Study of oil biodegrading by expanded perlite Loaded by oil-eating bacteria

Study of oil biodegrading by expanded perlite Loaded by oil-eating bacteria | 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 Study of oil biodegrading by expanded perlite Loaded by oil-eating bacteria Seyyed Reza Mortazavi, Amir H. M. Sarrafi, Afshar Alihosseini, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3703177/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bio sorption and biodegrading capacity as well as sorption of oil onto expanded perlites modified with oil-eating bacteria were studied. This investigation leveraged perlite as an oil absorbent, subsequently laden with oil-degrading micro-organisms, aiming to study not only oil spill absorption but also the eradication of oil spill. Findings from experiments with three different adsorbents - one devoid of microbes, one featuring perlite loaded with microbes, and one containing expanded perlite laden with microbes-indicate that expanded perlite, due to its large surface area and low density, presents an optimal environment for microbial growth and proliferation. Upon microbial colonization, the amount of oil absorption and removal escalated by 58% and 80.45%, respectively, compared to pre-expansion. Furthermore, microbial activity mitigated some oil contamination and decreased the surface tension between water and oil via production of surface active substances, thereby facilitating further separation of residual oil in the water. Biodegrading Expanded Perlite Oil Spill Adsorption Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Extensive water pollution is a consequence of various operations involved in the crude oil industry, namely extraction, transportation, refinement, and storage. Spillage usually contaminates the aquatic environment, triggering physical and chemical changes such as evaporation, decomposition, photochemical oxidation, bioremediation and absorption onto suspended materials, thereby exacerbating aquatic pollution (Saad et al. 2019 ; Sharma et al. 2023 ). Despite preventative measures, catastrophic incidents, such as the 2021 Orange County oil spill, persist and introduce oil contaminants into the water (Tanee et al. 2015). Petroleum hydrocarbons inflict several detrimental effects on living organisms, including toxicity, mutagenicity, potential carcinogenesis, and disruption of life cycles, necessitating their removal from the environment (Muthamilselvi et al. 2021 ; Yadav et al. 2023 ; Qaria et al. 2023 ). Three major strategies “physical, chemical, and biological” exist to eradicate oil pollutants. Among them, using micro-organisms in bio-remediation techniques is safe and economical. This process is conducted by natural environmental micro-organisms under natural conditions and can reduce pollution to acceptable levels (Tabari et al. 2009; Bouchez et al. 2016 ). Various methodologies have been conceived to combat oil spills and their associated problems. Relying on diverse principles of removal (Tabari et al. 2009; Bhardwaj et al. 2018), these techniques, especially adsorbents, are categorized as combined systems as they can either contain (passively) or remove (actively) pollutants. These strategies have proven particularly effective for the recovery of oil traces from the environment (Bhardwaj et al. 2018; Scarlett et al., 2021 ; Narimani et al., 2015 ; Piterlli et al. 2020). Absorption, in fact, is recognized as a straightforward and potent solution. A plethora of organic (Lee et al. 2020 ; Wang et al. 2014 ; Moradi et al. 2023 ) and inorganic pollutants populate aqueous solutions. However, in open waters, due to the transport of crude oil, the prevalence of organic pollutants and associated biodegradability impacts are heightened (Verma et al, 2022 ; Moradi et al. 2023 ; Zhang et al. 2018). Perlite, an abundant volcanic rock found in mines, has piqued industrial interest due to its favorable characteristics, which include low density, extensive pore space, a layered structure, swift oil spread, high permeability, and ease of collection (Allen, 1990 ; Wang et al. 2014 ). Following oil absorption, perlite floats as clumps on the water surface, simplifying its extraction. The process of oil stain absorption by perlite represents a type of surface absorption strongly influenced by factors such as the absorbent material type, contact surface, structure, and the amount and composition of organic matter. The physical nature of absorption in this process is facilitated by the small size of perlite grains, which increases their contact surface area and absorption capacity. Furthermore, as a neutral and environmentally friendly chemical compound, perlite does not pose any threats to the sea when introduced (Allen, 1990 ; Wang et al. 2014 ). Several factors can impact the amount of absorption, including the type and hydrophobicity of the absorbent and environmental conditions such as temperature. Notably, absorption tends to decrease with increasing temperature (Zhang et al. 2018; Zhang et al. 2019). Key information must be gathered for areas contaminated with oil compounds, including oil concentration, density, the population of oil-degrading micro-organisms, and the potential for biological decomposition. Additional influential factors include the porosity of the absorbent, oxygen availability in the pores, nutrients required for microbial growth (such as phosphorus and nitrogen), environmental temperature, and pH (Olivera et al. 2003 ; ‏ Masoudi et al. 2018 ). Studies on the biological analysis of petroleum compounds have demonstrated the feasibility of this method and its potential as one of the most cost-effective means of eradicating petroleum compounds from aquatic environments (Sabir 2015 ; Riser-Roberts 2020 ). Similar research in this field has been undertaken. For instance, Lee and colleagues examined the biological analysis of production wastes in oil-rich regions and identified several micro-organisms capable of consuming crude oil dissolved in water or present as fine droplets (Wang et al. 2014 , Hassanshahian et al. 2014 , Hedayati Moghaddam. 2023). Leveraging these micro-organisms, Lee achieved the decomposition of 85% of the crude oil in wastewater within seven days (Townsend et al. 2004; Tellez et al. 2002 ; Vocciante et al. 2022 ; Alihosseini, et al. 2015 ). BET method was used to determine its surface and porosity. The method is based on the physical absorption of nitrogen gas at a temperature of 77 degrees Kelvin. In this method, the samples were first heated to 200 degrees Celsius for degassing. Then, at a constant temperature, the amount of nitrogen absorbed at different pressures was checked using the nitrogen absorption and removal system. These isotherms are used to determine the specific surface area, volume of microporous, mesoporous, total pore volume and particle size distribution (Ding et al. 2012). Said analysis using the analyzer surface, model Bel Co., Belsorp mini done. Field emission scanning electron microscope (FE SEM) was used to study the morphology of the absorbent. Using the results of this stage, the characteristics of the surface, shape, size, how the particles are placed on the surface of the object and the composition of the components of the absorbent are shown (Yahya et al. 2015) This analysis was done using an electron microscope model KYKY EM8000. After adding an adsorbent to open water in order to absorb oil pollutants and due to the turbulence of the water, some adsorbent materials are always scattered in the water and cannot be collected. For this purpose, we can hope that the microorganisms loaded on the absorbents will remove the pollutants and prevent them from spreading further. In light of these findings, the present research aims to examine processes that effectively reduce water-based oil pollution and optimize these processes. This was accomplished by examining biological analysis methods, the isolation and selection of an appropriate microbial consortium, and applying the mineral adsorbent perlite. Consequently, the ability to absorb and remove oil pollution from the water was scrutinized. 2. Materials and Methods 2.1. Preparing the bioreactor The bioreactors (Fig. 1 ) in use comprised three 2.5-liter glass containers, each equipped with tightly sealed lids and two inserted tubes. One tube facilitated the introduction of nutrients or the extraction of samples, while the other was designed for the introduction of air via an aquarium pump. Prior to use, these components were sterilized in an autoclave for five minutes. 2.2. Preparation of Mineral Culture Medium The laboratory-prepared culture medium complied with the specifications of Table 1 and was sourced from Merck, Germany. Each liter of mineral culture medium contained 975 ml of main component and 25 ml of minor component. Table 1 Necessary minerals, including main and minor components, along with their concentration for preparation of culture medium (Bastani et al. 2006 ; Pires et al. 2021 ). Main elements KH 2 PO4 K2HPO 4 NaNO3 MgCl 2 .7H 2 O CaCl 2 .2H 2 O FeSO 4 Concentration(g/l) 3.40 4.30 4.00 0.20 0.04 0.03 Trace Elements MnCl 2 NaMoO 4 CuSO 4 H 3 BO 3 ZnSO 4 - Concentration(g/l) 0.400 0.080 0.006 0.013 0.060 - The culture medium, as specified in Table 1 , encompassed both primary and secondary elements in accordance with their relevance for microbial growth. The pH of the sample was adjusted to 7.2 using a NaOH solution (Martinsson et al. 2021 ). Approximately three liters of this solution were initially prepared and stirred for ten minutes. It was then autoclaved at a temperature of 120°C for 15 minutes and subsequently cooled. Thereafter, one liter of the prepared liquid and 10 gram of crude oil as a pollutant were added to each of the assembled bioreactors. The carbon source utilized for microbial growth was crude oil with API degree of 20 (heavy oil of Iran) and a density (g/cm 3 ) of 0.87. This was employed as a pollutant on a thin film of crude oil. 2.3. Bacterial preparation The present study employed a halotolerant, gram-positive bacteria strains from the Streptomyces genus, previously isolated from one of Ahvaz's Asmari reservoirs. Demonstrating significant growth in 5.7% salt concentration, this isolate proved capable of using oil as the sole carbon source. Furthermore, it managed to reduce 46.2% of hydrocarbons in a saline-based environment within six days. These bacteria are not able to grow in anaerobic conditions and this shows that the bacteria are aerobic. Their morphological and physiological traits were examined in nutrient agar to evaluate and prepare the isolated micro-organisms. The cultivation results on the agar medium revealed that the colonies formed were yellow and white, exhibiting smooth and convex borders. 2.4. Absorbent processing The perlite sample examined in this study was sourced from the Azar kani Mahabad company (Bastani et al. 2006 ). Perlite is a form of volcanic rock that experiences an expansion of 4 to 20 times its original volume when subjected to sudden heating in the furnace above 800 O C, causing a color shift from snow white to a greyish hue. Initially, the expanded perlite sample's dimensions were reduced to less than 5 mm using a jaw crusher. Half of the jaw crusher's output was then removed for additional crushing. In a roller crusher, the sample was crushed to a size less than 2mm. A separate quantity of the product was then selected for chemical analysis, mineralogy, and granulation. The chemical analysis presented in Table 2 closely aligned with the average chemical analysis typically reported for perlite minerals. Table 2 Chemical analysis of perlite samples by XRF Compounds SiO 2 Al 2 O 3 K2O Na 2 0 Fe 2 O 3 , FeO CaO MgO TiO 2 etc Weight percent 74.9 14.10 5.9 3.52 1.21 0.73 0.21 0.16 0.08 An oven capable of reaching a nominal temperature of 1250 O C was used to expand the perlite in the laboratory. Given the predominance of silica in perlite's structure and the presence of covalent Si-O bonds, these bonds can form hydrogen bonds with polar water molecules, enabling increased water absorption. Meanwhile, experimental data revealed that the expanded perlite's capacity to absorb 35% of the oil was not dependent (Bastani et al. 2006 ) on the timing of the perlite's contact with the water-oil mixture. This suggests that a portion of perlite's pore space is selectively available for non-polar molecules. The data obtained from the nitrogen adsorption isotherm including BET and surface and volume of pores for pearlite before and after expansion are summarized in Table (3). As it is known, the properties related to the pores and their surface are significantly increased for expanded pearlite. So that the BET level for the adsorbent has reached from about 6 to about 26.39/m 2 /g for expanded perlite, which shows the effect of temperature on the structure of raw materials. Table 3. Surface and pore characteristics obtained from nitrogen absorption isotherms for pearlite before and after expansion. Sample specific surface area of the sample (m 2 /g) Volume of pores (m 3 /g) Primary perlite 6 0.007 Expanded perlite 39.26 0.032 Figure 2 (B to F) depicts the microscopic structure of perlite grains prior to expansion, microbial loading, and oil absorption. These images were captured using a field emission scanning electron microscope (FESEM) at varying magnifications. As depicted in the figure, the absorbent grains' structure exhibited minimal voids and porosity. However, as shown in Figure 3, the cross-sectional area and porosity of the perlite grains increased after the expansion of the absorbent grains. This allowed them to function as greater oil absorbers and as habitats for microbial growth. 2.5. Processing The extent of oil adsorption was determined through two separate tests: 2.5.1. Oil sorption levels using expanded perlite: Samples of expanded perlite prepared prior to microbial cultivation, were used (Figures 4). Initially, one liter of water was poured into each container, followed by the addition of 10 grams of heavy crude oil to serve as an oil pollutant. Subsequently, 20 grams of expanded perlite were introduced into the oil-water mixture. Following a 5-minute absorption and saturation process, the moistened absorbent was placed on a specialized filter to drain unabsorbed, potentially trapped liquid. As depicted in illustrate 4 perlite transforms into clumps and sludge post-absorption. 2.5.2. Comparison of oil absorption with adsorbent and removal with oil-eating Bactria in a bioreactor: After Tests were conducted on the absorption rates of the perlite samples, sans microbes and reactors, to measure the rate of absorption and oil stain removal aided by oil-eating Bactria. Subsequently, the oil absorption and removal process was examined within the bioreactor, both with and without the presence of Bactria. As depicted in Fig. 1 , one liter of the previously prepared culture solution was poured into bioreactors A, B, and C. Ten grams of light crude oil was then added as an oil contaminant, visible as a thin film, in all three containers. In the next step, 20 grams of perlite was introduced to bioreactor A, while 20 grams of microbe-impregnated perlite were added to bioreactor B, and 20 grams of microbe-impregnated expanded perlite were added to bioreactor C. The cultivation period concluded after four days at a maintained temperature of 21 ± 3°C, with continuous aeration provided by an aquarium pump. Samples were taken every eight hours under uniform conditions to measure the surface tension of the solutions. 3. Results and discussion 3.1. Results of measurement of interfacial tension The results of measuring interfacial tension with a tensometer (KRUSS K11 model) during four days of culture for three bioreactors are shown in Fig. 5 (Average of four stages of microbial culture). It was observed that the surface tension remained constant at 71 mN/m in culture A, a consequence of microbial absence and, therefore, no bio-surfactant production. However, cultures B and C exhibited a decrease in surface tension, registering 33.8 mN/m and 30.3 mN/m, respectively. Culture A's lack of microbes automatically ruled out any possibility of oil consumption or removal. In contrast, Culture B, despite having a smaller quantity and diversity of microbes, demonstrated a lower rate of microbial proliferation and bio-surfactant production compared to Culture C. Further influencing these differences is the effect of expanding pearlite grains. This phenomenon increases porosity, enhancing oil and mineral absorption in the substrate. Such a condition proves beneficial in fostering microbial proliferation, leading to greater surfactant production and consequently lowering surface tension. 3.2. Measurement results of oil absorption rate After the completion of the cultivation phase, an assessment of oil absorption by the perlite adsorbents was conducted. Perlite samples were extracted from each bioreactor for this purpose. The absorption capacity of the perlite was quantified using a weighing method. Initially, the dry perlite was weighed prior to the experiment. Following the absorption and saturation process, the wet perlite was placed on a dedicated filter for four minutes, allowing any non-absorbed, entrapped liquid to be filtered out. Afterward, the perlite was weighed again. The amount of oil absorbed per gram of perlite was then calculated using the following equation (Eq. 1 ) (Hedayati Moghaddam et al. 2013 ; Lazim, A et al. 2019; Nourmoradi 2014; Hassanzadeh, et al. 2021 , Pelletier et al. 2004 ). In addition, because heavy oil at low temperature (21 ± 3°C) was used as a pollutant, the amount of evaporation of hydrocarbons was insignificant and it was ignored in the calculations. $$S={S}_{0}-{S}_{w}-{S}_{i}$$ 1 The parameters considered in this equation were as follows: weight of the absorbed oil (S), the weight of the perlite including the absorbed liquid (S o ), the weight of water absorbed with the oil (S w ), and the weight of the dry perlite before the experiment (S i ). The first set of experiments yielded intriguing results. The absorption of oil from the water was measured at 1.3 g/l in the presence of regular perlite and 2.7 g/l in the context of expanded perlite. Furthermore, the separated water content in regular perlite was 2.9 g/l, while that in expanded perlite grains was recorded at 6.4 g/l. Given the higher water absorption in both instances, it can be inferred that perlite grains exhibit stronger hydrophilic characteristics. However, the absorption results disclose that expanded perlite absorbed approximately 179% more crude oil than its regular counterpart. This dramatic increase can be attributed to the perlite grains' heightened porosity and enlarged surface area following expansion. The results of four stages of cultivation of oil-eating Bactria in order to check the weight of oil absorbed (S), the weight of oil removed by bacteria (O r ), in two bioreactors R B (granules perlite), and R C bioreactor (expanded perlite) in Table 3 is given. Each cultivation stage was carried out for four days, and each time the mass of adsorbent added (S i ) was 20 grams and the total mass of oil added to the bioreactor (O t ) was 10 grams. In the second series of tests, reactor A separated 1.17 g/l of oil. As the perlite used in this reactor demonstrated greater hydrophilicity, the oil absorption level was relatively lower than the other two reactors. Yet, from cultures B and C, 3.280 ± 0.106 g/l and 4.045 ± 0.260 g/l of oil were respectively separated. This relative increase, when compared to culture A, can be ascribed to bio-surfactant production by microbes, inducing a change in the perlite grains' wettability from hydrophilic to lipophilic. Moreover, the increased absorption in culture C compared to B can be traced back to the expansion of perlite grains and the resulting increase in surface area (Fig. 6 ). The standard deviation of oil absorption in bioreactor B and C is 0.106 and 0.260 g/l respectively, and the standard deviation of oil removal in bioreactor B and C is 0.093 and 0.121 g/l respectively. Table 4 The results of four stages of cultivation in order to check the weight of oil absorbed (S) and the weight of oil removed by bacteria (O r ), in two bioreactors R B (primary perlite) and bioreactor R C (expanded perlite). Microbial culture order Bioreactors S o (g) S w (g) S(g) O S (g) O r (g) No1 R B 26.03 2.86 3.17 4.34 2.49 R C 30.49 6.52 3.97 2.39 3.64 No2 R B 25.96 2.74 3.22 4.15 2.63 R C 30.60 6.77 3.83 2.41 3.71 No3 R B 26.42 3.01 3.41 4.08 2.51 R C 31.09 6.97 4.12 2.32 3.56 N04 R B 26.45 3.13 3.32 4.73 2.41 R C 31.34 7.08 4.26 2.27 3.47 weighted average R B 26.21 2.93 3.28 4.32 2.51 R C 30.88 6.83 4.04 2.34 3.59 3.3. Results of oil removal rate by oil-eating Bactria The remaining oil content in the culture medium was documented post-centrifugation for 10 minutes at 5000 rpm using a separating funnel. And along with the amount of oil separated from the absorbents, it was subtracted from the amount of 10 grams of oil added to the culture medium (O t ), and the amount of oil used as an organic source for the growth of microbes was measured (Eq. 2 ). This allowed for the measurement of the amount of oil utilized as an organic growth source for the microbes. $${\text{S}}_{\text{r}}={\text{S}}_{\text{t}}-\text{S} -{\text{S}}_{\text{s}}$$ 2 In this regard, the oil removed by bacteria ( \({\text{S}}_{\text{r}}\) ), the total weight of oil added to the bioreactor (S t ), the weight of absorbed oil (S) and the weight of dissolved and remaining oil in the bioreactor (S S ). The data revealed negligible oil consumption by microbes in bioreactor A due to the absence of microbial growth and synthesis. However, in bioreactors B and C, the average amount of oil removal by microorganisms was 2.520 ± 0.093 and 3.78 ± 0.121 grams per liter, respectively. These findings align with the previously recorded surface tension measurements across all three bioreactors, as oil consumption directly correlates with the bio-surfactant production level. Reactor C exhibited enhanced absorption due to the increased surface area and porosity, facilitating a greater microbial load and, consequently, improved potential for growth and proliferation. 4. Conclusion In the combined approach utilizing both adsorbents and microbes, the dispersion and subsequent non-collection of adsorbents from open water can potentially be mitigated as the microbes consume and eliminate a portion of the pollutants. Given that perlite grains function as both water and oil absorbers, and with their inherent hydrophilic nature, their expansion and subsequent increase in volume can create beneficial voids. These voids can serve as growth substrates for microbes, aiding in removing and absorbing oil stains from polluted water. The procedure was executed by introducing dry perlite seeds contaminated with microbes and expanded adsorbents impregnated with microbes. The findings indicated that with the first method using dry, microbe-contaminated adsorbents the removal and absorption of oil stains from water were boosted by 25.2% and 32.8%, respectively. Moreover, with the employment of expanded adsorbents, the removal of oil stains from water saw an enhancement of 35.95%, while oil absorption from water surged by 44.50%. The reasons for these increases in oil absorption are twofold. Firstly, the bio-surfactant synthesized by microbes modified the wettability of the adsorbent, transitioning it from hydrophilic to lipophilic. Secondly, the expansion of the adsorbent not only amplified the contact surface but also increased the porosity of the adsorbents. This change, in turn, enhanced the microbial load, boosting the absorption of oil and minerals required for microbial proliferation. Declarations Conflicts of interests On behalf of all author, the corresponding author states that there is no conflict of interest. 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Performance evaluation of an activated sludge system for removing petroleum hydrocarbons from oilfield produced water. Advances in Environmental Research , 6 (4), 455-470. ‏ https://doi.org/10.1016/S1093-0191(01)00073-9. Verma, S., Lee, T., Sahle-Demessie, E., Ateia, M., & Nadagouda, M. N. (2022). Recent advances on PFAS degradation via thermal and nonthermal methods. Chemical engineering journal advances , 100421. https://doi.org/10.1016/j.ceja.2022.100421 ‏ Vocciante, M., De Folly D’Auris, A., & Reverberi, A. P. (2022). A Novel Graphite-Based Sorbent for Oil Spill Cleanup. Materials , 15 (2), 609. https://doi.org/10.3390/ma15020609. ‏ Wang, J., Zheng, Y., & Wang, A. (2014). Kinetic and thermodynamic studies on the removal of oil from water using superhydrophobic kapok fiber. Water Environment Research , 86(4), 360-365. https://doi.org/10.2175/106143013x13807328849693. Yadav, S., Kumar, S., & Haritash, A. K. (2023). A comprehensive review of chlorophenols: Fate, toxicology and its treatment. Journal of Environmental Management, 342, 118254. ‏ https://doi.org/10.1016/j.jenvman.2023.118254 Yahya, M., Chen, Y. W., Lee, H. V., & Hassan, W. H. W. (2018). Reuse of selected lignocellulosic and processed biomasses as sustainable sources for the fabrication of nanocellulose via Ni (II)-catalyzed hydrolysis approach: a comparative study. Journal of Polymers and the Environment , 26, 2825-2844. Zhang, T., & Silverstein, M. S. (2018). Microphase-separated macroporous polymers from an emulsion-templated reactive triblock copolymer. Macromolecules , 51(10), 3828-3835. Zhang, T., & Silverstein, M. S. (2019). Robust, highly porous hydrogels templated within emulsions stabilized using a reactive, crosslinking triblock copolymer. Polymer , 168 , 146-154. https://doi.org/10.1016/j.polymer.2019.02.010. 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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-3703177","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267560444,"identity":"5a96f4dc-d1ce-44f2-a8b1-4270e16fea06","order_by":0,"name":"Seyyed Reza Mortazavi","email":"","orcid":"","institution":"Islamic Azad University Central Tehran Branch","correspondingAuthor":false,"prefix":"","firstName":"Seyyed","middleName":"Reza","lastName":"Mortazavi","suffix":""},{"id":267560445,"identity":"8f50ad24-4a1a-49fa-97e0-065b9552e282","order_by":1,"name":"Amir H. M. Sarrafi","email":"","orcid":"","institution":"Islamic Azad University Central Tehran Branch","correspondingAuthor":false,"prefix":"","firstName":"Amir","middleName":"H. M.","lastName":"Sarrafi","suffix":""},{"id":267560446,"identity":"7b60ecf6-d6ae-489e-bded-86ce16a022b6","order_by":2,"name":"Afshar Alihosseini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABH0lEQVRIie2SP0vDQBTA3xG4LIFbnyDNV7gQKB0Uv0qODl2iCF10KYHCdSlmjd+ibhkTCukScXUzQdClQ4tLBQcvEcngha4O9xse94778f5wAAbDf8TKVLjGoE0CwAFtDiTq4h9o85h3it8q+XEFgt9cRJ2ih0m7ggMfXTG2qas6HU3u7NL72KfgssiSO42ChcPJkuP0JAl9Lkq8lE7oY16Cl2RknujKvCwBHI5i9exQFFIpEA4hl0BWQOY6wy3sinw1ytPm/aCUCWXbVrnoU3gB3GqrZMEQlBJQ/Kki+hRPzbI+Vcq9mqVpzJP4NsXHEsfJWq8MCruutzczEauN7T/lzHXj8cPuNj07jxeLV/2aATLdJaqP0ScYDAaD4RjfYVhf7XCrz2AAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-6750-720X","institution":"Islamic Azad University of Tehran: Islamic Azad University Central Tehran Branch","correspondingAuthor":true,"prefix":"","firstName":"Afshar","middleName":"","lastName":"Alihosseini","suffix":""},{"id":267560447,"identity":"c2a18568-a4d5-4674-94a2-e01efac62e22","order_by":3,"name":"Ali Niazi","email":"","orcid":"","institution":"Islamic Azad University Central Tehran Branch","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Niazi","suffix":""}],"badges":[],"createdAt":"2023-12-04 01:37:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3703177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3703177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49806976,"identity":"b4b9419b-5301-4518-b621-6dac6adff5a5","added_by":"auto","created_at":"2024-01-18 10:43:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":142269,"visible":true,"origin":"","legend":"\u003cp\u003eThree bioreactors for microbial culture.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/1a5a87009503276525357f7d.png"},{"id":49806981,"identity":"504e38bc-42c6-4afe-bca9-9fb5355b7437","added_by":"auto","created_at":"2024-01-18 10:43:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":549646,"visible":true,"origin":"","legend":"\u003cp\u003ePerlite grains before expansion (A, B): Microscopic structure of perlite prior to microbial loading and oil absorption, captured via field emission scanning electron microscope (FESEM) at different magnifications: (C) 200 micrometers, (D) 30 µm, (E) 7 µm, (F) 3 µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/0631e12cdf80aa73423161f3.png"},{"id":49807308,"identity":"6fda2e69-3805-4925-a70d-ca0aa7be0512","added_by":"auto","created_at":"2024-01-18 10:51:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":482651,"visible":true,"origin":"","legend":"\u003cp\u003ePerlite grains after expansion (A, B): Microscopic structure of expanded perlite after microbial loading and oil absorption, captured via field emission scanning electron microscope (FE-SEM) at different magnifications: (C) 200 micrometers, (D) 30 µm, (E) 7 µm, (F) 3 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/39c810b6adfd765b3985463e.png"},{"id":49806980,"identity":"91fb1b78-0af8-411c-9f84-ba406e3f0f96","added_by":"auto","created_at":"2024-01-18 10:43:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":257795,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic observation of oil sorption by expanded pearlite.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/776d88f4429160bb3d26b910.png"},{"id":49806977,"identity":"d5ce23b9-67b5-4aa8-afa7-2606e9b11727","added_by":"auto","created_at":"2024-01-18 10:43:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":18944,"visible":true,"origin":"","legend":"\u003cp\u003eResults of water and oil interfacial tension in oil-contaminated cultivation in two reactors B and C.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/995bbed447c10efc33c01406.png"},{"id":49806978,"identity":"cf3e4b96-c5ba-4ed4-91a7-2e5de6f72519","added_by":"auto","created_at":"2024-01-18 10:43:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8372,"visible":true,"origin":"","legend":"\u003cp\u003eWeighted average of absorption (oil and water) and removal of impurities by microorganisms during four stages of cultivation in three bioreactors.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/097e32c7a41e26bc937981db.png"},{"id":50890055,"identity":"0557eac1-3c5c-4642-a90e-5dc6ec4dd77a","added_by":"auto","created_at":"2024-02-09 04:06:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1970305,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/d8002930-f7a9-4841-ad20-991c8aee02d8.pdf"},{"id":49806982,"identity":"80262b54-89a3-48b5-a1b9-2a079faad229","added_by":"auto","created_at":"2024-01-18 10:43:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":334784,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-3703177/v1/0cb492776f82bfe065522399.docx"}],"financialInterests":"","formattedTitle":"Study of oil biodegrading by expanded perlite Loaded by oil-eating bacteria","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eExtensive water pollution is a consequence of various operations involved in the crude oil industry, namely extraction, transportation, refinement, and storage. Spillage usually contaminates the aquatic environment, triggering physical and chemical changes such as evaporation, decomposition, photochemical oxidation, bioremediation and absorption onto suspended materials, thereby exacerbating aquatic pollution (Saad et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite preventative measures, catastrophic incidents, such as the 2021 Orange County oil spill, persist and introduce oil contaminants into the water (Tanee et al. 2015). Petroleum hydrocarbons inflict several detrimental effects on living organisms, including toxicity, mutagenicity, potential carcinogenesis, and disruption of life cycles, necessitating their removal from the environment (Muthamilselvi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yadav et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Qaria et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Three major strategies \u0026ldquo;physical, chemical, and biological\u0026rdquo; exist to eradicate oil pollutants. Among them, using micro-organisms in bio-remediation techniques is safe and economical. This process is conducted by natural environmental micro-organisms under natural conditions and can reduce pollution to acceptable levels (Tabari et al. 2009; Bouchez et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Various methodologies have been conceived to combat oil spills and their associated problems. Relying on diverse principles of removal (Tabari et al. 2009; Bhardwaj et al. 2018), these techniques, especially adsorbents, are categorized as combined systems as they can either contain (passively) or remove (actively) pollutants. These strategies have proven particularly effective for the recovery of oil traces from the environment (Bhardwaj et al. 2018; Scarlett et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Narimani et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Piterlli et al. 2020). Absorption, in fact, is recognized as a straightforward and potent solution. A plethora of organic (Lee et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Moradi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and inorganic pollutants populate aqueous solutions. However, in open waters, due to the transport of crude oil, the prevalence of organic pollutants and associated biodegradability impacts are heightened (Verma et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Moradi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. 2018). Perlite, an abundant volcanic rock found in mines, has piqued industrial interest due to its favorable characteristics, which include low density, extensive pore space, a layered structure, swift oil spread, high permeability, and ease of collection (Allen, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Following oil absorption, perlite floats as clumps on the water surface, simplifying its extraction. The process of oil stain absorption by perlite represents a type of surface absorption strongly influenced by factors such as the absorbent material type, contact surface, structure, and the amount and composition of organic matter. The physical nature of absorption in this process is facilitated by the small size of perlite grains, which increases their contact surface area and absorption capacity. Furthermore, as a neutral and environmentally friendly chemical compound, perlite does not pose any threats to the sea when introduced (Allen, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Several factors can impact the amount of absorption, including the type and hydrophobicity of the absorbent and environmental conditions such as temperature. Notably, absorption tends to decrease with increasing temperature (Zhang et al. 2018; Zhang et al. 2019). Key information must be gathered for areas contaminated with oil compounds, including oil concentration, density, the population of oil-degrading micro-organisms, and the potential for biological decomposition. Additional influential factors include the porosity of the absorbent, oxygen availability in the pores, nutrients required for microbial growth (such as phosphorus and nitrogen), environmental temperature, and pH (Olivera et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; \u0026rlm; Masoudi et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Studies on the biological analysis of petroleum compounds have demonstrated the feasibility of this method and its potential as one of the most cost-effective means of eradicating petroleum compounds from aquatic environments (Sabir \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Riser-Roberts \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similar research in this field has been undertaken. For instance, Lee and colleagues examined the biological analysis of production wastes in oil-rich regions and identified several micro-organisms capable of consuming crude oil dissolved in water or present as fine droplets (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Hassanshahian et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Hedayati Moghaddam. 2023). Leveraging these micro-organisms, Lee achieved the decomposition of 85% of the crude oil in wastewater within seven days (Townsend et al. 2004; Tellez et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Vocciante et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Alihosseini, et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). BET method was used to determine its surface and porosity. The method is based on the physical absorption of nitrogen gas at a temperature of 77 degrees Kelvin. In this method, the samples were first heated to 200 degrees Celsius for degassing. Then, at a constant temperature, the amount of nitrogen absorbed at different pressures was checked using the nitrogen absorption and removal system. These isotherms are used to determine the specific surface area, volume of microporous, mesoporous, total pore volume and particle size distribution (Ding et al. 2012). Said analysis using the analyzer surface, model Bel Co., Belsorp mini done. Field emission scanning electron microscope (FE SEM) was used to study the morphology of the absorbent. Using the results of this stage, the characteristics of the surface, shape, size, how the particles are placed on the surface of the object and the composition of the components of the absorbent are shown (Yahya et al. 2015) This analysis was done using an electron microscope model KYKY EM8000. After adding an adsorbent to open water in order to absorb oil pollutants and due to the turbulence of the water, some adsorbent materials are always scattered in the water and cannot be collected. For this purpose, we can hope that the microorganisms loaded on the absorbents will remove the pollutants and prevent them from spreading further. In light of these findings, the present research aims to examine processes that effectively reduce water-based oil pollution and optimize these processes. This was accomplished by examining biological analysis methods, the isolation and selection of an appropriate microbial consortium, and applying the mineral adsorbent perlite. Consequently, the ability to absorb and remove oil pollution from the water was scrutinized.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Preparing the bioreactor\u003c/h2\u003e\n \u003cp\u003eThe bioreactors (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) in use comprised three 2.5-liter glass containers, each equipped with tightly sealed lids and two inserted tubes. One tube facilitated the introduction of nutrients or the extraction of samples, while the other was designed for the introduction of air via an aquarium pump. Prior to use, these components were sterilized in an autoclave for five minutes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Preparation of Mineral Culture Medium\u003c/h2\u003e\n \u003cp\u003eThe laboratory-prepared culture medium complied with the specifications of Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and was sourced from Merck, Germany. Each liter of mineral culture medium contained 975 ml of main component and 25 ml of minor component.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNecessary minerals, including main and minor components, along with their concentration for preparation of culture medium (Bastani et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pires et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMain elements\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eKH\u003csub\u003e2\u003c/sub\u003ePO4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eK2HPO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNaNO3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConcentration(g/l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTrace Elements\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMnCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNaMoO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCuSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZnSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConcentration(g/l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe culture medium, as specified in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, encompassed both primary and secondary elements in accordance with their relevance for microbial growth. The pH of the sample was adjusted to 7.2 using a NaOH solution (Martinsson et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Approximately three liters of this solution were initially prepared and stirred for ten minutes. It was then autoclaved at a temperature of 120\u0026deg;C for 15 minutes and subsequently cooled. Thereafter, one liter of the prepared liquid and 10 gram of crude oil as a pollutant were added to each of the assembled bioreactors. The carbon source utilized for microbial growth was crude oil with API degree of 20 (heavy oil of Iran) and a density (g/cm\u003csup\u003e3\u003c/sup\u003e) of 0.87. This was employed as a pollutant on a thin film of crude oil.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Bacterial preparation\u003c/h2\u003e\n \u003cp\u003eThe present study employed a halotolerant, gram-positive bacteria strains from the Streptomyces genus, previously isolated from one of Ahvaz\u0026apos;s Asmari reservoirs. Demonstrating significant growth in 5.7% salt concentration, this isolate proved capable of using oil as the sole carbon source. Furthermore, it managed to reduce 46.2% of hydrocarbons in a saline-based environment within six days. These bacteria are not able to grow in anaerobic conditions and this shows that the bacteria are aerobic.\u003c/p\u003e\n \u003cp\u003eTheir morphological and physiological traits were examined in nutrient agar to evaluate and prepare the isolated micro-organisms. The cultivation results on the agar medium revealed that the colonies formed were yellow and white, exhibiting smooth and convex borders.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Absorbent processing\u003c/h2\u003e\n \u003cp\u003eThe perlite sample examined in this study was sourced from the Azar kani Mahabad company (Bastani et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Perlite is a form of volcanic rock that experiences an expansion of 4 to 20 times its original volume when subjected to sudden heating in the furnace above 800 \u003csup\u003eO\u003c/sup\u003eC, causing a color shift from snow white to a greyish hue. Initially, the expanded perlite sample\u0026apos;s dimensions were reduced to less than 5 mm using a jaw crusher. Half of the jaw crusher\u0026apos;s output was then removed for additional crushing. In a roller crusher, the sample was crushed to a size less than 2mm. A separate quantity of the product was then selected for chemical analysis, mineralogy, and granulation. The chemical analysis presented in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e closely aligned with the average chemical analysis typically reported for perlite minerals.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eChemical analysis of perlite samples by XRF\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAl\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eK2O\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNa\u003csub\u003e2\u003c/sub\u003e0\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, FeO\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCaO\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMgO\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTiO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eetc\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeight percent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e74.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAn oven capable of reaching a nominal temperature of 1250 \u003csup\u003eO\u003c/sup\u003eC was used to expand the perlite in the laboratory. Given the predominance of silica in perlite\u0026apos;s structure and the presence of covalent Si-O bonds, these bonds can form hydrogen bonds with polar water molecules, enabling increased water absorption. Meanwhile, experimental data revealed that the expanded perlite\u0026apos;s capacity to absorb 35% of the oil was not dependent (Bastani et al. \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e) on the timing of the perlite\u0026apos;s contact with the water-oil mixture. This suggests that a portion of perlite\u0026apos;s pore space is selectively available for non-polar molecules.\u003c/p\u003e\n \u003cp\u003eThe data obtained from the nitrogen adsorption isotherm including BET and surface and volume of pores for pearlite before and after expansion are summarized in Table\u0026nbsp;(3). As it is known, the properties related to the pores and their surface are significantly increased for expanded pearlite. So that the BET level for the adsorbent has reached from about 6 to about 26.39/m\u003csup\u003e2\u003c/sup\u003e/g for expanded perlite, which shows the effect of temperature on the structure of raw materials.\u003c/p\u003e\n \u003cp\u003eTable 3. Surface and pore characteristics obtained from nitrogen absorption isotherms for pearlite before and after expansion.\u003c/p\u003e\n \u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"501\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.355289421157686%\" valign=\"top\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.30339321357285%\" valign=\"top\"\u003e\n \u003cp\u003especific surface area of the sample (m\u003csup\u003e2\u003c/sup\u003e/g)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.34131736526946%\" valign=\"top\"\u003e\n \u003cp\u003eVolume of pores (m\u003csup\u003e3\u003c/sup\u003e/g)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.355289421157686%\" valign=\"top\"\u003e\n \u003cp\u003ePrimary perlite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.30339321357285%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.34131736526946%\" valign=\"top\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.355289421157686%\" valign=\"top\"\u003e\n \u003cp\u003eExpanded perlite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.30339321357285%\" valign=\"top\"\u003e\n \u003cp\u003e39.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.34131736526946%\" valign=\"top\"\u003e\n \u003cp\u003e0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp skip=\"true\"\u003eFigure 2 (B to F) depicts the microscopic structure of perlite grains prior to expansion, microbial loading, and oil absorption. These images were captured using a field emission scanning electron microscope (FESEM) at varying magnifications. As depicted in the figure, the absorbent grains\u0026apos; structure exhibited minimal voids and porosity. However, as shown in Figure 3, the cross-sectional area and porosity of the perlite grains increased after the expansion of the absorbent grains. This allowed them to function as greater oil absorbers and as habitats for microbial growth.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.5. \u0026nbsp;Processing\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThe extent of oil adsorption was determined through two separate tests:\u003c/p\u003e\n \u003cp\u003e2.5.1. \u0026nbsp; Oil sorption levels using expanded perlite:\u003c/p\u003e\n \u003cp\u003eSamples of expanded perlite prepared prior to microbial cultivation, were used (Figures 4). Initially, one liter of water was poured into each container, followed by the addition of 10 grams of heavy crude oil to serve as an oil pollutant. Subsequently, 20 grams of expanded perlite were introduced into the oil-water mixture. Following a 5-minute absorption and saturation process, the moistened absorbent was placed on a specialized filter to drain unabsorbed, potentially trapped liquid. As depicted in illustrate 4 perlite transforms into clumps and sludge post-absorption.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.5.2. Comparison of oil absorption with adsorbent and removal with oil-eating Bactria in a bioreactor:\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAfter Tests were conducted on the absorption rates of the perlite samples, sans microbes and reactors, to measure the rate of absorption and oil stain removal aided by oil-eating Bactria. Subsequently, the oil absorption and removal process was examined within the bioreactor, both with and without the presence of Bactria. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, one liter of the previously prepared culture solution was poured into bioreactors A, B, and C. Ten grams of light crude oil was then added as an oil contaminant, visible as a thin film, in all three containers. In the next step, 20 grams of perlite was introduced to bioreactor A, while 20 grams of microbe-impregnated perlite were added to bioreactor B, and 20 grams of microbe-impregnated expanded perlite were added to bioreactor C. The cultivation period concluded after four days at a maintained temperature of 21\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C, with continuous aeration provided by an aquarium pump. Samples were taken every eight hours under uniform conditions to measure the surface tension of the solutions.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Results of measurement of interfacial tension\u003c/h2\u003e\n \u003cp\u003eThe results of measuring interfacial tension with a tensometer (KRUSS K11 model) during four days of culture for three bioreactors are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (Average of four stages of microbial culture). It was observed that the surface tension remained constant at 71 mN/m in culture A, a consequence of microbial absence and, therefore, no bio-surfactant production. However, cultures B and C exhibited a decrease in surface tension, registering 33.8 mN/m and 30.3 mN/m, respectively. Culture A\u0026apos;s lack of microbes automatically ruled out any possibility of oil consumption or removal. In contrast, Culture B, despite having a smaller quantity and diversity of microbes, demonstrated a lower rate of microbial proliferation and bio-surfactant production compared to Culture C. Further influencing these differences is the effect of expanding pearlite grains. This phenomenon increases porosity, enhancing oil and mineral absorption in the substrate. Such a condition proves beneficial in fostering microbial proliferation, leading to greater surfactant production and consequently lowering surface tension.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Measurement results of oil absorption rate\u003c/h2\u003e\n \u003cp\u003eAfter the completion of the cultivation phase, an assessment of oil absorption by the perlite adsorbents was conducted. Perlite samples were extracted from each bioreactor for this purpose. The absorption capacity of the perlite was quantified using a weighing method. Initially, the dry perlite was weighed prior to the experiment. Following the absorption and saturation process, the wet perlite was placed on a dedicated filter for four minutes, allowing any non-absorbed, entrapped liquid to be filtered out. Afterward, the perlite was weighed again. The amount of oil absorbed per gram of perlite was then calculated using the following equation (Eq.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) (Hedayati Moghaddam et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lazim, A et al. 2019; Nourmoradi 2014; Hassanzadeh, et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e, Pelletier et al. \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e). In addition, because heavy oil at low temperature (21\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C) was used as a pollutant, the amount of evaporation of hydrocarbons was insignificant and it was ignored in the calculations.\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$S={S}_{0}-{S}_{w}-{S}_{i}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eThe parameters considered in this equation were as follows: weight of the absorbed oil (S), the weight of the perlite including the absorbed liquid (S\u003csub\u003eo\u003c/sub\u003e), the weight of water absorbed with the oil (S\u003csub\u003ew\u003c/sub\u003e), and the weight of the dry perlite before the experiment (S\u003csub\u003ei\u003c/sub\u003e).\u003c/p\u003e\n \u003cp\u003eThe first set of experiments yielded intriguing results. The absorption of oil from the water was measured at 1.3 g/l in the presence of regular perlite and 2.7 g/l in the context of expanded perlite. Furthermore, the separated water content in regular perlite was 2.9 g/l, while that in expanded perlite grains was recorded at 6.4 g/l. Given the higher water absorption in both instances, it can be inferred that perlite grains exhibit stronger hydrophilic characteristics. However, the absorption results disclose that expanded perlite absorbed approximately 179% more crude oil than its regular counterpart. This dramatic increase can be attributed to the perlite grains\u0026apos; heightened porosity and enlarged surface area following expansion. The results of four stages of cultivation of oil-eating Bactria in order to check the weight of oil absorbed (S), the weight of oil removed by bacteria (O\u003csub\u003er\u003c/sub\u003e), in two bioreactors R\u003csub\u003eB\u003c/sub\u003e (granules perlite), and R\u003csub\u003eC\u003c/sub\u003e bioreactor (expanded perlite) in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e is given. Each cultivation stage was carried out for four days, and each time the mass of adsorbent added (S\u003csub\u003ei\u003c/sub\u003e) was 20 grams and the total mass of oil added to the bioreactor (O\u003csub\u003et\u003c/sub\u003e) was 10 grams. In the second series of tests, reactor A separated 1.17 g/l of oil. As the perlite used in this reactor demonstrated greater hydrophilicity, the oil absorption level was relatively lower than the other two reactors. Yet, from cultures B and C, 3.280\u0026thinsp;\u0026plusmn;\u0026thinsp;0.106 g/l and 4.045\u0026thinsp;\u0026plusmn;\u0026thinsp;0.260 g/l of oil were respectively separated. This relative increase, when compared to culture A, can be ascribed to bio-surfactant production by microbes, inducing a change in the perlite grains\u0026apos; wettability from hydrophilic to lipophilic. Moreover, the increased absorption in culture C compared to B can be traced back to the expansion of perlite grains and the resulting increase in surface area (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The standard deviation of oil absorption in bioreactor B and C is 0.106 and 0.260 g/l respectively, and the standard deviation of oil removal in bioreactor B and C is 0.093 and 0.121 g/l respectively.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe results of four stages of cultivation in order to check the weight of oil absorbed (S) and the weight of oil removed by bacteria (O\u003csub\u003er\u003c/sub\u003e), in two bioreactors R\u003csub\u003eB\u003c/sub\u003e (primary perlite) and bioreactor R\u003csub\u003eC\u003c/sub\u003e (expanded perlite).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMicrobial culture order\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBioreactors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS\u003csub\u003eo\u003c/sub\u003e(g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS\u003csub\u003ew\u003c/sub\u003e(g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS(g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eO\u003csub\u003eS\u003c/sub\u003e(g)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eO\u003csub\u003er\u003c/sub\u003e(g)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNo1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNo2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eNo3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eN04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eweighted average\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eC\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Results of oil removal rate by oil-eating Bactria\u003c/h2\u003e\n \u003cp\u003eThe remaining oil content in the culture medium was documented post-centrifugation for 10 minutes at 5000 rpm using a separating funnel. And along with the amount of oil separated from the absorbents, it was subtracted from the amount of 10 grams of oil added to the culture medium (O\u003csub\u003et\u003c/sub\u003e), and the amount of oil used as an organic source for the growth of microbes was measured (Eq. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This allowed for the measurement of the amount of oil utilized as an organic growth source for the microbes.\u003c/p\u003e\n \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e$${\\text{S}}_{\\text{r}}={\\text{S}}_{\\text{t}}-\\text{S} -{\\text{S}}_{\\text{s}}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eIn this regard, the oil removed by bacteria (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{S}}_{\\text{r}}\\)\u003c/span\u003e\u003c/span\u003e), the total weight of oil added to the bioreactor (S\u003csub\u003et\u003c/sub\u003e), the weight of absorbed oil (S) and the weight of dissolved and remaining oil in the bioreactor (S\u003csub\u003eS\u003c/sub\u003e). The data revealed negligible oil consumption by microbes in bioreactor A due to the absence of microbial growth and synthesis. However, in bioreactors B and C, the average amount of oil removal by microorganisms was 2.520\u0026thinsp;\u0026plusmn;\u0026thinsp;0.093 and 3.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.121 grams per liter, respectively. These findings align with the previously recorded surface tension measurements across all three bioreactors, as oil consumption directly correlates with the bio-surfactant production level. Reactor C exhibited enhanced absorption due to the increased surface area and porosity, facilitating a greater microbial load and, consequently, improved potential for growth and proliferation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn the combined approach utilizing both adsorbents and microbes, the dispersion and subsequent non-collection of adsorbents from open water can potentially be mitigated as the microbes consume and eliminate a portion of the pollutants. Given that perlite grains function as both water and oil absorbers, and with their inherent hydrophilic nature, their expansion and subsequent increase in volume can create beneficial voids. These voids can serve as growth substrates for microbes, aiding in removing and absorbing oil stains from polluted water. The procedure was executed by introducing dry perlite seeds contaminated with microbes and expanded adsorbents impregnated with microbes. The findings indicated that with the first method using dry, microbe-contaminated adsorbents the removal and absorption of oil stains from water were boosted by 25.2% and 32.8%, respectively. Moreover, with the employment of expanded adsorbents, the removal of oil stains from water saw an enhancement of 35.95%, while oil absorption from water surged by 44.50%. The reasons for these increases in oil absorption are twofold. Firstly, the bio-surfactant synthesized by microbes modified the wettability of the adsorbent, transitioning it from hydrophilic to lipophilic. Secondly, the expansion of the adsorbent not only amplified the contact surface but also increased the porosity of the adsorbents. This change, in turn, enhanced the microbial load, boosting the absorption of oil and minerals required for microbial proliferation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConflicts of interests\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eOn behalf of all author, the corresponding author states that there is no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding and Competing Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAllen, M. J. (1990). The Perlite Institute Annual Meeting and Conference. \u003cem\u003eIndustrial Minerals, September\u003c/em\u003e, 69-73.\u003cspan dir=\"RTL\"\u003e \u003c/span\u003ehttps://doi.org/10.1016/S1353-2561 (02)00066-X.\u003cspan dir=\"RTL\"\u003e\u0026rlm;\u003c/span\u003e \u003c/li\u003e\n\u003cli\u003eAlihosseini, A., Dadfar, E., Aibod, Saeid. (2015). Synthesis and Characterization of Novel Poly (Amide-Imide) Nanocomposite/Silicate Particles Based on N-Pyromellitimido-L-Phenyl Alanine Containing Sulfone Moieties. \u003cem\u003eJournal of Applied Chemical Science International\u003c/em\u003e, 84-92\u003c/li\u003e\n\u003cli\u003eBastani, D., Safekordi, A. A., Alihosseini, A., \u0026amp; Taghikhani, V. (2006). Study of oil sorption by expanded perlite at 298.15 K. \u003cem\u003eSeparation and Purification Technology\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e(2), 295-300. https://doi.org/10.1016/j.seppur.2006.05.004.\u003c/li\u003e\n\u003cli\u003eBhardwaj, N.\u0026amp; Bhaskarwar, A. N. (2018). A review on sorbent devices for oil-spill control. \u003cem\u003eEnvironmental Pollution\u003c/em\u003e, \u003cem\u003e243\u003c/em\u003e, 1758-1771. https://academic.oup.com/jimb/article/30/9/542/5992141.\u003cspan dir=\"RTL\"\u003e \u0026rlm;\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eBouchez T., Blieux A. L. , Dequiedt S. , Domaizon I. , Dufresne A. , Ferreira S. , Godon J. J. , Hellal J. , Joulian C. , Quaise A. r, Martin-Laurent F. , Mauffret A. , Monier J. M. , Peyret P. , Schmitt-Koplin P. , Sibourg O. , D\u0026rsquo;oiron E. , Bispo A. , Deportes I. , Grand C. , Cuny P. , Maron P. 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Robust, highly porous hydrogels templated within emulsions stabilized using a reactive, crosslinking triblock copolymer. \u003cem\u003ePolymer\u003c/em\u003e, \u003cem\u003e168\u003c/em\u003e, 146-154. https://doi.org/10.1016/j.polymer.2019.02.010. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biodegrading, Expanded Perlite, Oil Spill, Adsorption","lastPublishedDoi":"10.21203/rs.3.rs-3703177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3703177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBio sorption and biodegrading capacity as well as sorption of oil onto expanded perlites modified with oil-eating bacteria were studied. This investigation leveraged perlite as an oil absorbent, subsequently laden with oil-degrading micro-organisms, aiming to study not only oil spill absorption but also the eradication of oil spill. Findings from experiments with three different adsorbents - one devoid of microbes, one featuring perlite loaded with microbes, and one containing expanded perlite laden with microbes-indicate that expanded perlite, due to its large surface area and low density, presents an optimal environment for microbial growth and proliferation. Upon microbial colonization, the amount of oil absorption and removal escalated by 58% and 80.45%, respectively, compared to pre-expansion. Furthermore, microbial activity mitigated some oil contamination and decreased the surface tension between water and oil via production of surface active substances, thereby facilitating further separation of residual oil in the water.\u003c/p\u003e","manuscriptTitle":"Study of oil biodegrading by expanded perlite Loaded by oil-eating bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 10:43:02","doi":"10.21203/rs.3.rs-3703177/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6d9ed2b-55f5-48a3-8730-99e50423a986","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-09T03:57:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 10:43:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3703177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3703177","identity":"rs-3703177","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}