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A novel biosurfactant producing Kocuria rosea ABR6 as potential strain in oil sludge recovery and lubrication

A novel biosurfactant producing Kocuria rosea ABR6 as potential strain in oil sludge recovery and... Biosurfactants are amphiphilic molecules composed of a hydrophilic and hydrophobic moiety and had the ability to penetrate into different phases to reduce the surface tension. This features caused to oil recovery, lubrication and facilities of crude oil in pipeline. In current research Biosurfactant‑producing strain was isolated from the storage tanks of the Isfahan Oil Refining Company in Iran, and screened by oil expansion test, droplet collapse, and surface tension reduction measurement. Hydrocarbon recovery from crude oil sludge was measured under constant conditions. The effect of factoring biosource lubrication on crude oil in pipelines was investigated in vitro. Also, the optimization of biosurfactant production in different conditions was measured as a single factor and using Response Surface Method (RSM). The best biosurfactant‑producing bacterium was identified as Kocuria rosea ABR6, and its sequence was regis‑ tered in the gene bank with access number of MK100469. Chemical analysis proved that the produced biosurfactant was a lipopeptide. 7% of crude oil was recovered from petroleum sludge by biosurfactant obtained from Kocuria rosea ABR6. Also, the speed of crude oil transfer in pipelines was upgraded as it could be said that for a certain distance the transfer time reduced from 64 to 35 s. The highest biosurfactant production was measured at pH 9, aeration rate of 120 rpm and 96 h after incubation. The use of biosurfactants produced by Kocuria rosea ABR6 is recommended to remove oil sludge and lubricate oil in pipelines recommended in the oil industry. Keywords: Biosurfactant, Kocuria rosea, Lubrication, Oil Sludge, Response Surface Methodology Key points Introduction In recent years, the control and prevention of chemical pollutants in the petroleum industry always have been • To improve recovery and prevent waste accumula- a worldwide issue (Varjani and Upasani 2017). There tion in crude oil storage tank, application of biosur- are three physical, chemical, and biological methods for factant suggested. treating these pollutants. However, these mechanisms • The isolated bacteria have high lubrication capacity. have numerous disadvantages e.g. in most cases the final • One of the significant problems in petroleum indus - product is usually toxic. In addition, there is an urgent try is biodegradability of Oil derivatives like oil need for sustainable cognition and eco-friendly meth- sludge. ods which require a lesser amount of chemicals, are economically viable, and produce non toxic natural final products. One of the chemical pollutants in the petro- *Correspondence: rasekhb@ripi.ir leum industry is emulsion of different petroleum hydro - Environment and Biotechnology Research Division, Research Institute carbons, solid particles, heavy metals, and highly toxic of Petroleum Industry (RIPI), P.O. Box: 14665‑137, Tehran, Iran water, named oil sludge. Removal of these components Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Akbari et al. AMB Expr (2021) 11:131 Page 2 of 10 requires expensive and time-consuming processes. Due Material and methods to its hazardous nature and increased quantities around Sample collection the world, it’s necessary to clarify this problem (Lima The sample, collected from crude oil storage tanks et  al. 2011). Expulsion of oily sludge from storage tanks located in Isfahan Oil Refining Company, Isfahan, Iran. can be performed by using biosurfactants to decline vis- Afterwards, transferred to the laboratory under con- cosity and recovery and refinery of oil (Banat et al. 1991). trolled conditions. Other treatments of oily sludge such as composting and land farming may have considerable applicability and a Isolation of biosurfactant producing bacteria low operating budget for large-scale treatment, but their In order to isolate the bacteria, Bushnell Hass culture −1 microbial degradation process is time-consuming. Selec- medium was used. Culture medium contains (g L ): tion of proper method depends on sludge features, dis- KH2PO4, 1; 0.2; K2HPO1; NH4NO3, MgSO4. 7H2O 1; posal regulatory requirements, costs, time constraints FeCl3, 0.002; CaCl2, 0.02; and crude oil, 1% (v/v), and (Hu et  al. 2013). The use of Biosurfactants in the indus - the pH was adjusted to 7 using NaOH 1  M. The culture try is the most important achievement of the twenty-first medium was sterilized by autoclave; 10  ml of each sam- century (Singh et al. 2018). Biosurfactants are amphiphi- ple were inoculated in a 500  ml medium and incubated lic surface-active agents secreted from microorganisms. for 10 days at 30 C and 100 rpm. Then 1 ml of inoculum −1 Bioactive molecules which are able to decrease the sur- was added to the culture medium (1.8 g L agar). In the face tension between an aqueous mixture and hydrocar- next step, bacteria which grown on the specific medium bon (Shekhar et al. 2015). In recent years, the application were transferred to an olive broth with the following −1 of biosurfactants has increased significantly, due to the compounds (g L ): MgSO4. 7H2O, 0.2; K2HPO4, 1; non-toxic, inexpensive economic value, biodegradable NH4NO3, 1; FeCl3, 0.002; CaCl2, 0.02; yeast extract and nature. Bioremediation process is based on an integrated olive oil, 1% (v/v). Olive oil was added, and the pH was approach employing microbial communities such as adjusted to 7. After autoclaving, 1% of pre culture was fungi, actinomycetes, bacteria, and earthworms (Sugan- shifted to the olive broth and incubated for 4 days at 30° thi et al. 2018). On the other hand, the global market for C and 100 rpm (Cameotra and Singh 2008; EI-Sheshtawy surfactant is amazing and, the market is also, expected to and Doheim 2014). grow to USD 39.86 Billion by 2021 (Markets and Markets 2017; Hart 2014). It is considered as a viable process for Identification of bacterial isolate administration of organic pollutants-rich solid wastes To better and certain identification, based on the other and wastewater. Many studies have suggested using bac- studies, positive or negative to gram stain test and some teria as a biosurfactant to recovery and removing oily routine biochemistry tests (Jessim et  al. 2020), like ure- sludge. Many studies have used bacteria as biosurfactant ase, oxidase, utilization of inulin, citrate utilization test, to recovery oily sludge, Pseudomonas aeruginosa F-2 phosphatase tests, gelatinase, nitrate reduction tests (Yan et  al. 2012) from refinery oily sludge, Bacillus sub - arabinose, and N-acetyl-l-glutamic acid amylase (Kandi tilis 3KP (Ni’matuzahroh et al. 2016) from the petroleum et al. 2016). sludge, Pseudomonas balearica strain Z8 (Nejad et  al. To molecular test, Bacterial genomic DNA was 2020) from oily sludge wastes. According to many stud- extracted from the bacterial isolate grown on nutrient ies, Oily sludge is one of the most important solid wastes broth using standard protocol. PCR was carried out as generated in the petroleum industry, and understand- formerly described (Akbari et al. 2016). 16S rDNA ampli- ing the mechanism of degradation it would be signifi - con from isolated strain was sequenced, and the infor- cant, so we decided to investigate biosurfactants as the mation was searched using NCBI-BLAST search tool for best choice (Hu et  al. 2013). The utilize of environmen - identification of the strain type. The sequence was sub - tal nanotechnologies (E_nano) is a complex process to mitted to the NCBI Gene Bank (Akbari et al. 2018). resolve the problems as best as associated with the petro- leum industry, (Younis et  al. 2020) we carried out ZnO Evaluation of biosurfactant production by isolated bacteria as E-Nano in our project. There aren’t many documents Possibility of biosurfactant presence was investigated to investigate about biosurfactant production by Kocuria using oil displacement method. In short, 50  μl of crude rosea. The main aims of this study is introduce suitable oil was mixed to a 10 cm diameter plate containing 10 ml native biosurfactant producing bacteria from petroleum of distilled water. The cell free culture broth (15  μl) was reservoir tanks and improve biosurfactant production then added to the oil surface. The plates with clear zone by optimization. Then lubrication of crude oil in pipeline were scored as positive indicative of the present of bio- model and oil recovery from oily sludge by this biosur- surfactant production (Mousavi et  al. 2015; Nejad et  al. factant have been evaluated. 2020; Liu et al. 2020). Surface tension of supernatant was A kbari et al. AMB Expr (2021) 11:131 Page 3 of 10 measured by tensiometer (KRÜSS KIOT K9, Switzerland) acetic acid with a ratio of 65/15/2 v/v/v was selected. and reported as mN/m. The surface tension of the water For staining solution containing 15 Ursinol and 8.2  ml was considered as a control. Also extraction of biosur- of 60% sulfuric acid in 42  ml of distilled water sprayed factant from isolated bacteria was performed according on paper and dried at 100  °C for 10  min and the spots to the technique mentioned by Jorfi et  al. (2013). After were examined (Camacho-Chab et al. 2013). centrifugation at 10,000g for 15 min in order to eliminate the bacterial cells, and the pH was adjusted by adding 2.0  ml of HCL to the biosurfactant. The precipitate was Product analysis by Fourier‑transform infrared spectroscopy separated by centrifugation (10,000g for 20 min) and then (FTIR) extracted with chloroform: methanol (2:1, v/v) mixture. FTIR (BRUKER,) was performed to show the presence In order to have crude biosurfactant, solvent evaporation of chemical functional groups in bio surfactant pro- under vacuum was used. duced by isolated bacteria. Lyophilize was performed to extracted biosurfactant and biosurfactant analysis was −1 performed in assay range of 400–4000  cm (Yaraguppi Treatment of oily sludge with biosurfactant et al. 2020). To preparate the crude oil from oil sludge in laboratory conditions, the present process was performed according to the method introduced by Lima et  al. 40  g of petro- Biodegradation analysis of crude oil leum sludge obtained from the bottom of crude oil stor- BH media selected to evaluate the ability of bacteria age tanks autoclaved from Khark refinery. Then 50  ml isolated from crude oil (100  ml) supplemented with 1% of sterilized distilled water was added to it. Then 10  ml crude oil. Upon incubation at pH 7 and 30  °C, 160  rpm of the medium culture of biosurfactant were inoculated for 72 h, the grown cultures were centrifuged at 100 rpm, after 96 h of incubation. Positive control was prepared by 4  °C for 10  min to pellet down the cells. For Isolated adding 10  ml of Twin 80 and negative control was pre- bacteria, crude oil was used as carbon source. This was pared without adding chemical surfactant and biosur- investigated by the weighing the residual crude oil based factant. After 5  days Incubation at ambient temperature on a gravimetric method wherein the residual crude oil and aeration at 100 rpm, oily sludge was emulsified. Then in the cell-free supernatant was extracted in an equal vol- emulsion by adding 1  ml of magnesium nitrate solution ume of n-hexane and separated using a rotary vacuum was broken, the aqueous phase was separated and the evaporator (Sharma et al. 2019). amount of recycled oil was measured (Lima et  al. 2011; Cameotra and Singh 2008; Akbari et al. 2020). Optimization of culture conditions for biosurfactant Crude oil lubrication by biosurfactant production In present study, the isolated strain was cultured in Bush- In order to determine biosurfactant production ability of nell Hass medium at 30 °C and 100 rpm. After 96 h incu- the isolated bacteria, we decided to carry out single factor bation, in order to ensure from biosurfactant production, optimization and multi-factor optimization by Response emulsification index, and oil displacement method were Surfaces methodology (RSM). In the first section, we developed. The pipeline was designed under laboratory overviewed 40 experiments, and according to the results, condition. Gross biosurfactant was poured in the crude we detected 15 experiments, which showed in Table  1. oil from the collected sample in a ratio of one-fifth as In this study, for performing single factor optimization the optimal ratio and it was performed under mixing for biosurfactant production, the five factors including with a specified speed, and the system output was then pH, temperature, carbon and nitrogen sources, agitation passed through a 40 cm duct at a 65° angle. Duct passage and time condition were considered. The culture condi - time was recorded before and after mixing (Amani and tions were based on pH (7, 8, 9, and 10), temperature1 Kariminezhad 2016). and carbon source (oil olive, glucose, tribotirin, crude oil) and nitrogen source (peptone, NH4NO3, NaNO3, Chemical analysis TLC and FTIR triptone, yeast extract), the biosurfactant production was Product analysis by thin‑layer chromatography (TLC) also evaluated under agitation (80, 100, 120, 140  rpm) In order to chromatograph, a thin layer of silica 60 and time conditions. During the experiments, when each paper with dimensions of 15.5 cm was arranged. 0.1 mg factor was examined, the other four were maintained as of dried biosurfactant dissolved in 10 μl of 90% ethanol constant. To determine the importance of the factors and and 5 µl of sample was dotted at a distance of 1 cm from to better understand interactions between them analysis the edge of the paper. The solvent system that was used using ANOVA table was developed (Liu et al. 2020). as the mobile phase including chloroform, methanol, Akbari et al. AMB Expr (2021) 11:131 Page 4 of 10 Table 1 Experiments of second step designed by response biosurfactant Produced by W11 was positive. Isolated surface analysis bacteria were measured by standard screening technique like, oil displacement method, oil drop collapse method Experiment Factor number1 Factor number2 Factor Agitation rate Incubation number3 (DCM), surface tension (SFT) measurement and emul- pH sification index (Soltanighias et al. 2019). This proved its ability to produce biosurfactants and selected for further 1 140 120 9 oil recovery analysis, optimization, and Biodegradation 2 140 72 8 analysis. 3 140 96 10 4 100 120 9 Isolation of Biosurfactant Producing Bacteria 5 100 96 10 The isolated bacteria showed a positive growth in the 6 100 72 8 selective culture medium (olive broth and Buschnel 7 120 96 9 Hass) and this strain we named, W11. To certain iden- 8 120 96 8 tification, biochemistry tests were helpful, e.g. PCR of 9 120 120 10 purified bacterial DNA with universal primers presented 10 120 72 10 the 1500  bp band in gel electrophoresis. The analysis 11 120 96 9 of the genomic sequence of 16S rDNA using Finch TV 12 120 96 8 and thence BLASTN approved that W11 from oil sludge 13 120 72 9 sample has been associated with Kocuria rosea and was 14 120 96 9 named Kocuria rosea ABR6.The genomic sequence of 15 120 120 10 16S rDNA was deposited in the NCBI under the acces- sion number of MK100469. Also this strain deposited in Petroleum Biotechnology Culture Collection as kocuria Result rosea PBCC1167. In current research, several bacteria strains were iso- lated. Because the dominant strain in enrichment cul- Treatment of oily sludge with biosurfactant ture (Bushnell Hass medium) was W11 isolate, bacterial The medium culture of Kocuria rosea ABR6 after 72  h production was examined. The screening revealed that incubation with 100  rpm at 30  °C was performed to W11 produced a considerable amount of biosurfactant, decrease the oily sludge with sharp viscosity in crude and reduced the surface tension from 72 mN/m to 31.6 oil storage tank. As a result, the treatment of petroleum mN/m. Also; the diameter of the oil expansion halo was sludge with biosurfactant from the isolated Kocuria measured at 9 cm (Fig.  1A). Drop collapse in less than a rosea ABR6, 50% of crude oil was recycled in labora- minute pointed up the presence of biosurfactant in the tory conditions (Fig.  1B). In the positive control sample, Bushnell Hass medium. The drop collapse test to survey 75% of crude oil was recovered from petroleum sludge, Fig. 1 A Oil displacement test by supernatant obtained from Kocuria rosea ABR6. B The emulsion stability of oily sludge obtained from culture medium of Kocuria rosea ABR6 after 7 days’ incubation A kbari et al. AMB Expr (2021) 11:131 Page 5 of 10 provided, in the negative control sample, only 3% of the demonstrated the biosurfactant produced by the Kocuria crude oil is recycled. rosea ABR6 was the lipopeptide. Crude oil lubrication by biosurfactant Biodegradation analysis of crude oil For a better describing the effect of Crude oil lubrication Following the research, we found out the biosurfactant using Biosurfactant, we designed an experiment on crude production utilizes various carbons as energy source. In oil in pipelines in  vitro. The Biosurfactant produced by this study, the ability of Kocuria rosea ABR6 in utilizing isolated bacteria accelerated the movement of crude oil, crude oil as a carbon source and producing biosurfactant as the crude oil movement time decreased from 66 to was explored. The percentage of crude oil biodegradation 39 s. by biosurfactant sounds fast. This may be because that the microorganisms in the oil sludge have the ability of Chemical analysis TLC and FTIR using the remaining crude oil as a source of carbon and Thin-layer chromatography (TLC) was used to separate energy. In sum, degradation of crude oil was reached as non-volatile mixtures. After accomplishment of 72 h, the 22%. rest of oil and biosurfactant in the supernatant was con- trolled with chromatographic technique. This study was performed with abiotic sample introduced as a control. Optimization of bacterial growth in order to maximize As illustrated in Fig. 2B, Rf 0.81 was observed. biosurfactant production FTIR analysis was performed to characterize the bio- Single factor test3 surfactant type secreted from Kocuria rosea ABR6. Impact of pH: To increase the amount of biosurfactant According to spectrum FTIR (Fig. 2A), A stretch around production by the Kocuria rosea ABR6 during the selec- −1 1380  cm corresponds to the presence of –CH and – tive condition, five parameters need to be optimized. CH groups in aliphatic chains of lipids. A broad band According to the results, the highest emulsification −1 at 2926  cm represents the O–H stretching vibrations was related to pH 9 and around 80% (Fig.  3A) respec- −1 from free hydroxyl groups. Regions around 2926  cm tively, emulsification of the isolated bacteria in pH 7, signify alcohols and phenols. Peak in the region of 8, 9, and 10 were 70%, 75%, 80%, and 65%. Impact of −1 518  cm may be likely due to the presence of disulfides carbon sources: The most production of biosurfactant −1 in the molecule. Peak around 2402  cm may be repre- resulted to be 80% and was related to olive oil about −1 senting the P–H in the phosphine. Peak around 948  cm 80%, also fermentation of glucose, tributyrin, crude oil may be due to occurrence of P–O–R stretch of ester by isolated strain were 40%, 53%, 75% (Fig. 3B). Impact −1 group. Peak around 3398  cm reveals the presence of of nitrogen sources: In order to find the best nitrogen −1 RCONH2 related to amino acids. Peak near 1659  cm sources, numbers indicated yeast extract with 75% was indicate to the C=C from alkene of bacteria protein, at the best nitrogen source. Other sources include, pep- −1 −1 −1 also, peak near 832  cm, 616  cm and 716  cm due tone, NH4NO3, NaNO3, and tryptone were 70%, 73%, −1 to the presence of alkene. Peak around 936  cm may be 52%, and 55%. (Fig.  3C). Impact of agitation: results attributed to OH, Carboxylic group. The FTIR analysis show the best agitation was 120  rpm, the rest of the Fig. 2 A Characterization of biosurfactan using FTIR analysis from Kocuria rosea ABR6. B Chromatogram biosurfactant produced by Kocuria rosea ABR6 Akbari et al. AMB Expr (2021) 11:131 Page 6 of 10 Fig. 3 A Impact of pH in biosurfactant produced by Kocuria rosea ABR6, B impact of carbon sources biosurfactant produced by Kocuria rosea ABR6. C Impact of nitrogen sources biosurfactant produced by Kocuria rosea ABR6. D Impact of agitation biosurfactant produced by Kocuria rosea ABR6 results of agitation under 80  rpm, 100  rpm, 120  rpm, The effect of different factors in three dimensions and 140 rpm were, 75%, 78%, 83%, and 80% (Fig. 3D). Statistical analysis and analysis of variance (ANOVA) in the second stage of optimizing biosurfactant pro- duction by Kocuria rosea ABR6 was proceed. Figure  5 Response surface analysis shows, the statistical analysis and analysis of variance The response surface analysis results are showed in (ANOVA) that for each variable, the corresponding Fig.  4. The highest growth was related to 72  h after correlation coefficient is P-value and F-value. Based incubation; hence, the highest emulsification was on regression analysis and based on the evidences, it around 96 percentages. According to graph after 96  h, was found that pH and incubation time were positive isolated strain showed the most biosurfactant produc- factors and the P-value was less than 0.05. In general, tion. It shows, Kocuria rosea had maximum biosur- the model was significant and significantly affected the factant production in stationary phase. According to production of biosurfactants. Figure 6 illustrated actual Fig.  4, pH 9 provided the best condition for biosur- and predicted amounts of biosurfactant production factant production in this case study. Based on the found to be in linear form. Figure  7 shows, the effect emulsification activity around 97.38, aeration speed of pH, agitation rate and incubation time factors on optimally reported to be 120  rpm. In order to find the producing biosurfactant and emulsification index. As highest level of biosurfactant production by Kocuria the slope of the invoice line increases, the effect of that rosea ABR6, a two-stage experiment was designed. In factor on the response will be steeper, as well as, the the first stage, 40 tests were performed and after that Smoother line slope shows the effect of that factor on 15 tests. Variable factors in these tests were pH, aera- response was less. In this experiment, the representa- tion speed and incubation. Based on these results, if tive factor, pH had a steep line slope and other factors the conditions are pH 9, aeration speed 120  rpm and like incubation time and agitation rate had slower line incubation 72 h, the production of biosurfactant will be slope, therefore, they have less effect on the produc - 100%. tion of biosurfactants. Also, the production equation of A kbari et al. AMB Expr (2021) 11:131 Page 7 of 10 Fig. 4 A Contour curve the effect of aeration speed factors and incubation time kocuria rosea ABR6, B three ‑ dimensional (3D) curve of the effect of aeration velocity factors and incubation time, C contour curve the effect of pH and aeration rate factors kocuria rosea ABR6, D three ‑ dimensional (3D) curve of the effect of pH and aeration factors kocuria rosea ABR6, E contour curve the effect of pH factors and incubation time kocuria rosea ABR6, F three‑ dimensional (3D) curve of the influence of pH factors and incubation time biosurfactant based on the tested factors is as follows. According to this equation and the selection of other values, the amount of production can be predicted. Y = 93/71 − 71/0A + 26/3B − 77/8C − 29/4AB + 48/7A Y = Biosurfactant production; A = pH; B = Incubation time; C = Agitation rate. Fig. 5 the analysis of variance ANOVA in biosurfactant production by kocuria rosea ABR6 Akbari et al. AMB Expr (2021) 11:131 Page 8 of 10 oil and oil sludge, but obviously the bacteria that are native to the refinery they carry out this process bet - ter. Therefore, in this study, we isolated the native bac - teria of the reservoir from storage tank and checked out the ability to produce biosurfactants, biodegradation and lubrication. In the present current study, a pipeline was designed in laboratory condition, biosurfactant that produced by Kocuria rosea ABR6 led to increas- ing movement of the crude oil in the pipelines. There are not many articles about effects of biosurfactant on crude oil lubrication by bacteria. In present study, we succeed to isolate 30 strains from tanks located in Isfa- han Petroleum Refinery, by which 11 strains had the ability to produce biosurfactant. The Isolate named W11 was known as the best biosurfactant producer. The result of 16s rDNA shows, isolated W11 had been Kocuria rosea. The accession number of MK100469 confirmed by gene bank global and named Kocuria rosea ABR6. In order to identify species of bacteria, some routine biochemistry test was done, as well as, Fig. 6 Actual and predicted values of biosurfactant production in Jessim et  al. (2020). the biochemistry test were help- RSM software by Kocuria rosea ABR6 ful but they weren’t determiner, and because has also been observed that various species of Kocuria react dif- ferently to routine biochemical tests, so we had needed an advance molecular method and selected 16s rDNA (Kandi 2016). Advance molecular method amplicon 16s rDNA from isolated strain was sequenced, and the information was searched using NCBI-BLAST search tool to identify of the strain type. Based on results TLC and FTIR and the variety of materials present in the biosurfactant, its chemical nature was investigated and these results pointed to the lipopeptide nature. Dhasayan et al. (2015) demonstrated that in FTIR anal- ysis, the biosurfactant obtained from B. amyloliquefa- ciens MB-101 was similar to the lipopeptide structures. Nature of biosurfactant demonstrate molecular char- acteristics and different applications, in this study, we introduce Kocuria rosea ABR6, as lubricant for crude oil, which reduced movement time of xrude oil. Ren et  al. (2020) introduced biosurfactant SWPUEN-1 as a cleaning agent to treat oily sludge. During differ - ent experiments, we found there are the correlations among variable factors and decided to perform a sta- tistical technique, named RSM to analyze them. RSM Fig. 7 Impact of factors, A: pH, B: Incubation time, C: Agitation rate Analysis demonstrated, efficiency of biodegradation depends on the pH, agitation, aeration, carbon and nitrogen sources. The presence of heavy mental in oil Discussion sludge can be a hazardous and negative effect on the Nowadays, the importance of oil pollution is obvious. environment and human health (Hu et  al. 2013). The Oil, and especially oil sludge, is contamination sources potential of bacterial isolate in crude oil lubrication in water and soil areas. For this purpose, a biological indicates the importance of biological usage Kocuria solution to remove oil sludge seems necessary and logi- rosea ABR6 in petroleum industry. In this research, the cal way. The diversity of bacteria are able to degrade designed pipeline applied to enhance the lubrication A kbari et al. AMB Expr (2021) 11:131 Page 9 of 10 Consent for publication. of crude oil through pipelines. It was explored this There is agreement among all the authors of this article. method employed to reduce viscosity and pressure drop to aid lubrication of crude oil pipeline, and it was Competing interests There are no conflict of interest with authors or university and institution in successful. However, designing a pipeline to crude oil this project. transport is depend on some factors e.g. the properties of the crude oil, distance dimension, cost, environmen- Author details Department of Microbiology, Faculty of Biological Sciences, Falavarjan tal problems and local and international regulations Branch, Islamic Azad University, Isfahan, Iran. Environment and Biotechnol‑ (Hart 2014). Many studies reported the isolation of ogy Research Division, Research Institute of Petroleum Industry (RIPI), P.O. Box: biosurfactant-producing bacteria from sea sample, e.g. 14665‑137, Tehran, Iran. Department of Microbiology, Faculty of Microbiol‑ ogy Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran. Bacillus amyloliquefaciens SH20 and Bacillus thuring- Faculty of New Science and Technology, University of Tehran, Tehran, Iran. iensis SH24 by Barakat et al. (2017), soil sample, Acine- Biotechnology Group, Department of Chemical Engineering, Tarbiat Modares tobacter junii B6 by Ohadi et  al. (2017) and solar salt University, Tehran, Iran. works, Kocuria marina BS-15 by Sarafin et  al. (2014). Received: 20 June 2021 Accepted: 19 August 2021 Due to the fact that the oil industry has a lot of bio-pol- lution, so the application of biological methods seems to be necessary. 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Inter Biode Biodeg 62(3):274–280 The authors are grateful to the Dean of Graduate Studies, Falavarjan Branch, Dhasayan A, Selvin J, Kiran S (2015) Biosurfactant production from marine bac‑ Islamic Azad University, Isfahan, Iran, for providing assistance with the experi‑ teria associated with sponge Callyspongia diffusa. 3 Biotech 5(4):443–454 ments, and to the Research Institute of Petroleum Industry, Tehran, Iran for the EI‑Sheshtawy HS, Doheim M. Selection of Pseudomonas aeruginosa for biosur ‑ valuable discussions. factant production and studies of its antimicrobial studies of its activity. Egypt J petrol. 2014;23:1‑6 Authors’ contributions Hart A (2014) A review of technologies for transporting heavy crude oil and EA carried out the experiments, KBM and BR analyzed the data and supervised bitumen via pipelines. J Petro Expl Produc Techn 4(3):327–336 the research. FK prepared the manuscript. ZEK designed RSM and FY designed Hu G, Li J, Zeng G (2013) Recent development in the treatment of oily sludge pipeline. RP edited the manuscript. All authors read and approved the from petroleum industry: a review. J Haza Mate 261:470–490 manuscript. Jessim AI, Hassan RK, Salman AS (2020) Isolation and identification of some pathogenic bacteria species from contaminated ambient with oily hydro‑ Funding carbons. Jbmoa. https:// doi. org/ 10. 15406/ jbmoa. 2020. 08. 00282 Present project is not funded by any institution. Jorfi S, Rezaee A, Mobeh‑Ali GA, Jaafarzadeh NA (2013) Application of biosur ‑ factants produced by Pseudomonas aeruginosa SP4 for bioremediation Availability of data and materials of soils contaminated by pyrene. Soil Sediment Contam. 22(8):890–911. 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Bioresourc technol. 116:24–28 Forootanfar H (2017) Isolation, characterization, and optimization of bio‑ Yaraguppi DA, Bagewadi ZK, Muddapur UM, Mulla SI (2020) Response surface surfactant production by an oil‑ degrading Acinetobacter junii B6 isolated methodology‑based optimization of biosurfactant production from from an Iranian oil excavation site. Biocat Agri Biotech 12:1–9 isolated Bacillus aryabhattai strain ZDY2. J Petrol Explor Prod Technol. Ren H, Zhou S, Wang B, Peng L, Li X (2020) Treatment mechanism of sludge 10(6):2483–99 containing highly viscous heavy oil using biosurfactant. Colloids Surf A Younis SA, Maitlo HA, Lee J, Kim KH (2020) Nanotechnology‑based sorption Physicochem Eng Aspects. 585:124117 and membrane technologies for the treatment of petroleum‑based Sakshi, Singh SK, Haritash AK.,2020. Evolutionary Relationship of Polycyclic pollutants in natural ecosystems and wastewater streams. 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A novel biosurfactant producing Kocuria rosea ABR6 as potential strain in oil sludge recovery and lubrication

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Abstract

Biosurfactants are amphiphilic molecules composed of a hydrophilic and hydrophobic moiety and had the ability to penetrate into different phases to reduce the surface tension. This features caused to oil recovery, lubrication and facilities of crude oil in pipeline. In current research Biosurfactant‑producing strain was isolated from the storage tanks of the Isfahan Oil Refining Company in Iran, and screened by oil expansion test, droplet collapse, and surface tension reduction measurement. Hydrocarbon recovery from crude oil sludge was measured under constant conditions. The effect of factoring biosource lubrication on crude oil in pipelines was investigated in vitro. Also, the optimization of biosurfactant production in different conditions was measured as a single factor and using Response Surface Method (RSM). The best biosurfactant‑producing bacterium was identified as Kocuria rosea ABR6, and its sequence was regis‑ tered in the gene bank with access number of MK100469. Chemical analysis proved that the produced biosurfactant was a lipopeptide. 7% of crude oil was recovered from petroleum sludge by biosurfactant obtained from Kocuria rosea ABR6. Also, the speed of crude oil transfer in pipelines was upgraded as it could be said that for a certain distance the transfer time reduced from 64 to 35 s. The highest biosurfactant production was measured at pH 9, aeration rate of 120 rpm and 96 h after incubation. The use of biosurfactants produced by Kocuria rosea ABR6 is recommended to remove oil sludge and lubricate oil in pipelines recommended in the oil industry. Keywords: Biosurfactant, Kocuria rosea, Lubrication, Oil Sludge, Response Surface Methodology Key points Introduction In recent years, the control and prevention of chemical pollutants in the petroleum industry always have been • To improve recovery and prevent waste accumula- a worldwide issue (Varjani and Upasani 2017). There tion in crude oil storage tank, application of biosur- are three physical, chemical, and biological methods for factant suggested. treating these pollutants. However, these mechanisms • The isolated bacteria have high lubrication capacity. have numerous disadvantages e.g. in most cases the final • One of the significant problems in petroleum indus - product is usually toxic. In addition, there is an urgent try is biodegradability of Oil derivatives like oil need for sustainable cognition and eco-friendly meth- sludge. ods which require a lesser amount of chemicals, are economically viable, and produce non toxic natural final products. One of the chemical pollutants in the petro- *Correspondence: rasekhb@ripi.ir leum industry is emulsion of different petroleum hydro - Environment and Biotechnology Research Division, Research Institute carbons, solid particles, heavy metals, and highly toxic of Petroleum Industry (RIPI), P.O. Box: 14665‑137, Tehran, Iran water, named oil sludge. Removal of these components Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Akbari et al. AMB Expr (2021) 11:131 Page 2 of 10 requires expensive and time-consuming processes. Due Material and methods to its hazardous nature and increased quantities around Sample collection the world, it’s necessary to clarify this problem (Lima The sample, collected from crude oil storage tanks et  al. 2011). Expulsion of oily sludge from storage tanks located in Isfahan Oil Refining Company, Isfahan, Iran. can be performed by using biosurfactants to decline vis- Afterwards, transferred to the laboratory under con- cosity and recovery and refinery of oil (Banat et al. 1991). trolled conditions. Other treatments of oily sludge such as composting and land farming may have considerable applicability and a Isolation of biosurfactant producing bacteria low operating budget for large-scale treatment, but their In order to isolate the bacteria, Bushnell Hass culture −1 microbial degradation process is time-consuming. Selec- medium was used. Culture medium contains (g L ): tion of proper method depends on sludge features, dis- KH2PO4, 1; 0.2; K2HPO1; NH4NO3, MgSO4. 7H2O 1; posal regulatory requirements, costs, time constraints FeCl3, 0.002; CaCl2, 0.02; and crude oil, 1% (v/v), and (Hu et  al. 2013). The use of Biosurfactants in the indus - the pH was adjusted to 7 using NaOH 1  M. The culture try is the most important achievement of the twenty-first medium was sterilized by autoclave; 10  ml of each sam- century (Singh et al. 2018). Biosurfactants are amphiphi- ple were inoculated in a 500  ml medium and incubated lic surface-active agents secreted from microorganisms. for 10 days at 30 C and 100 rpm. Then 1 ml of inoculum −1 Bioactive molecules which are able to decrease the sur- was added to the culture medium (1.8 g L agar). In the face tension between an aqueous mixture and hydrocar- next step, bacteria which grown on the specific medium bon (Shekhar et al. 2015). In recent years, the application were transferred to an olive broth with the following −1 of biosurfactants has increased significantly, due to the compounds (g L ): MgSO4. 7H2O, 0.2; K2HPO4, 1; non-toxic, inexpensive economic value, biodegradable NH4NO3, 1; FeCl3, 0.002; CaCl2, 0.02; yeast extract and nature. Bioremediation process is based on an integrated olive oil, 1% (v/v). Olive oil was added, and the pH was approach employing microbial communities such as adjusted to 7. After autoclaving, 1% of pre culture was fungi, actinomycetes, bacteria, and earthworms (Sugan- shifted to the olive broth and incubated for 4 days at 30° thi et al. 2018). On the other hand, the global market for C and 100 rpm (Cameotra and Singh 2008; EI-Sheshtawy surfactant is amazing and, the market is also, expected to and Doheim 2014). grow to USD 39.86 Billion by 2021 (Markets and Markets 2017; Hart 2014). It is considered as a viable process for Identification of bacterial isolate administration of organic pollutants-rich solid wastes To better and certain identification, based on the other and wastewater. Many studies have suggested using bac- studies, positive or negative to gram stain test and some teria as a biosurfactant to recovery and removing oily routine biochemistry tests (Jessim et  al. 2020), like ure- sludge. Many studies have used bacteria as biosurfactant ase, oxidase, utilization of inulin, citrate utilization test, to recovery oily sludge, Pseudomonas aeruginosa F-2 phosphatase tests, gelatinase, nitrate reduction tests (Yan et  al. 2012) from refinery oily sludge, Bacillus sub - arabinose, and N-acetyl-l-glutamic acid amylase (Kandi tilis 3KP (Ni’matuzahroh et al. 2016) from the petroleum et al. 2016). sludge, Pseudomonas balearica strain Z8 (Nejad et  al. To molecular test, Bacterial genomic DNA was 2020) from oily sludge wastes. According to many stud- extracted from the bacterial isolate grown on nutrient ies, Oily sludge is one of the most important solid wastes broth using standard protocol. PCR was carried out as generated in the petroleum industry, and understand- formerly described (Akbari et al. 2016). 16S rDNA ampli- ing the mechanism of degradation it would be signifi - con from isolated strain was sequenced, and the infor- cant, so we decided to investigate biosurfactants as the mation was searched using NCBI-BLAST search tool for best choice (Hu et  al. 2013). The utilize of environmen - identification of the strain type. The sequence was sub - tal nanotechnologies (E_nano) is a complex process to mitted to the NCBI Gene Bank (Akbari et al. 2018). resolve the problems as best as associated with the petro- leum industry, (Younis et  al. 2020) we carried out ZnO Evaluation of biosurfactant production by isolated bacteria as E-Nano in our project. There aren’t many documents Possibility of biosurfactant presence was investigated to investigate about biosurfactant production by Kocuria using oil displacement method. In short, 50  μl of crude rosea. The main aims of this study is introduce suitable oil was mixed to a 10 cm diameter plate containing 10 ml native biosurfactant producing bacteria from petroleum of distilled water. The cell free culture broth (15  μl) was reservoir tanks and improve biosurfactant production then added to the oil surface. The plates with clear zone by optimization. Then lubrication of crude oil in pipeline were scored as positive indicative of the present of bio- model and oil recovery from oily sludge by this biosur- surfactant production (Mousavi et  al. 2015; Nejad et  al. factant have been evaluated. 2020; Liu et al. 2020). Surface tension of supernatant was A kbari et al. AMB Expr (2021) 11:131 Page 3 of 10 measured by tensiometer (KRÜSS KIOT K9, Switzerland) acetic acid with a ratio of 65/15/2 v/v/v was selected. and reported as mN/m. The surface tension of the water For staining solution containing 15 Ursinol and 8.2  ml was considered as a control. Also extraction of biosur- of 60% sulfuric acid in 42  ml of distilled water sprayed factant from isolated bacteria was performed according on paper and dried at 100  °C for 10  min and the spots to the technique mentioned by Jorfi et  al. (2013). After were examined (Camacho-Chab et al. 2013). centrifugation at 10,000g for 15 min in order to eliminate the bacterial cells, and the pH was adjusted by adding 2.0  ml of HCL to the biosurfactant. The precipitate was Product analysis by Fourier‑transform infrared spectroscopy separated by centrifugation (10,000g for 20 min) and then (FTIR) extracted with chloroform: methanol (2:1, v/v) mixture. FTIR (BRUKER,) was performed to show the presence In order to have crude biosurfactant, solvent evaporation of chemical functional groups in bio surfactant pro- under vacuum was used. duced by isolated bacteria. Lyophilize was performed to extracted biosurfactant and biosurfactant analysis was −1 performed in assay range of 400–4000  cm (Yaraguppi Treatment of oily sludge with biosurfactant et al. 2020). To preparate the crude oil from oil sludge in laboratory conditions, the present process was performed according to the method introduced by Lima et  al. 40  g of petro- Biodegradation analysis of crude oil leum sludge obtained from the bottom of crude oil stor- BH media selected to evaluate the ability of bacteria age tanks autoclaved from Khark refinery. Then 50  ml isolated from crude oil (100  ml) supplemented with 1% of sterilized distilled water was added to it. Then 10  ml crude oil. Upon incubation at pH 7 and 30  °C, 160  rpm of the medium culture of biosurfactant were inoculated for 72 h, the grown cultures were centrifuged at 100 rpm, after 96 h of incubation. Positive control was prepared by 4  °C for 10  min to pellet down the cells. For Isolated adding 10  ml of Twin 80 and negative control was pre- bacteria, crude oil was used as carbon source. This was pared without adding chemical surfactant and biosur- investigated by the weighing the residual crude oil based factant. After 5  days Incubation at ambient temperature on a gravimetric method wherein the residual crude oil and aeration at 100 rpm, oily sludge was emulsified. Then in the cell-free supernatant was extracted in an equal vol- emulsion by adding 1  ml of magnesium nitrate solution ume of n-hexane and separated using a rotary vacuum was broken, the aqueous phase was separated and the evaporator (Sharma et al. 2019). amount of recycled oil was measured (Lima et  al. 2011; Cameotra and Singh 2008; Akbari et al. 2020). Optimization of culture conditions for biosurfactant Crude oil lubrication by biosurfactant production In present study, the isolated strain was cultured in Bush- In order to determine biosurfactant production ability of nell Hass medium at 30 °C and 100 rpm. After 96 h incu- the isolated bacteria, we decided to carry out single factor bation, in order to ensure from biosurfactant production, optimization and multi-factor optimization by Response emulsification index, and oil displacement method were Surfaces methodology (RSM). In the first section, we developed. The pipeline was designed under laboratory overviewed 40 experiments, and according to the results, condition. Gross biosurfactant was poured in the crude we detected 15 experiments, which showed in Table  1. oil from the collected sample in a ratio of one-fifth as In this study, for performing single factor optimization the optimal ratio and it was performed under mixing for biosurfactant production, the five factors including with a specified speed, and the system output was then pH, temperature, carbon and nitrogen sources, agitation passed through a 40 cm duct at a 65° angle. Duct passage and time condition were considered. The culture condi - time was recorded before and after mixing (Amani and tions were based on pH (7, 8, 9, and 10), temperature1 Kariminezhad 2016). and carbon source (oil olive, glucose, tribotirin, crude oil) and nitrogen source (peptone, NH4NO3, NaNO3, Chemical analysis TLC and FTIR triptone, yeast extract), the biosurfactant production was Product analysis by thin‑layer chromatography (TLC) also evaluated under agitation (80, 100, 120, 140  rpm) In order to chromatograph, a thin layer of silica 60 and time conditions. During the experiments, when each paper with dimensions of 15.5 cm was arranged. 0.1 mg factor was examined, the other four were maintained as of dried biosurfactant dissolved in 10 μl of 90% ethanol constant. To determine the importance of the factors and and 5 µl of sample was dotted at a distance of 1 cm from to better understand interactions between them analysis the edge of the paper. The solvent system that was used using ANOVA table was developed (Liu et al. 2020). as the mobile phase including chloroform, methanol, Akbari et al. AMB Expr (2021) 11:131 Page 4 of 10 Table 1 Experiments of second step designed by response biosurfactant Produced by W11 was positive. Isolated surface analysis bacteria were measured by standard screening technique like, oil displacement method, oil drop collapse method Experiment Factor number1 Factor number2 Factor Agitation rate Incubation number3 (DCM), surface tension (SFT) measurement and emul- pH sification index (Soltanighias et al. 2019). This proved its ability to produce biosurfactants and selected for further 1 140 120 9 oil recovery analysis, optimization, and Biodegradation 2 140 72 8 analysis. 3 140 96 10 4 100 120 9 Isolation of Biosurfactant Producing Bacteria 5 100 96 10 The isolated bacteria showed a positive growth in the 6 100 72 8 selective culture medium (olive broth and Buschnel 7 120 96 9 Hass) and this strain we named, W11. To certain iden- 8 120 96 8 tification, biochemistry tests were helpful, e.g. PCR of 9 120 120 10 purified bacterial DNA with universal primers presented 10 120 72 10 the 1500  bp band in gel electrophoresis. The analysis 11 120 96 9 of the genomic sequence of 16S rDNA using Finch TV 12 120 96 8 and thence BLASTN approved that W11 from oil sludge 13 120 72 9 sample has been associated with Kocuria rosea and was 14 120 96 9 named Kocuria rosea ABR6.The genomic sequence of 15 120 120 10 16S rDNA was deposited in the NCBI under the acces- sion number of MK100469. Also this strain deposited in Petroleum Biotechnology Culture Collection as kocuria Result rosea PBCC1167. In current research, several bacteria strains were iso- lated. Because the dominant strain in enrichment cul- Treatment of oily sludge with biosurfactant ture (Bushnell Hass medium) was W11 isolate, bacterial The medium culture of Kocuria rosea ABR6 after 72  h production was examined. The screening revealed that incubation with 100  rpm at 30  °C was performed to W11 produced a considerable amount of biosurfactant, decrease the oily sludge with sharp viscosity in crude and reduced the surface tension from 72 mN/m to 31.6 oil storage tank. As a result, the treatment of petroleum mN/m. Also; the diameter of the oil expansion halo was sludge with biosurfactant from the isolated Kocuria measured at 9 cm (Fig.  1A). Drop collapse in less than a rosea ABR6, 50% of crude oil was recycled in labora- minute pointed up the presence of biosurfactant in the tory conditions (Fig.  1B). In the positive control sample, Bushnell Hass medium. The drop collapse test to survey 75% of crude oil was recovered from petroleum sludge, Fig. 1 A Oil displacement test by supernatant obtained from Kocuria rosea ABR6. B The emulsion stability of oily sludge obtained from culture medium of Kocuria rosea ABR6 after 7 days’ incubation A kbari et al. AMB Expr (2021) 11:131 Page 5 of 10 provided, in the negative control sample, only 3% of the demonstrated the biosurfactant produced by the Kocuria crude oil is recycled. rosea ABR6 was the lipopeptide. Crude oil lubrication by biosurfactant Biodegradation analysis of crude oil For a better describing the effect of Crude oil lubrication Following the research, we found out the biosurfactant using Biosurfactant, we designed an experiment on crude production utilizes various carbons as energy source. In oil in pipelines in  vitro. The Biosurfactant produced by this study, the ability of Kocuria rosea ABR6 in utilizing isolated bacteria accelerated the movement of crude oil, crude oil as a carbon source and producing biosurfactant as the crude oil movement time decreased from 66 to was explored. The percentage of crude oil biodegradation 39 s. by biosurfactant sounds fast. This may be because that the microorganisms in the oil sludge have the ability of Chemical analysis TLC and FTIR using the remaining crude oil as a source of carbon and Thin-layer chromatography (TLC) was used to separate energy. In sum, degradation of crude oil was reached as non-volatile mixtures. After accomplishment of 72 h, the 22%. rest of oil and biosurfactant in the supernatant was con- trolled with chromatographic technique. This study was performed with abiotic sample introduced as a control. Optimization of bacterial growth in order to maximize As illustrated in Fig. 2B, Rf 0.81 was observed. biosurfactant production FTIR analysis was performed to characterize the bio- Single factor test3 surfactant type secreted from Kocuria rosea ABR6. Impact of pH: To increase the amount of biosurfactant According to spectrum FTIR (Fig. 2A), A stretch around production by the Kocuria rosea ABR6 during the selec- −1 1380  cm corresponds to the presence of –CH and – tive condition, five parameters need to be optimized. CH groups in aliphatic chains of lipids. A broad band According to the results, the highest emulsification −1 at 2926  cm represents the O–H stretching vibrations was related to pH 9 and around 80% (Fig.  3A) respec- −1 from free hydroxyl groups. Regions around 2926  cm tively, emulsification of the isolated bacteria in pH 7, signify alcohols and phenols. Peak in the region of 8, 9, and 10 were 70%, 75%, 80%, and 65%. Impact of −1 518  cm may be likely due to the presence of disulfides carbon sources: The most production of biosurfactant −1 in the molecule. Peak around 2402  cm may be repre- resulted to be 80% and was related to olive oil about −1 senting the P–H in the phosphine. Peak around 948  cm 80%, also fermentation of glucose, tributyrin, crude oil may be due to occurrence of P–O–R stretch of ester by isolated strain were 40%, 53%, 75% (Fig. 3B). Impact −1 group. Peak around 3398  cm reveals the presence of of nitrogen sources: In order to find the best nitrogen −1 RCONH2 related to amino acids. Peak near 1659  cm sources, numbers indicated yeast extract with 75% was indicate to the C=C from alkene of bacteria protein, at the best nitrogen source. Other sources include, pep- −1 −1 −1 also, peak near 832  cm, 616  cm and 716  cm due tone, NH4NO3, NaNO3, and tryptone were 70%, 73%, −1 to the presence of alkene. Peak around 936  cm may be 52%, and 55%. (Fig.  3C). Impact of agitation: results attributed to OH, Carboxylic group. The FTIR analysis show the best agitation was 120  rpm, the rest of the Fig. 2 A Characterization of biosurfactan using FTIR analysis from Kocuria rosea ABR6. B Chromatogram biosurfactant produced by Kocuria rosea ABR6 Akbari et al. AMB Expr (2021) 11:131 Page 6 of 10 Fig. 3 A Impact of pH in biosurfactant produced by Kocuria rosea ABR6, B impact of carbon sources biosurfactant produced by Kocuria rosea ABR6. C Impact of nitrogen sources biosurfactant produced by Kocuria rosea ABR6. D Impact of agitation biosurfactant produced by Kocuria rosea ABR6 results of agitation under 80  rpm, 100  rpm, 120  rpm, The effect of different factors in three dimensions and 140 rpm were, 75%, 78%, 83%, and 80% (Fig. 3D). Statistical analysis and analysis of variance (ANOVA) in the second stage of optimizing biosurfactant pro- duction by Kocuria rosea ABR6 was proceed. Figure  5 Response surface analysis shows, the statistical analysis and analysis of variance The response surface analysis results are showed in (ANOVA) that for each variable, the corresponding Fig.  4. The highest growth was related to 72  h after correlation coefficient is P-value and F-value. Based incubation; hence, the highest emulsification was on regression analysis and based on the evidences, it around 96 percentages. According to graph after 96  h, was found that pH and incubation time were positive isolated strain showed the most biosurfactant produc- factors and the P-value was less than 0.05. In general, tion. It shows, Kocuria rosea had maximum biosur- the model was significant and significantly affected the factant production in stationary phase. According to production of biosurfactants. Figure 6 illustrated actual Fig.  4, pH 9 provided the best condition for biosur- and predicted amounts of biosurfactant production factant production in this case study. Based on the found to be in linear form. Figure  7 shows, the effect emulsification activity around 97.38, aeration speed of pH, agitation rate and incubation time factors on optimally reported to be 120  rpm. In order to find the producing biosurfactant and emulsification index. As highest level of biosurfactant production by Kocuria the slope of the invoice line increases, the effect of that rosea ABR6, a two-stage experiment was designed. In factor on the response will be steeper, as well as, the the first stage, 40 tests were performed and after that Smoother line slope shows the effect of that factor on 15 tests. Variable factors in these tests were pH, aera- response was less. In this experiment, the representa- tion speed and incubation. Based on these results, if tive factor, pH had a steep line slope and other factors the conditions are pH 9, aeration speed 120  rpm and like incubation time and agitation rate had slower line incubation 72 h, the production of biosurfactant will be slope, therefore, they have less effect on the produc - 100%. tion of biosurfactants. Also, the production equation of A kbari et al. AMB Expr (2021) 11:131 Page 7 of 10 Fig. 4 A Contour curve the effect of aeration speed factors and incubation time kocuria rosea ABR6, B three ‑ dimensional (3D) curve of the effect of aeration velocity factors and incubation time, C contour curve the effect of pH and aeration rate factors kocuria rosea ABR6, D three ‑ dimensional (3D) curve of the effect of pH and aeration factors kocuria rosea ABR6, E contour curve the effect of pH factors and incubation time kocuria rosea ABR6, F three‑ dimensional (3D) curve of the influence of pH factors and incubation time biosurfactant based on the tested factors is as follows. According to this equation and the selection of other values, the amount of production can be predicted. Y = 93/71 − 71/0A + 26/3B − 77/8C − 29/4AB + 48/7A Y = Biosurfactant production; A = pH; B = Incubation time; C = Agitation rate. Fig. 5 the analysis of variance ANOVA in biosurfactant production by kocuria rosea ABR6 Akbari et al. AMB Expr (2021) 11:131 Page 8 of 10 oil and oil sludge, but obviously the bacteria that are native to the refinery they carry out this process bet - ter. Therefore, in this study, we isolated the native bac - teria of the reservoir from storage tank and checked out the ability to produce biosurfactants, biodegradation and lubrication. In the present current study, a pipeline was designed in laboratory condition, biosurfactant that produced by Kocuria rosea ABR6 led to increas- ing movement of the crude oil in the pipelines. There are not many articles about effects of biosurfactant on crude oil lubrication by bacteria. In present study, we succeed to isolate 30 strains from tanks located in Isfa- han Petroleum Refinery, by which 11 strains had the ability to produce biosurfactant. The Isolate named W11 was known as the best biosurfactant producer. The result of 16s rDNA shows, isolated W11 had been Kocuria rosea. The accession number of MK100469 confirmed by gene bank global and named Kocuria rosea ABR6. In order to identify species of bacteria, some routine biochemistry test was done, as well as, Fig. 6 Actual and predicted values of biosurfactant production in Jessim et  al. (2020). the biochemistry test were help- RSM software by Kocuria rosea ABR6 ful but they weren’t determiner, and because has also been observed that various species of Kocuria react dif- ferently to routine biochemical tests, so we had needed an advance molecular method and selected 16s rDNA (Kandi 2016). Advance molecular method amplicon 16s rDNA from isolated strain was sequenced, and the information was searched using NCBI-BLAST search tool to identify of the strain type. Based on results TLC and FTIR and the variety of materials present in the biosurfactant, its chemical nature was investigated and these results pointed to the lipopeptide nature. Dhasayan et al. (2015) demonstrated that in FTIR anal- ysis, the biosurfactant obtained from B. amyloliquefa- ciens MB-101 was similar to the lipopeptide structures. Nature of biosurfactant demonstrate molecular char- acteristics and different applications, in this study, we introduce Kocuria rosea ABR6, as lubricant for crude oil, which reduced movement time of xrude oil. Ren et  al. (2020) introduced biosurfactant SWPUEN-1 as a cleaning agent to treat oily sludge. During differ - ent experiments, we found there are the correlations among variable factors and decided to perform a sta- tistical technique, named RSM to analyze them. RSM Fig. 7 Impact of factors, A: pH, B: Incubation time, C: Agitation rate Analysis demonstrated, efficiency of biodegradation depends on the pH, agitation, aeration, carbon and nitrogen sources. The presence of heavy mental in oil Discussion sludge can be a hazardous and negative effect on the Nowadays, the importance of oil pollution is obvious. environment and human health (Hu et  al. 2013). The Oil, and especially oil sludge, is contamination sources potential of bacterial isolate in crude oil lubrication in water and soil areas. For this purpose, a biological indicates the importance of biological usage Kocuria solution to remove oil sludge seems necessary and logi- rosea ABR6 in petroleum industry. In this research, the cal way. The diversity of bacteria are able to degrade designed pipeline applied to enhance the lubrication A kbari et al. AMB Expr (2021) 11:131 Page 9 of 10 Consent for publication. of crude oil through pipelines. It was explored this There is agreement among all the authors of this article. method employed to reduce viscosity and pressure drop to aid lubrication of crude oil pipeline, and it was Competing interests There are no conflict of interest with authors or university and institution in successful. However, designing a pipeline to crude oil this project. transport is depend on some factors e.g. the properties of the crude oil, distance dimension, cost, environmen- Author details Department of Microbiology, Faculty of Biological Sciences, Falavarjan tal problems and local and international regulations Branch, Islamic Azad University, Isfahan, Iran. Environment and Biotechnol‑ (Hart 2014). Many studies reported the isolation of ogy Research Division, Research Institute of Petroleum Industry (RIPI), P.O. Box: biosurfactant-producing bacteria from sea sample, e.g. 14665‑137, Tehran, Iran. Department of Microbiology, Faculty of Microbiol‑ ogy Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran. Bacillus amyloliquefaciens SH20 and Bacillus thuring- Faculty of New Science and Technology, University of Tehran, Tehran, Iran. iensis SH24 by Barakat et al. (2017), soil sample, Acine- Biotechnology Group, Department of Chemical Engineering, Tarbiat Modares tobacter junii B6 by Ohadi et  al. (2017) and solar salt University, Tehran, Iran. works, Kocuria marina BS-15 by Sarafin et  al. (2014). Received: 20 June 2021 Accepted: 19 August 2021 Due to the fact that the oil industry has a lot of bio-pol- lution, so the application of biological methods seems to be necessary. 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Journal

AMB Express – Springer Journals

Published: Sep 22, 2021

Keywords: Biosurfactant; Kocuria rosea; Lubrication; Oil Sludge; Response Surface Methodology

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