to other remediation agents. Therefore, biosurfactants hold great potential for enhancing bioremediation processes and are extensively utilized in environment protection efforts[20]. Several factors influence the process of hydrocarbon degradation facilitated by biosurfactants[21]. Beyond temperature, agitation speed of culture, pH and salinity, the availability of limiting nutrients also plays a crucial role in the production and effectiveness of biosurfactants[22-25], impacting both the quantity and quality of the biosurfactants produced, which in turn affects their efficacy in degrading hydrocarbons. Furthermore, high cost of the raw materials required for fermentation can be a significant barrier to the practical application of biosurfactants[26]. To address this problem，researchers are exploring the use of waste or low-cost substrate as renewable substrates to increase the production of biosurfactant, such as crude palm oil, molasses, cashew apple juice[27-28]. However, there is limited research on the utilization of industrial waste oil and by-products from food production as substrates for biosurfactant production. Bacillus cereus GX7, isolated from oil sludge, was identified as a notably proficient producer of surfactant producer in our preceding research[29]. In this research, we have explored the feasibility of using industrial and agricultural waste as nutrient source for the biosurfactant production with B acillus cereus GX7, reducing production costs and environmental impact. Additionally, we investigated the potential of employing GX7 and its produced surfactants in the remediation of oil pollution in soil and seawater environments. 2. Materials and methods 2.1Materials collection Chemical reagents used in this study including hydrochloric acid, chloroform, methanol, sodium chloride, glucose were of analytical grade. The crude oil was obtained from Venezuela(10°3' N, 66°58' W), sterilize by ultraviolet lamp before using. The waste cooking oil used was collected from a local restaurant in the city of Qingdao, China. The waste motor oil was provided by a local repair center in Qingdao, China. The starch hydrolysate, wheat bran juice and corn soak liquid were provided by Qingdao Haidar Starch Co., LTD. All collected samples were sealed in the sterile glass bottle and kept in a cool and dry place for further use. 2.2Bacterial strain and growth conditions The bacterium ( Bacillus cereus GX7) was previously isolated from the bottom sludge of the oil storage tank in Shengli Oilfield, China. The Luria-Bertani (LB) culture utilized for fermentation was composed of 10 g/L peptone, 5 g/L yeast, 5 g/L NaCl, 10-15 g/L agar (solid medium) and 4 mL/L trace element solution. The trace element solution contains 2 mg/L of CaCl 2 , 50 mg/L of FeCl 3 ·6H 2 O, 2 mg/L of CuSO 4 ; 10 mg/L of ZnSO 4 ·7H 2 O. After adjusting the pH to 7.5, the medium was sterilized at 121℃ for 20 min. GX7 was cultured in LB medium at 150 r/min and 30 ℃ for 2d to obtain the fermentation liquid. 2.3Experimental design 2.3.1 Utilization of industrial and agricultural waste to produce biosurfactants During the fermentation process of biosurfactants, waste cooking oil and waste motor-oil were employed as oil-soluble carbon sources, while starch hydrolysate was utilized as water-soluble carbon sources, and corn steep liquid was used as a nitrogen sources, respectively. After filtration under reduced pressure to remove solid impurities and bacteria, the samples were added to the medium to obtain the desired concentrations (0.1%, 0.5%, 1%, 2%, 3% w/v) as the carbon/nitrogen source, without any chemical supplementation. The bacterial solution was inoculated into the medium at a 1% (v/v) inoculation rate, and the medium without a carbon source or nitrogen source was used as a blank control. The emulsification index (E24) assay and surface tension measurement of fermentation liquid was carried out to evaluate the feasibility of GX7 producing biosurfactants from various substances. Three parallel samples were prepared for all experiments. 2.3.2 Remediation of crude oil-contaminated beach sand Beach sand with a diameter of 0.150-0.180 mm was washed 6 times with 1 mol/L HCl solution and then rinsed with tap water until the pH was neutral. Two grams of crude oil was added into 50 g beach sand and thoroughly mixed in a 100 mL conical flask, with the initial weight of crude oil denoted G1. The fermentation broth of GX7 was centrifuged to remove the bacteria, and subsequently, 50mL of supernatant was addedto the oil-contaminated beach sand system for a crude oil degradation experiment conducted at 30℃, 150 rpm. Simultaneously, 50 mL of five commercially available chemical surfactants at their critical micelle concentrations (CMC) were added to the oil-contaminated beach sand system: Tween 80 (0.013 g/L), Triton X-100 (0.15 g/L), sodium dodecyl sulfonate (3.015 g/L), sodium dodecyl benzene sulfonate (0.418 g/L), and sodium dodecyl sulfate (2.304 g/L). Samples were periodically collected to assess the degradation efficiency of crude oil. 2.3.3 Remediation of diesel oil contaminated seawater To investigate the mechanism of biosurfactant produced by strain GX7 during hydrocarbon degradation, four groups of remediation experiments were conducted to simulate diesel oil pollution in seawater: (1) Marine indigenous bacteria (hereinafter referred to as BD) only, (2) GX7 only, (3) GX7 and BD, (4) The fermentation liquid of GX7 after centrifugation (hereinafter referred to as FJ) and BD. The seawater utilized was collected from Jiaozhou Bay, China. For group (2), the seawater was filtrated by 0.45 μm membrane to remove the impurity and bacteria. The concentration of diesel oil in simulated seawater was 10 g/L, and recorded as C 0 . After 7 days of seawater remediation experiment at 30℃ and 150rpm, the residual diesel content was measured to calculate the diesel degradation rate, while the microbial community was analyzed. 2.4Analytical method 2.4.1Crude oil/Diesel oil degradation rate The supernatant from the conical bottle was decanted from the sand, and n-hexane was added to the supernatant, thoroughly shaken, and then transferred to a separatory funnel for extraction. This process was repeated three times until the organic phase is completely clarified, after which the n-hexane phases are combined. After removing the n-hexane and water using rotary evaporation at 35℃,weighing the remaining crude oil and denoted as G2. The removal rate of crude oil is calculated as G2/G1×100%. The remaining diesel oil in the seawater is also extracted with n-hexane, the diesel concentration (C t ) in organic phase was detected by ultraviolet spectrophotometry[30]. The diesel oil degradation efficiency was calculated by the following equation: R (%)=(C 0 −C t )/C 0 ×100%. 2.4.2 Determination of surface tension and emulsification index The surface tension of the fermentation solution was determined by the platinum ring method with an automatic surface tensiometer (BZY-102, Shanghai Fangrui Instrument Co., LTD.). Before measuring the surface tension, the platinum ring is thoroughly rinsed with acetone and dried at 40℃. The emulsification index (E24) was determined according to the procedure outlined by Satpute et al. [31]. Specifically, 5mL of diesel oil was added to an equal volume of cell-free supernatant, which was then vigorously vortexed for two minutes at high speed. The mixture was subsequently allowed to stand at room temperature for 24 hours. After this period, the height of the emulsion layer was measured relative to the total height of the mixture to calculate the emulsification index. 2.4.3 Identification of intermediates in seawater by GC – MS Diesel oil in seawater was analyzed using an Agilent 7890A gas chromatograph coupled with an Agilent 7000 mass spectrometer. Helium was used as the carrier gas at a flow rate of 1 mL/min. The temperature program started at 70℃ for 2 min, ramped up at 20 ℃/min to 230℃, further increased at 40℃/min to 300℃, and held at 300℃ for 10 min. Identification of components was performed by comparing their mass spectra with those in the NIST mass spectrometry library. 2.4.4 Characteristics of microbial community Microbial diversity and community composition of different group after 7 days of degradation were analyzed by 16 S rRNA gene amplification，The hypervariable V3-V4 region of the 16 S rRNA gene was selected for amplification using primers 341 F (CCTACGGGNGGCWGCAG) and 806 R (GGACTACHVGGGTWTCTAAT)[32]. The sequence was determined by Shanghai Meiji Technology Co., Ltd. 3. Results and discussion 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 d egradation 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 Achromobacte r (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. 4. Conclusion Bacillus cereus GX7 can effectively utilize water-soluble organic wastes as carbon and nitrogen sources to enhance surfactant production but cannot effectively use oil-soluble organic waste as nutrients. The biosurfactant produced by GX7 has a removal rate of 14.63% to crude oil in oily sand, which is slightly lower than that of chemical surfactants. In the remediation of marine diesel pollution, GX7 and its fermentation broth can collaborate with indigenous bacteria to improve the degradation rate of diesel oil, reaching 94.38% and 70.36% respectively in seawater supplemented with fermentation supernate and strain GX7. The results of community structure showed that the addition of GX7 and its fermentation solution affected the community structure and increase the abundance of dominant strains ( Pseudomonas and Stenotrophomonas ). This study is an important reference for the utilization of biosurfactant strategies in addressing water and soil contamination. Declarations Author Contribution A.wrote the main manuscript text and prepared figures 1-6B.Methodology and prepared figure 7C.D.Methodology and Formal analysisE.Writing – review &editing, Validation, Resources, Project administration, SupervisionF.G.H.Methodology and Formal analysis Acknowledgments This work was supported by the National Natural Science Foundation of China [grant number 52374174]; Project of Shandong Province Higher Educational Young Innovative Talent Introduction and Cultivation Team [Wastewater treatment and resource innovation team]. References Sean Hill. Four countries could account for most near-term petroleum liquids supply growth[N]. Today in Energy, 2024-3.14.https://www.eia.gov/todayinenergy/detail.php?id=61583. Kartik Patel;Mitesh Patel.Improving bioremediation process of petroleum wastewater using biosurfactants producing Stenotrophomonas sp. S1VKR-26 and assessment of phytotoxicity[J].Bioresource technology,2020,Vol.315: 123861 Ping Guo;Wei-Wei Xu;Dan-Na Wei;Man-Xia Zhang;Jin Zhang;Shi Tang;Bin-Xia Cao;Jian-Guo Lin;Wei Li.Potential Application of Biosurfactant-Producing Bacteria for Bioremediation of Oil Polluted Marine Intertidal Sediments[J].Journal of Marine Science and Engineering,2022,Vol.10(6): 731 Chuan Yin;Huan Yan;Yuancheng Cao;Huanfang Gao.Enhanced bioremediation performance of diesel-contaminated soil by immobilized composite fungi on rice husk biochar[J].Environmental Research,2023,Vol.226: 115663 Israa Abdulwahab Al-Baldawi;Siti Rozaimah Sheikh Abdullah;Nurina Anuar;Fatihah Suja;Idris Mushrifah.Phytodegradation of total petroleum hydrocarbon (TPH) in diesel-contaminated water using Scirpus grossus.[J].Ecological Engineering,2015,Vol.74: 463-473 I. Moumed;J. Arrar;A. Namane & …F. Bentahar.Effects of surfactant and oxidant on bioremediation of contaminated soil by total petroleum hydrocarbons using indigenous bacteria[J].International Journal of Environmental Science and Technology,Vol.20(8): 8863-8874 Tahir Haneef;Muhammad Raza Ul Mustafa;Kashif Rasool;Yeek Chia Ho;Shamsul Rahman Mohamed Kutty.Removal of Polycyclic Aromatic Hydrocarbons in a Heterogeneous Fenton Like Oxidation System Using Nanoscale Zero-Valent Iron as a Catalyst[J].Water,2020,Vol.12(2430): 2430 Xin Sui;Xuemei Wang;Yuhuan Li;Hongbing Ji.Remediation of Petroleum-Contaminated Soils with Microbial and Microbial Combined Methods: Advances, Mechanisms, and Challenges[J].Sustainability,2021,Vol.13(9267): 9267 Molaei S;Moussavi G;Talebbeydokhti N;Shekoohiyan S.Biodegradation of the petroleum hydrocarbons using an anoxic packed-bed biofilm reactor with in-situ biosurfactant-producing bacteria.[J].Journal of hazardous materials,2022,Vol.421: 126699 Phan, Chia-Wei;Abu Bakar, NurFaizah;Hamzah, Ainon.A Comparative Study on Biosurfactant Activity of Crude Oil–Degrading Bacteria and Its Correlation to Total Petroleum Hydrocarbon Degradation.[J].Bioremediation journal,2013,Vol.17(4): 240-251 M. Gloria Pereira; Stephen M. Mudge.Cleaning oiled shores: laboratory experiments testing the potential use of vegetable oil biodiesels[J].Chemosphere,2004,Vol.54(3): 297-304 Brakstad, Odd G.( [email protected] );Bonaunet, Kristin.Biodegradation of petroleum hydrocarbons in seawater at low temperatures (0-5 °C) and bacterial communities associated with degradation.[J].Biodegradation,2006,Vol.17(1): 71-82 Jenyffer M. Campos;Tânia L. M. Stamford;Leonie A. Sarubbo.Production of a bioemulsifier with potential application in the food industry.[J].Appl Biochem Biotechnol,2014,Vol.172(6): 3234-3252 Ashis K Mukherjee;Kishore Das.Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat.[J].FEMS microbiology ecology,2005,Vol.54(3): 479-489 Ali Khan, Aqib Hassan;Tanveer, Sundus;Alia, Shagufta;Anees, Mariam;Sultan, Aneesa;Iqbal, Mazhar;Yousaf, Sohail.Role of nutrients in bacterial biosurfactant production and effect of biosurfactant production on petroleum hydrocarbon biodegradation[J].ECOLOGICAL ENGINEERING,2017,Vol.104: 158-164 Joy, Sam.Biosurfactant producing bacteria from hydrocarbon contaminated environment[J].Environmental Footprints and Eco-Design of Products and Processes,2017,: 259-305 Luna, Juliana M;Rufino, Raquel D;Sarubbo, Leonie A;Bezerra, Karen Gercyane O.Saponins and microbial biosurfactants: Potential raw materials for the formulation of cosmetics[J].Biotechnology Progress,2018,Vol.34(6): 1482-1493 Swati Sharma;Rahul Verma;Lalit M. Pandey.Crude oil degradation and biosurfactant production abilities of isolated Agrobacterium fabrum SLAJ731(Article)[J].Biocatalysis and Agricultural Biotechnology,2019,Vol.21: 101322 Xuliang Zhuang;Yaxin Wang;Haoyu Wang;Yuzhu Dong;Xianglong Li;Shijie Wang;Haonan Fan;Shanghua Wu.Comparison of the efficiency and microbial mechanisms of chemical- and bio-surfactants in remediation of petroleum hydrocarbon[J].Environmental Pollution,2022,Vol.314: 120198 Qingguo Chen; Jingjing Li; Liu Mei; Huiling Sun; Mutai Bao.Study on the biodegradation of crude oil by free and immobilized bacterial consortium in marine environment.[J].PLoS ONE,2017,Vol.12(3): 1-23 Soumen Mukherjee;Palashpriya Das;Ramkrishna Sen.Towards commercial production of microbial surfactants.[J].Trends in biotechnology,2006,Vol.24(11): 509-515 C Amézcua-Vega;H M Poggi-Varaldo;F Esparza-García;E Ríos-Leal;R Rodríguez-Vázquez.Effect of culture conditions on fatty acids composition of a biosurfactant produced by Candida ingens and changes of surface tension of culture media.[J].Bioresource technology,2007,Vol.98(1): 237-240 Ana Eliza Zeraik;Marcia Nitschke.Biosurfactants as Agents to Reduce Adhesion of Pathogenic Bacteria to Polystyrene Surfaces: Effect of Temperature and Hydrophobicity.[J].Current Microbiology,2010,Vol.61(6): 554-559 F.J.S. Oliveira;L. Vazquez;N.P. de Campos;F.P. de Frana.Production of rhamnolipids by a Pseudomonas alcaligenes strain.[J].Process Biochemistry,2009,Vol.44(4): 383-389 Abouseoud, M;Yataghene, A;Amrane, A;Maachi, R.Effect of pH and salinity on the emulsifying capacity and naphthalene solubility of a biosurfactant produced by Pseudomonas fluorescens[J].Journal of Hazardous Materials,2010,Vol.180(1): 131-136 Verma, Rahul;Sharma, Swati;Kundu, Lal Mohan;Pandey, Lalit Mohan.Experimental investigation of molasses as a sole nutrient for the production of an alternative metabolite biosurfactant(Article)[J].Journal of Water Process Engineering,2020,Vol.38: 101632 Rocha, MVP;Souza, MCM;Benedicto, SCL;Bezerra, MS;Macedo, GR;Pinto, GAS;Goncalves, LRB.Production of biosurfactant by Pseudomonas aeruginosa grown on cashew apple juice[J].Applied Biochemistry and Biotechnology,2007,Vol.137-140(1-12): 185-194 Suryanti, V.;Handayani, D.S.;Masykur, A.;Lindasari.Optimization Production of Biosurfactant by Pseudomonas putida Using Crude Palm Oil (CPO) as Substrate[J].IOP Conference Series: Earth and Environmental Science,2017,Vol.75(1): 012013 Xiaoyan Wang, Jin Gao, Yu Gao*, Linlin Zhang, Congchao Xu, Qintong Li, Lin Li, Jianliang Xue. Analysis of surfactant production by Bacillus cereus GX7 and optimization of fermentation conditions. [J] Colloids and Surfaces B: Biointerfaces, 2024, 233: 113629. DOI10.1016/j.colsurfb.2023.113629 Shi K, Xue JL, Xiao XF, Qiao YL, Wu YN, Gao Y. Mechanism of degrading petroleum hydrocarbons by compound marine petroleum-degrading bacteria: surface adsorption, cell uptake, and biodegradation. Energy Fuels. 2019;33:11373–9. Satpute, S.K., Banpurkar, A.G., Dhakephalkar, P.K., Banat, I.M., Chopade, B.A., 2010.Methods for investigating biosurfactants and bioemulsifiers: a review. Crit. Rev.Biotechnol. 30, 127–144. Suxiang Liu，Rui Sun，Mengmeng Cai，Yue Kong，Yu Gao*，Tonghuan Zhang， Xinfeng Xiao, Yanlu Qiao, Jianliang Xue, Guofu Huang. Petroleum spill bioremediation by an indigenous constructed bacterial consortium in marine environments, Ecotoxicology and Environmental Safety, 2022, 241: 113769. August 2022. 10.1016/j.ecoenv.2022.113769. Wadekar;S B Kale;A M Lali;D N Bhowmick;A P Pratap.MICROBIAL SYNTHESIS OF RHAMNOLIPIDS BY Pseudomonas aeruginosa (ATCC 10145) ON WASTE FRYING OIL AS LOW COST CARBON SOURCE.[J].Preparative biochemistry & biotechnology,2012,Vol.42(3): 249-266 Agarwal AK;Sharma M;Singh SK.Experimental investigations of heavy metal addition in lubricating oil and soot deposition in an EGR operated engine[J].Applied Thermal Engineering,2006,Vol.26(2): 259-266 Ramadass, Kavitha;Megharaj, Mallavarapu;Venkateswarlu, Kadiyala;Naidu, Ravi.Toxicity and oxidative stress induced by used and unused motor oil on freshwater microalga, Pseudokirchneriella subcapitata.[J].ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH,2015,Vol.22(12): 8890-8901 M.P. Soumya;Keerthi Sasikumar;Ashok Pandey;K. Madhavan Nampoothiri.Cassava starch hydrolysate as sustainable carbon source for exopolysaccharide production by Lactobacillus plantarum[J].Bioresource Technology Reports,2019,Vol.6: 85-88 Tatyana Patrício;Lucas Vitor de Araújo Santos;Ruan Dionízio da Silva;Renata Torres dos Santos e Santos;Normando Ribeiro-Filho;Sharline Florentino de Melo Santos;Flávio Luiz Honorato da Silva.Pectinases production using an exotic Caatinga passion fruit waste and wheat bran as a substrate for Clarification of grape juice[J].Food Bioscience,2024,: 104163 Sergio Valdivia-Rivera; Elizabeth Del Carmen Varela-Santos; Tannia Alexandra Quiñ.Production of hydrocarbon-degrading microorganisms using agricultural residues of Mangifera indica L. and Carica papaya as carbon source.[J].3 Biotech,2019,Vol.9(2): 43 Liu, Xiaoyan; Wang, Xinfeng; Xu, Jiaxing; Xia, Jun; Lv, Jinshun.Citric acid production by Yarrowia lipolytica SWJ-1b using corn steep liquor as a source of organic nitrogen and vitamins[J].Industrial Crops and Products,2015,Vol.78: 154-160 Yang, TW (Yang, Tao-Wei);Rao, ZM (Rao, Zhi-Ming);Zhang, X (Zhang, Xian);Xu, MJ (Xu, Mei-Juan);Xu, ZH (Xu, Zheng-Hong);Yang, ST (Yang, Shang-Tian).Effects of corn steep liquor on production of 2,3-butanediol and acetoin by Bacillus subtilis.[J].Process Biochemistry,2013,Vol.48(11): 1610-1617 Garcia, M. Teresa;Ribosa, Isabel ;Kowalczyk, Iwona ;Pakiet, Marta ;Brycki, Bogumil .Biodegradability and aquatic toxicity of new cleavable betainate cationic oligomeric surfactants.[J].Journal of Hazardous Materials,2019,Vol.371: 108-114 Ao Wang, Wenxian Fu, Yu Feng, Zhimin Liu, Donghui Song.Synergetic effects of microbial-phytoremediation reshape microbial communities and improve degradation of petroleum contaminants[J].Journal of hazardous materials,2022,Vol.429: 128396 Muhammad Zohaib Nawaz, Chunyan Xu, Majjid A Qaria, Syed Zeeshan Haider, Hafiz Rameez Khalid, Huda Ahmed Alghamdi, Iqrar Ahmad Khan, Daochen Zhu.Genomic and biotechnological potential of a novel oil-degrading strain Enterobacter kobei DH7 isolated from petroleum-contaminated soil[J].Chemosphere,2023,Vol.340: 139815 Louella Concepta Goveas, Raja Selvaraj, Ramesh Vinayagam, Ahad Amer Alsaiari, Nahed S Alharthi, Shyama Prasad Sajankila.Nitrogen dependence of rhamnolipid mediated degradation of petroleum crude oil by indigenous Pseudomonas sp. WD23 in seawater[J].Chemosphere,2022,Vol.304: 135235 Temidayo Oluyomi Elufisan[a][b];Luis Lozano[c];Patricia Bustos[c];Isabel Cristina Rodríguez-Luna[a];Alejandro Sánchez-Varela[a];Omotayo Opemipo Oyedara[d];Miguel ngel Villalobos-López[e];Xianwu Guo[a].Complete Genome Sequence of Stenotrophomonas maltophilia Strain SVIA2,Isolated from Crude Oil-Contaminated Soil in Tabasco,Mexico[J].Genome Announcements,2019,Vol.8(30): e00529-19 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 03 Oct, 2024 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 21 Jul, 2024 Reviews received at journal 21 Jul, 2024 Reviews received at journal 21 Jul, 2024 Reviews received at journal 21 Jul, 2024 Reviews received at journal 19 Jul, 2024 Reviewers agreed at journal 14 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers agreed at journal 11 Jul, 2024 Reviewers invited by journal 11 Jul, 2024 Editor assigned by journal 11 Jul, 2024 Submission checks completed at journal 11 Jul, 2024 First submitted to journal 10 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4721567","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329833305,"identity":"3a753a32-0263-4032-ac0a-926d097d21c3","order_by":0,"name":"Yunyun Zhang","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yunyun","middleName":"","lastName":"Zhang","suffix":""},{"id":329833306,"identity":"710d7f63-71f0-450a-b2c2-c91e1b2fa210","order_by":1,"name":"Jin Gao","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Gao","suffix":""},{"id":329833307,"identity":"ddb3716a-d42b-4dd5-9cbe-d43dbd0ebd5e","order_by":2,"name":"Qintong Li","email":"","orcid":"","institution":"College of Engineering, Shibaura Institute of Technology, Tokyo 1358548, Japan","correspondingAuthor":false,"prefix":"","firstName":"Qintong","middleName":"","lastName":"Li","suffix":""},{"id":329833308,"identity":"9c39e243-dc3a-4c60-902c-1cd43163c22e","order_by":3,"name":"Jingjing Yang","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Yang","suffix":""},{"id":329833309,"identity":"70602f1c-3680-4fd6-aec4-ed006136c39c","order_by":4,"name":"Yu Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYNCCCgbGBjCDjWgtZ0BamEnRwthGihZ598PHpHnn1cluOH/+AMOHssMM/LMb8GsxPJOWJs277bDxhhvJDIwzzh1mkLhzgICWhhwzoJYDiRtuMDMw87YdZjCQSCCgpf8NUMucusQN5w8zMP8lRou8BMiWBubEDQeSGZgZidFiIPEs2XLOscPGM28kGxzsOZfOI3GDkC39yQdvvKmpk+07f/Dhgx9l1nL8MwjZcoCBRQLGOQDEPPjVg2wBRuEHgqpGwSgYBaNgZAMAnBxEic/CVhMAAAAASUVORK5CYII=","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Gao","suffix":""},{"id":329833310,"identity":"c7dddc26-a832-4a6e-85ef-4b8ecc851d9b","order_by":5,"name":"Jianliang Xue","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jianliang","middleName":"","lastName":"Xue","suffix":""},{"id":329833311,"identity":"3c1940fd-c01b-40fb-96a6-7e02935fa6fc","order_by":6,"name":"Lin Li","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Li","suffix":""},{"id":329833312,"identity":"b993a4f3-3096-49af-b172-a27726228a8d","order_by":7,"name":"Yiting Ji","email":"","orcid":"","institution":"Shandong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yiting","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2024-07-11 03:56:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4721567/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4721567/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11274-024-04115-7","type":"published","date":"2024-10-03T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61775060,"identity":"348242c7-723c-492f-87c4-5704d80b085b","added_by":"auto","created_at":"2024-08-05 12:24:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32956,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of surfactants produced by GX7 using different concentrations of oil-soluble carbon sources: (a)Waste motor oil, and (b) Waste cooking oil.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/ab4d071fdcaa59b57c29ed54.png"},{"id":61775057,"identity":"cf1c4020-b4e2-43d8-bd89-c09f2e8769b1","added_by":"auto","created_at":"2024-08-05 12:24:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74396,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of surfactants produced by GX7 using different concentrations of water-soluble carbon sources: (a)Starch hydrolysate, and (b)wheat bran juice.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/933959bda8d10d71b23650e1.png"},{"id":61775054,"identity":"2496b731-3797-4817-8ab0-afe68cc3911c","added_by":"auto","created_at":"2024-08-05 12:24:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25110,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of surfactants produced by GX7 using different concentrations of corn steep liquor as nitrogen source\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/ba482867b68acf2a33c6d104.png"},{"id":61775056,"identity":"cd8a882f-2316-4cc7-a80a-e5425a23e786","added_by":"auto","created_at":"2024-08-05 12:24:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58897,"visible":true,"origin":"","legend":"\u003cp\u003eRemoval efficiency of crude oil from oily sand by different surfactants: (a)Fermentation broth produced by GX7, (b)Chemical surfactants.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/207a4a37b5b046859b966435.png"},{"id":61775840,"identity":"30d0e163-16e9-49b1-a653-c3ffc4cba74e","added_by":"auto","created_at":"2024-08-05 12:32:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28237,"visible":true,"origin":"","legend":"\u003cp\u003eRemoval rate of diesel oil in seawater by adding different substances.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/c85c04f2519ca13a8f759b8c.png"},{"id":61775061,"identity":"f3e2199e-a2c1-4d0f-b698-88895b795172","added_by":"auto","created_at":"2024-08-05 12:24:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72709,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of diesel composition in different groups: (a) TIC; (b)Alkanes degradation\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/f66efc999afd705240c18584.png"},{"id":61775058,"identity":"0ba88740-cec7-4076-8a62-d6094e856fa1","added_by":"auto","created_at":"2024-08-05 12:24:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":288921,"visible":true,"origin":"","legend":"\u003cp\u003eComposition of contaminated seawater microorganisms at the genus level: (a) Shannon index; (b)Relative abundance\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/899306e8fe244a41b94a2ba4.png"},{"id":66096963,"identity":"d2e7e8e1-9c56-4bde-a89a-79ce83eef374","added_by":"auto","created_at":"2024-10-07 16:12:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1047115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4721567/v1/53abb5ed-6a33-4c9f-9dbc-ee83f7cd8485.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biosurfactant production by Bacillus cereus GX7 utilizing organic waste and its application in the remediation of hydrocarbon-contaminated environments","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e GX7 effectively utilize water-soluble organic wastes as the sole carbon and nitrogen sources to produce surfactant .\u003c/li\u003e\n \u003cli\u003eGX7 and its biosurfactant efficiently degrade petroleum hydrocarbons in polluted seawater collaborated with indigenous bacteria.\u003c/li\u003e\n \u003cli\u003eGX7 and its biosurfactant optimized the microbial community structure for remediating diesel-contaminated seawater.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eGlobally, petroleum production and utilization serve as pivotal elements in the energy landscape, underpinning various sectors including transportation, manufacturing, and energy generation. In 2023, the world\u0026apos;s petroleum production was estimated at approximately 100 million barrels per day, catering to a vast array of energy demands across the globe[1]. Despite its critical role in economic development, the extensive use of petroleum has precipitated substantial environmental and health challenges[2]. Petroleum hydrocarbon pollution primarily arises from oil spills, oily wastewater discharge, pipeline leaks, and extraction activities, significantly contaminating soil and water[3-4]. Aromatic hydrocarbons in petroleum, particularly polycyclic aromatic hydrocarbons (PAHs) such as bicyclic and tricyclic hydrocarbons, are highly toxic to humans and animals. PAHs can enter the body through inhalation, skin contact, and dietary intake, adversely affecting the normal functioning of vital organs like the liver and kidneys, and may even lead to cancer[5]. Therefore, the development of effective remediation techniques of petroleum pollution is urgently needed[6].\u003c/p\u003e\n\u003cp\u003eCompared to conventional physio-chemical treatment methods which incur high maintenance cost, consume significant and generate toxic intermediate products during the remediation process[7-8], bioremediation techniques are environmentally friendlyand cost-effective[9]. Therefore, bioremediation is considered as the most promising technology in petroleum contamination remediation. Bioremediation techniques include biostimulation, which enhances the activity of indigenous microorganisms, and bioaugmentation, which involves adding specific degrading microbes in environments[6]. In essence, it is the decomposition of petroleum hydrocarbons by petroleum degrading microorganisms during their growth and metabolism. However, petroleum bioavailability and microbial activities are usually limited due to the low solubility and hydrophobicity of petroleum hydrocarbons[10]. The sticky nature of oil, combined with its ability to be easily absorbed by rocks and sand, makes it particularly challenging to remove once discharged into the environment[11]. Therefore, improving the solubility of petroleum hydrocarbons is crucial for enhancing their biodegradation[12].Some bacteria possess specific enzymatic abilities that enable them to produce surfactants, which facilitate the emulsification of hydrocarbon[13]. The utilization of biosurfactants has been effective addressing the issue of bioavailability and is often recommended to enhance remediation efforts[6]. This can be achieved by introducing biosurfactant-producing microorganisms or applying microbial biosurfactants directly to contaminated sites[3]. This process significantly accelerates the degradation rate of petroleum hydrocarbons by increasing their bioavailability to microbial action[14].\u003c/p\u003e\n\u003cp\u003eBiosurfactants were first discovered in the late 1960s as extracellular amphiphilic compounds produced during hydrocarbon fermentation[15]. These molecules are amphipathic, containing both hydrophilic and hydrophobic groups, which enable them to significantly reduce the surface and interfacial tensions. By attaching to various hydrophobic compounds, biosurfactants increase the solubility of petroleum, facilitating its degradation in environmental remediation processes[16-17]. In addition, biosurfactants possess distinct advantages over chemical surfactants, which have increasingly been recognized for their poor environmental stability and the potential to contribute to chemical pollution. In contrast, biosurfactants are biodegradable and generally exhibit lower toxicity, making them more environmentally friendly alternatives[18]. In the remediation of petroleum contaminated soil, the use of biosurfactants significantly enhance biological functions within the soil, such as soil conductivity and core enzyme activities involved in the degradation of petroleum hydrocarbon. Furthermore, they facilitate quorum sensing among different species[19]. This suggests that biosurfactants have a less negative impact on the microbial community in the soil compared to other remediation agents. Therefore, biosurfactants hold great potential for enhancing bioremediation processes and are extensively utilized in environment protection efforts[20].\u003c/p\u003e\n\u003cp\u003eSeveral factors influence the process of hydrocarbon degradation facilitated by biosurfactants[21]. Beyond temperature, agitation speed of culture, pH and salinity, the availability of limiting nutrients also plays a crucial role in the production and effectiveness of biosurfactants[22-25], impacting both the quantity and quality of the biosurfactants produced, which in turn affects their efficacy in degrading hydrocarbons. Furthermore, high cost of the raw materials required for fermentation can be a significant barrier to the practical application of biosurfactants[26]. To address this problem，researchers are exploring the use of waste or low-cost substrate as renewable substrates to increase the production of biosurfactant, such as crude palm oil, molasses, cashew apple juice[27-28]. However, there is limited research on the utilization of industrial waste oil and by-products from food production as substrates for biosurfactant production.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e GX7, isolated from oil sludge, was identified as a notably proficient producer of surfactant producer in our preceding research[29]. In this research, we have explored the feasibility of\u0026nbsp;using industrial and agricultural waste as nutrient source for the biosurfactant production\u0026nbsp;with\u0026nbsp;\u003cem\u003eB\u003c/em\u003e\u003cem\u003eacillus cereus\u003c/em\u003e GX7, reducing production costs and environmental impact. Additionally, we investigated the potential of employing GX7 and its produced surfactants in the remediation of oil pollution in soil and seawater environments.\u003c/p\u003e"},{"header":"2. Materials and methods ","content":"\u003cp\u003e\u003cstrong\u003e2.1Materials collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemical reagents used in this study including hydrochloric acid, chloroform, methanol, sodium chloride, glucose were of analytical grade. The crude oil was obtained from Venezuela(10\u0026deg;3\u0026apos; N, 66\u0026deg;58\u0026apos; W), sterilize by ultraviolet lamp before using. The waste cooking oil used was collected from a local restaurant in the city of Qingdao, China. The waste motor oil was provided by a local repair center in Qingdao, China. The starch hydrolysate, wheat bran juice and corn soak liquid were provided by Qingdao Haidar Starch Co., LTD. All collected samples were sealed in the sterile glass bottle and kept in a cool and dry place for further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2Bacterial strain and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterium (\u003cem\u003eBacillus cereus\u003c/em\u003e GX7) was previously isolated from the bottom sludge of the oil storage tank in Shengli Oilfield, China. The Luria-Bertani (LB) culture utilized for fermentation was composed of 10 g/L peptone, 5 g/L yeast, 5 g/L NaCl, 10-15 g/L agar (solid medium) and 4 mL/L trace element solution. The trace element solution contains 2 mg/L of CaCl\u003csub\u003e2\u003c/sub\u003e, 50 mg/L of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, 2 mg/L of CuSO\u003csub\u003e4\u003c/sub\u003e; 10 mg/L of ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO. After adjusting the pH to 7.5, the medium was sterilized at 121℃ for 20 min. GX7 was cultured in LB medium at 150 r/min and 30 ℃\u0026nbsp;for 2d to obtain the fermentation liquid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3Experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1 Utilization of industrial and agricultural waste to produce biosurfactants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the fermentation process of biosurfactants, waste cooking oil and waste motor-oil were employed as oil-soluble carbon sources, while starch hydrolysate was utilized as water-soluble carbon sources, and corn steep liquid was used as a nitrogen sources, respectively. After filtration under reduced pressure to remove solid impurities and bacteria, the samples were added to the medium to obtain the desired concentrations (0.1%, 0.5%, 1%, 2%, 3% w/v) as the carbon/nitrogen source, without any chemical supplementation. The bacterial solution was inoculated into the medium at a 1% (v/v) inoculation rate, and the medium without a carbon source or nitrogen source was used as a blank control. The emulsification index (E24) assay and surface tension measurement of fermentation liquid was carried out to evaluate the feasibility of GX7 producing biosurfactants from various substances. Three parallel samples were prepared for all experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2 Remediation of crude oil-contaminated beach sand\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeach sand with a diameter of 0.150-0.180 mm was washed 6 times with 1 mol/L HCl solution and then rinsed with tap water until the pH was neutral. Two grams of crude oil was added into 50 g beach sand and thoroughly mixed in a 100 mL conical flask, with the initial weight of crude oil denoted G1. The fermentation broth of GX7 was centrifuged to remove the bacteria, and subsequently, 50mL of supernatant was addedto the oil-contaminated beach sand system for a crude oil degradation experiment conducted at 30℃, 150 rpm. Simultaneously, 50 mL of five commercially available chemical surfactants at their critical micelle concentrations (CMC) were added to the oil-contaminated beach sand system: Tween 80 (0.013 g/L), Triton X-100 (0.15 g/L), sodium dodecyl sulfonate (3.015 g/L), sodium dodecyl benzene sulfonate (0.418 g/L), and sodium dodecyl sulfate (2.304 g/L). Samples were periodically collected to assess the degradation efficiency of crude oil.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.3 Remediation of diesel oil contaminated seawater\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mechanism of biosurfactant produced by strain GX7 during hydrocarbon degradation, four groups of remediation experiments were conducted to simulate diesel oil pollution in seawater: (1) Marine indigenous bacteria (hereinafter referred to as BD) only, (2) GX7 only, (3) GX7 and BD, (4) The fermentation liquid of GX7 after centrifugation (hereinafter referred to as FJ) and BD. The seawater utilized was collected from Jiaozhou Bay, China. For group (2), the seawater was filtrated by 0.45 \u0026mu;m membrane to remove the impurity and bacteria. The concentration of diesel oil in simulated seawater was 10 g/L, and recorded as C\u003csub\u003e0\u003c/sub\u003e. After 7 days of seawater remediation experiment at 30℃\u0026nbsp;and 150rpm, the residual diesel content was measured to calculate the diesel degradation rate, while the microbial community was analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4Analytical method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1Crude oil/Diesel oil degradation rate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supernatant from the conical bottle was decanted from the sand, and n-hexane was added to the supernatant, thoroughly shaken, and then transferred to a separatory funnel for extraction. This process was repeated three times until the organic phase is completely clarified, after which the n-hexane phases are combined. After removing the n-hexane and water using rotary evaporation at 35℃,weighing the remaining crude oil and denoted as G2. The removal rate of crude oil is calculated as G2/G1\u0026times;100%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe remaining diesel oil in the seawater is also extracted with n-hexane, the diesel concentration (C\u003csub\u003et\u003c/sub\u003e) in organic phase was detected by ultraviolet spectrophotometry[30]. The diesel oil degradation efficiency was calculated by the following equation: R (%)=(C\u003csub\u003e0\u003c/sub\u003e\u0026minus;C\u003csub\u003et\u003c/sub\u003e)/C\u003csub\u003e0\u003c/sub\u003e\u0026times;100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2 Determination of surface tension and emulsification index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface tension of the fermentation solution was determined by the platinum ring method with an automatic surface tensiometer (BZY-102, Shanghai Fangrui Instrument Co., LTD.). Before measuring the surface tension, the platinum ring is thoroughly rinsed with acetone and dried at 40℃.\u003c/p\u003e\n\u003cp\u003eThe emulsification index (E24) was determined according to the procedure outlined by Satpute et al.\u0026nbsp;[31]. Specifically, 5mL of diesel oil was added to an equal volume of cell-free supernatant, which was then vigorously vortexed for two minutes at high speed. The mixture was subsequently allowed to stand at room temperature for 24 hours. After this period, the height of the emulsion layer was measured relative to the total height of the mixture to calculate the emulsification index.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.3 Identification of intermediates in seawater by GC\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003cstrong\u003eMS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiesel oil in seawater was analyzed using an Agilent 7890A gas chromatograph coupled with an Agilent 7000 mass spectrometer. Helium was used as the carrier gas at a flow rate of 1 mL/min. The temperature program started at 70℃\u0026nbsp;for 2 min, ramped up at 20\u0026nbsp;℃/min to 230℃, further increased at 40℃/min to 300℃, and held at 300℃\u0026nbsp;for 10 min. Identification of components was performed by comparing their mass spectra with those in the NIST mass spectrometry library.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.4 Characteristics of microbial community\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrobial diversity and community composition of different group after 7 days of degradation were analyzed by 16\u0026thinsp;S rRNA gene amplification，The hypervariable V3-V4 region of the 16\u0026thinsp;S rRNA gene was selected for amplification using primers \u0026nbsp;341\u0026thinsp;F (CCTACGGGNGGCWGCAG) and 806\u0026thinsp;R (GGACTACHVGGGTWTCTAAT)[32]. The sequence was determined by Shanghai Meiji Technology Co., Ltd.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Production of biosurfactant\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1Availability of oil-soluble carbon sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUtilizing 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].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2Availability of water-soluble carbon sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen 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 \u003cem\u003eAspergillus niger\u003c/em\u003e 18FSDE16 via semi-solid-state fermentation (SSSF)[37]. Soumya investigated the production of EPS by a \u003cem\u003eLactobacilli\u003c/em\u003e 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.3 Availability of corn\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esteep liquor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;as a nitrogen source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u003cem\u003eBacillus cereus\u003c/em\u003e GX7 can efficiently utilize corn steep liquor as a nitrogen source for fermentation, thereby producing surfactant. In Yang\u0026apos; s study, corn steep liquor enhanced the growth and production of \u003cem\u003eB. subtilis\u003c/em\u003e, 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Application of GX7 and its surfactants in the remediation of oily sand\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Biosurfactant-Enhanced Bioremediation in diesel polluted seawater \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1 Diesel biodegradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.2 Analysis of d\u003c/strong\u003e\u003cstrong\u003eegradation product\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Microbial community analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 \u0026nbsp; 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 \u003cem\u003eEnterobacter\u003c/em\u003e (97.58%), with \u003cem\u003ePseudomonas\u003c/em\u003e also existed at a low abundance. \u003cem\u003eEnterobacter\u003c/em\u003e had several metabolic pathways and enzyme-encoding genes related to oil degradation by genome analysis[43]. In group BD+ GX7, the dominate genus were \u003cem\u003ePseudomonas\u003c/em\u003e(47.26%), \u003cem\u003eStenotrophomonas\u003c/em\u003e(33.78%) and \u003cem\u003eAchromobacte\u003c/em\u003e\u003cem\u003er\u003c/em\u003e(7.89%). The microbial community structure of the BD+fermentation supernatant group closely resembled that of BD+GX7. \u003cem\u003ePseudomonas\u003c/em\u003e (42.39%) and \u003cem\u003eStenotrophomonas\u003c/em\u003e (30.42%) were dominant, while \u003cem\u003eEnterobacter\u003c/em\u003e (14.82%) and \u003cem\u003eAchromobacter\u0026nbsp;\u003c/em\u003e(5.93%) also showed a high abundance, with a minor presence of \u003cem\u003eBrucella,\u003c/em\u003e \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e are genus that widely exist in soil and seawater. \u003cem\u003ePseudomonas\u003c/em\u003e had significant petroleum degradation potential in seawater under nutrient limited conditions assisted by biosurfactant with low CMC (15.0 g/L)[44]. \u003cem\u003eStenotrophomonas\u003c/em\u003e has shown promising potential in the degradation of polycyclic aromatic hydrocarbons (PAHs), possessing essential genes involved in the PAHs degradation[45]. Although \u003cem\u003eBacillus\u003c/em\u003e 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\u003cem\u003e\u0026nbsp;Bacillus\u003c/em\u003e, 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.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e GX7 can effectively utilize water-soluble organic wastes as carbon and nitrogen sources to enhance surfactant production but cannot effectively use oil-soluble organic waste as nutrients. The biosurfactant produced by GX7 has a removal rate of 14.63% to crude oil in oily sand, which is slightly lower than that of chemical surfactants. In the remediation of marine diesel pollution, GX7 and its fermentation broth can collaborate with indigenous bacteria to improve the degradation rate of diesel oil, reaching 94.38% and 70.36% respectively in seawater supplemented with fermentation supernate and strain GX7. The results of community structure showed that the addition of GX7 and its fermentation solution affected the community structure and increase the abundance of dominant strains (\u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e). This study is an important reference for the utilization of biosurfactant strategies in addressing water and soil contamination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.wrote the main manuscript text and prepared figures 1-6B.Methodology and prepared figure 7C.D.Methodology and Formal analysisE.Writing \u0026ndash; review \u0026amp;editing, Validation, Resources, Project administration, SupervisionF.G.H.Methodology and Formal analysis\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China [grant number 52374174]; Project of Shandong Province Higher Educational Young Innovative Talent Introduction and Cultivation Team [Wastewater treatment and resource innovation team].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSean Hill. Four countries could account for most near-term petroleum liquids supply growth[N]. Today in Energy, 2024-3.14.https://www.eia.gov/todayinenergy/detail.php?id=61583.\u003c/li\u003e\n \u003cli\u003eKartik Patel;Mitesh Patel.Improving bioremediation process of petroleum wastewater using biosurfactants producing Stenotrophomonas sp. S1VKR-26 and assessment of phytotoxicity[J].Bioresource technology,2020,Vol.315: 123861\u003c/li\u003e\n \u003cli\u003ePing Guo;Wei-Wei Xu;Dan-Na Wei;Man-Xia Zhang;Jin Zhang;Shi Tang;Bin-Xia Cao;Jian-Guo Lin;Wei Li.Potential Application of Biosurfactant-Producing Bacteria for Bioremediation of Oil Polluted Marine Intertidal Sediments[J].Journal of Marine Science and Engineering,2022,Vol.10(6): 731\u003c/li\u003e\n \u003cli\u003eChuan Yin;Huan Yan;Yuancheng Cao;Huanfang Gao.Enhanced bioremediation performance of diesel-contaminated soil by immobilized composite fungi on rice husk biochar[J].Environmental Research,2023,Vol.226: 115663\u003c/li\u003e\n \u003cli\u003eIsraa Abdulwahab Al-Baldawi;Siti Rozaimah Sheikh Abdullah;Nurina Anuar;Fatihah Suja;Idris Mushrifah.Phytodegradation of total petroleum hydrocarbon (TPH) in diesel-contaminated water using Scirpus grossus.[J].Ecological Engineering,2015,Vol.74: 463-473\u003c/li\u003e\n \u003cli\u003eI. Moumed;J. Arrar;A. Namane \u0026amp; \u0026hellip;F. Bentahar.Effects of surfactant and oxidant on bioremediation of contaminated soil by total petroleum hydrocarbons using indigenous bacteria[J].International Journal of Environmental Science and Technology,Vol.20(8): 8863-8874\u003c/li\u003e\n \u003cli\u003eTahir Haneef;Muhammad Raza Ul Mustafa;Kashif Rasool;Yeek Chia Ho;Shamsul Rahman Mohamed Kutty.Removal of Polycyclic Aromatic Hydrocarbons in a Heterogeneous Fenton Like Oxidation System Using Nanoscale Zero-Valent Iron as a Catalyst[J].Water,2020,Vol.12(2430): 2430\u003c/li\u003e\n \u003cli\u003eXin Sui;Xuemei Wang;Yuhuan Li;Hongbing Ji.Remediation of Petroleum-Contaminated Soils with Microbial and Microbial Combined Methods: Advances, Mechanisms, and Challenges[J].Sustainability,2021,Vol.13(9267): 9267\u003c/li\u003e\n \u003cli\u003eMolaei S;Moussavi G;Talebbeydokhti N;Shekoohiyan S.Biodegradation of the petroleum hydrocarbons using an anoxic packed-bed biofilm reactor with in-situ biosurfactant-producing bacteria.[J].Journal of hazardous materials,2022,Vol.421: 126699\u003c/li\u003e\n \u003cli\u003ePhan, Chia-Wei;Abu Bakar, NurFaizah;Hamzah, Ainon.A Comparative Study on Biosurfactant Activity of Crude Oil\u0026ndash;Degrading Bacteria and Its Correlation to Total Petroleum Hydrocarbon Degradation.[J].Bioremediation journal,2013,Vol.17(4): 240-251\u003c/li\u003e\n \u003cli\u003eM. Gloria Pereira; Stephen M. Mudge.Cleaning oiled shores: laboratory experiments testing the potential use of vegetable oil biodiesels[J].Chemosphere,2004,Vol.54(3): 297-304\u003c/li\u003e\n \u003cli\u003eBrakstad, Odd G.( [email protected]);Bonaunet, Kristin.Biodegradation of petroleum hydrocarbons in seawater at low temperatures (0-5 \u0026deg;C) and bacterial communities associated with degradation.[J].Biodegradation,2006,Vol.17(1): 71-82\u003c/li\u003e\n \u003cli\u003eJenyffer M. Campos;T\u0026acirc;nia L. M. Stamford;Leonie A. Sarubbo.Production of a bioemulsifier with potential application in the food industry.[J].Appl Biochem Biotechnol,2014,Vol.172(6): 3234-3252\u003c/li\u003e\n \u003cli\u003eAshis K Mukherjee;Kishore Das.Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat.[J].FEMS microbiology ecology,2005,Vol.54(3): 479-489\u003c/li\u003e\n \u003cli\u003eAli Khan, Aqib Hassan;Tanveer, Sundus;Alia, Shagufta;Anees, Mariam;Sultan, Aneesa;Iqbal, Mazhar;Yousaf, Sohail.Role of nutrients in bacterial biosurfactant production and effect of biosurfactant production on petroleum hydrocarbon biodegradation[J].ECOLOGICAL ENGINEERING,2017,Vol.104: 158-164\u003c/li\u003e\n \u003cli\u003eJoy, Sam.Biosurfactant producing bacteria from hydrocarbon contaminated environment[J].Environmental Footprints and Eco-Design of Products and Processes,2017,: 259-305\u003c/li\u003e\n \u003cli\u003eLuna, Juliana M;Rufino, Raquel D;Sarubbo, Leonie A;Bezerra, Karen Gercyane O.Saponins and microbial biosurfactants: Potential raw materials for the formulation of cosmetics[J].Biotechnology Progress,2018,Vol.34(6): 1482-1493\u003c/li\u003e\n \u003cli\u003eSwati Sharma;Rahul Verma;Lalit M. Pandey.Crude oil degradation and biosurfactant production abilities of isolated Agrobacterium fabrum SLAJ731(Article)[J].Biocatalysis and Agricultural Biotechnology,2019,Vol.21: 101322\u003c/li\u003e\n \u003cli\u003eXuliang Zhuang;Yaxin Wang;Haoyu Wang;Yuzhu Dong;Xianglong Li;Shijie Wang;Haonan Fan;Shanghua Wu.Comparison of the efficiency and microbial mechanisms of chemical- and bio-surfactants in remediation of petroleum hydrocarbon[J].Environmental Pollution,2022,Vol.314: 120198\u003c/li\u003e\n \u003cli\u003eQingguo\u0026nbsp;Chen;\u0026nbsp;Jingjing\u0026nbsp;Li;\u0026nbsp;Liu\u0026nbsp;Mei; Huiling\u0026nbsp;Sun;\u0026nbsp;Mutai\u0026nbsp;Bao.Study on the biodegradation of crude oil by free and immobilized bacterial consortium in marine environment.[J].PLoS ONE,2017,Vol.12(3): 1-23\u003c/li\u003e\n \u003cli\u003eSoumen Mukherjee;Palashpriya Das;Ramkrishna Sen.Towards commercial production of microbial surfactants.[J].Trends in biotechnology,2006,Vol.24(11): 509-515\u003c/li\u003e\n \u003cli\u003eC Am\u0026eacute;zcua-Vega;H M Poggi-Varaldo;F Esparza-Garc\u0026iacute;a;E R\u0026iacute;os-Leal;R Rodr\u0026iacute;guez-V\u0026aacute;zquez.Effect of culture conditions on fatty acids composition of a biosurfactant produced by Candida ingens and changes of surface tension of culture media.[J].Bioresource technology,2007,Vol.98(1): 237-240\u003c/li\u003e\n \u003cli\u003eAna Eliza Zeraik;Marcia Nitschke.Biosurfactants as Agents to Reduce Adhesion of Pathogenic Bacteria to Polystyrene Surfaces: Effect of Temperature and Hydrophobicity.[J].Current Microbiology,2010,Vol.61(6): 554-559\u003c/li\u003e\n \u003cli\u003eF.J.S. Oliveira;L. Vazquez;N.P. de Campos;F.P. de Frana.Production of rhamnolipids by a Pseudomonas alcaligenes strain.[J].Process Biochemistry,2009,Vol.44(4): 383-389\u003c/li\u003e\n \u003cli\u003eAbouseoud, M;Yataghene, A;Amrane, A;Maachi, R.Effect of pH and salinity on the emulsifying capacity and naphthalene solubility of a biosurfactant produced by Pseudomonas fluorescens[J].Journal of Hazardous Materials,2010,Vol.180(1): 131-136\u003c/li\u003e\n \u003cli\u003eVerma, Rahul;Sharma, Swati;Kundu, Lal Mohan;Pandey, Lalit Mohan.Experimental investigation of molasses as a sole nutrient for the production of an alternative metabolite biosurfactant(Article)[J].Journal of Water Process Engineering,2020,Vol.38: 101632\u003c/li\u003e\n \u003cli\u003eRocha, MVP;Souza, MCM;Benedicto, SCL;Bezerra, MS;Macedo, GR;Pinto, GAS;Goncalves, LRB.Production of biosurfactant by Pseudomonas aeruginosa grown on cashew apple juice[J].Applied Biochemistry and Biotechnology,2007,Vol.137-140(1-12): 185-194\u003c/li\u003e\n \u003cli\u003eSuryanti, V.;Handayani, D.S.;Masykur, A.;Lindasari.Optimization Production of Biosurfactant by Pseudomonas putida Using Crude Palm Oil (CPO) as Substrate[J].IOP Conference Series: Earth and Environmental Science,2017,Vol.75(1): 012013\u003c/li\u003e\n \u003cli\u003eXiaoyan Wang, Jin Gao, Yu Gao*, Linlin Zhang, Congchao Xu, Qintong Li, Lin Li, Jianliang Xue. Analysis of surfactant production by Bacillus cereus GX7 and optimization of fermentation conditions. [J] Colloids and Surfaces B: Biointerfaces, 2024, 233: 113629. DOI10.1016/j.colsurfb.2023.113629\u003c/li\u003e\n \u003cli\u003eShi K, Xue JL, Xiao XF, Qiao YL, Wu YN, Gao Y. Mechanism of degrading petroleum hydrocarbons by compound marine petroleum-degrading bacteria: surface adsorption, cell uptake, and biodegradation. Energy Fuels. 2019;33:11373\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eSatpute, S.K., Banpurkar, A.G., Dhakephalkar, P.K., Banat, I.M., Chopade, B.A., 2010.Methods for investigating biosurfactants and bioemulsifiers: a review. Crit. Rev.Biotechnol. 30, 127\u0026ndash;144.\u003c/li\u003e\n \u003cli\u003eSuxiang Liu，Rui Sun，Mengmeng Cai，Yue Kong，Yu Gao*，Tonghuan Zhang，\u0026nbsp;Xinfeng Xiao, Yanlu Qiao, Jianliang Xue, Guofu Huang. Petroleum spill bioremediation by an indigenous constructed bacterial consortium in marine environments, Ecotoxicology and Environmental Safety, 2022, 241: 113769. August 2022. 10.1016/j.ecoenv.2022.113769.\u003c/li\u003e\n \u003cli\u003eWadekar;S B Kale;A M Lali;D N Bhowmick;A P Pratap.MICROBIAL SYNTHESIS OF RHAMNOLIPIDS BY Pseudomonas aeruginosa (ATCC 10145) ON WASTE FRYING OIL AS LOW COST CARBON SOURCE.[J].Preparative biochemistry \u0026amp; biotechnology,2012,Vol.42(3): 249-266\u003c/li\u003e\n \u003cli\u003eAgarwal AK;Sharma M;Singh SK.Experimental investigations of heavy metal addition in lubricating oil and soot deposition in an EGR operated engine[J].Applied Thermal Engineering,2006,Vol.26(2): 259-266\u003c/li\u003e\n \u003cli\u003eRamadass, Kavitha;Megharaj, Mallavarapu;Venkateswarlu, Kadiyala;Naidu, Ravi.Toxicity and oxidative stress induced by used and unused motor oil on freshwater microalga, Pseudokirchneriella subcapitata.[J].ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH,2015,Vol.22(12): 8890-8901\u003c/li\u003e\n \u003cli\u003eM.P. Soumya;Keerthi Sasikumar;Ashok Pandey;K. Madhavan Nampoothiri.Cassava starch hydrolysate as sustainable carbon source for exopolysaccharide production by Lactobacillus plantarum[J].Bioresource Technology Reports,2019,Vol.6: 85-88\u003c/li\u003e\n \u003cli\u003eTatyana Patr\u0026iacute;cio;Lucas Vitor de Ara\u0026uacute;jo Santos;Ruan Dion\u0026iacute;zio da Silva;Renata Torres dos Santos e Santos;Normando Ribeiro-Filho;Sharline Florentino de Melo Santos;Fl\u0026aacute;vio Luiz Honorato da Silva.Pectinases production using an exotic Caatinga passion fruit waste and wheat bran as a substrate for Clarification of grape juice[J].Food Bioscience,2024,: 104163\u003c/li\u003e\n \u003cli\u003eSergio Valdivia-Rivera;\u0026nbsp;Elizabeth Del Carmen Varela-Santos;\u0026nbsp;Tannia Alexandra Qui\u0026ntilde;.Production of hydrocarbon-degrading microorganisms using agricultural residues of Mangifera indica L. and Carica papaya as carbon source.[J].3 Biotech,2019,Vol.9(2): 43\u003c/li\u003e\n \u003cli\u003eLiu, Xiaoyan; Wang, Xinfeng; Xu, Jiaxing; Xia, Jun; Lv, Jinshun.Citric acid production by Yarrowia lipolytica SWJ-1b using corn steep liquor as a source of organic nitrogen and vitamins[J].Industrial Crops and Products,2015,Vol.78: 154-160\u003c/li\u003e\n \u003cli\u003eYang, TW (Yang, Tao-Wei);Rao, ZM (Rao, Zhi-Ming);Zhang, X (Zhang, Xian);Xu, MJ (Xu, Mei-Juan);Xu, ZH (Xu, Zheng-Hong);Yang, ST (Yang, Shang-Tian).Effects of corn steep liquor on production of 2,3-butanediol and acetoin by Bacillus subtilis.[J].Process Biochemistry,2013,Vol.48(11): 1610-1617\u003c/li\u003e\n \u003cli\u003eGarcia, M. Teresa;Ribosa, Isabel ;Kowalczyk, Iwona ;Pakiet, Marta ;Brycki, Bogumil .Biodegradability and aquatic toxicity of new cleavable betainate cationic oligomeric surfactants.[J].Journal of Hazardous Materials,2019,Vol.371: 108-114\u003c/li\u003e\n \u003cli\u003eAo Wang, Wenxian Fu, Yu Feng, Zhimin Liu, Donghui Song.Synergetic effects of microbial-phytoremediation reshape microbial communities and improve degradation of petroleum contaminants[J].Journal of hazardous materials,2022,Vol.429: 128396\u003c/li\u003e\n \u003cli\u003eMuhammad Zohaib Nawaz,\u0026nbsp;Chunyan Xu,\u0026nbsp;Majjid A Qaria,\u0026nbsp;Syed Zeeshan Haider,\u0026nbsp;Hafiz Rameez Khalid,\u0026nbsp;Huda Ahmed Alghamdi,\u0026nbsp;Iqrar Ahmad Khan,\u0026nbsp;Daochen Zhu.Genomic and biotechnological potential of a novel oil-degrading strain Enterobacter kobei DH7 isolated from petroleum-contaminated soil[J].Chemosphere,2023,Vol.340: 139815\u003c/li\u003e\n \u003cli\u003eLouella Concepta Goveas,\u0026nbsp;Raja Selvaraj,\u0026nbsp;Ramesh Vinayagam,\u0026nbsp;Ahad Amer Alsaiari,\u0026nbsp;Nahed S Alharthi,\u0026nbsp;Shyama Prasad Sajankila.Nitrogen dependence of rhamnolipid mediated degradation of petroleum crude oil by indigenous Pseudomonas sp. WD23 in seawater[J].Chemosphere,2022,Vol.304: 135235\u003c/li\u003e\n \u003cli\u003eTemidayo Oluyomi Elufisan[a][b];Luis Lozano[c];Patricia Bustos[c];Isabel Cristina Rodr\u0026iacute;guez-Luna[a];Alejandro S\u0026aacute;nchez-Varela[a];Omotayo Opemipo Oyedara[d];Miguel ngel Villalobos-L\u0026oacute;pez[e];Xianwu Guo[a].Complete Genome Sequence of Stenotrophomonas maltophilia Strain SVIA2,Isolated from Crude Oil-Contaminated Soil in Tabasco,Mexico[J].Genome Announcements,2019,Vol.8(30): e00529-19\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biosurfactants , Bacillus cereus GX7 , Organic waste application , Petroleum hydrocarbons pollution, Bioremediation","lastPublishedDoi":"10.21203/rs.3.rs-4721567/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4721567/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe use of biosurfactants represents a promising technology for remediating hydrocarbon pollution in the environment. This study evaluated\u003cem\u003e \u003c/em\u003ea highly effective biosurfactant strain-\u003cem\u003eBacillus cereus\u003c/em\u003e GX7’s ability to produce biosurfactants from industrial and agriculture organic wastes. GX7 showed poor utilization capacity for oil soluble organic waste but effectively utilized of water soluble organic wastes such as starch hydrolysate and wheat bran juice as carbon sources to enhance biosurfactant production. This led to significant improvements in surface tension and emulsification index. Corn steep liquor was also effective as a nitrogen source for GX7 in biosurfactant production. The biosurfactants produced by strain GX7 demonstrated a remediation effect on oily beach sand, but are slightly inferior to chemical surfactants. Inoculation with GX7 (70.36%) or its fermentation solution (94.38%) can effectively enhanced the degradation efficiency of diesel oil in polluted seawater, surpassing that of indigenous degrading bacteria treatments (57.62%). Moreover, Inoculation with GX7’s fermentation solution notably improved the community structure by increasing the abundance of functional bacteria such as \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eStenotrophomonas\u003c/em\u003e in seawater. These findings suggest that the GX7 as a promising candidate for bioremediation of petroleum hydrocarbons.\u003c/p\u003e","manuscriptTitle":"Biosurfactant production by Bacillus cereus GX7 utilizing organic waste and its application in the remediation of hydrocarbon-contaminated environments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 12:24:34","doi":"10.21203/rs.3.rs-4721567/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-21T21:47:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-21T18:27:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-21T16:34:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-21T12:36:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-19T17:24:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132031966646752566947441631129003059967","date":"2024-07-14T05:35:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267720121191587404501185472224477995001","date":"2024-07-12T06:06:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102659359593826230870198340705704004994","date":"2024-07-12T04:38:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187377436007581719095264553286924959132","date":"2024-07-12T03:27:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298872995759331865561880109163192504598","date":"2024-07-11T13:10:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"214321035635519335822737593376627061951","date":"2024-07-11T11:07:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337628863139332894494575492115365127539","date":"2024-07-11T11:02:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88479002844524575022754800009338383565","date":"2024-07-11T10:57:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-11T10:53:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-11T07:38:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-11T05:22:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2024-07-11T03:55:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f41bc331-9c33-43ec-9216-0a3209f3dca2","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:03:51+00:00","versionOfRecord":{"articleIdentity":"rs-4721567","link":"https://doi.org/10.1007/s11274-024-04115-7","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2024-10-03 15:57:58","publishedOnDateReadable":"October 3rd, 2024"},"versionCreatedAt":"2024-08-05 12:24:34","video":"","vorDoi":"10.1007/s11274-024-04115-7","vorDoiUrl":"https://doi.org/10.1007/s11274-024-04115-7","workflowStages":[]},"version":"v1","identity":"rs-4721567","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4721567","identity":"rs-4721567","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}