In current research, several bacteria strains were isolated. Because the dominant strain in enrichment culture (Bushnell Hass medium) was W11 isolate, bacterial production was examined. The screening revealed that W11 produced a considerable amount of biosurfactant, and reduced the surface tension from 72 mN/m to 31.6 mN/m. Also; the diameter of the oil expansion halo was measured at 9 cm (Fig. 1A). Drop collapse in less than a minute pointed up the presence of biosurfactant in the Bushnell Hass medium. The drop collapse test to survey biosurfactant Produced by W11 was positive. Isolated bacteria were measured by standard screening technique like, oil displacement method, oil drop collapse method (DCM), surface tension (SFT) measurement and emulsification index (Soltanighias et al. 2019). This proved its ability to produce biosurfactants and selected for further oil recovery analysis, optimization, and Biodegradation analysis.
Isolation of Biosurfactant Producing Bacteria
The isolated bacteria showed a positive growth in the selective culture medium (olive broth and Buschnel Hass) and this strain we named, W11. To certain identification, biochemistry tests were helpful, e.g. PCR of purified bacterial DNA with universal primers presented the 1500 bp band in gel electrophoresis. The analysis of the genomic sequence of 16S rDNA using Finch TV and thence BLASTN approved that W11 from oil sludge sample has been associated with Kocuria rosea and was named Kocuria rosea ABR6.The genomic sequence of 16S rDNA was deposited in the NCBI under the accession number of MK100469. Also this strain deposited in Petroleum Biotechnology Culture Collection as kocuria rosea PBCC1167.
Treatment of oily sludge with biosurfactant
The medium culture of Kocuria rosea ABR6 after 72 h incubation with 100 rpm at 30 °C was performed to decrease the oily sludge with sharp viscosity in crude oil storage tank. As a result, the treatment of petroleum sludge with biosurfactant from the isolated Kocuria rosea ABR6, 50% of crude oil was recycled in laboratory conditions (Fig. 1B). In the positive control sample, 75% of crude oil was recovered from petroleum sludge, provided, in the negative control sample, only 3% of the crude oil is recycled.
Crude oil lubrication by biosurfactant
For a better describing the effect of Crude oil lubrication using Biosurfactant, we designed an experiment on crude oil in pipelines in vitro. The Biosurfactant produced by isolated bacteria accelerated the movement of crude oil, as the crude oil movement time decreased from 66 to 39 s.
Chemical analysis TLC and FTIR
Thin-layer chromatography (TLC) was used to separate non-volatile mixtures. After accomplishment of 72 h, the rest of oil and biosurfactant in the supernatant was controlled with chromatographic technique. This study was performed with abiotic sample introduced as a control. As illustrated in Fig. 2B, Rf 0.81 was observed.
FTIR analysis was performed to characterize the biosurfactant type secreted from Kocuria rosea ABR6. According to spectrum FTIR (Fig. 2A), A stretch around 1380 cm−1 corresponds to the presence of –CH3 and –CH2 groups in aliphatic chains of lipids. A broad band at 2926 cm−1 represents the O–H stretching vibrations from free hydroxyl groups. Regions around 2926 cm−1 signify alcohols and phenols. Peak in the region of 518 cm−1 may be likely due to the presence of disulfides in the molecule. Peak around 2402 cm−1 may be representing the P–H in the phosphine. Peak around 948 cm−1 may be due to occurrence of P–O–R stretch of ester group. Peak around 3398 cm−1 reveals the presence of RCONH2 related to amino acids. Peak near 1659 cm−1 indicate to the C=C from alkene of bacteria protein, also, peak near 832 cm−1, 616 cm−1 and 716 cm−1 due to the presence of alkene. Peak around 936 cm−1 may be attributed to OH, Carboxylic group. The FTIR analysis demonstrated the biosurfactant produced by the Kocuria rosea ABR6 was the lipopeptide.
Biodegradation analysis of crude oil
Following the research, we found out the biosurfactant production utilizes various carbons as energy source. In this study, the ability of Kocuria rosea ABR6 in utilizing crude oil as a carbon source and producing biosurfactant was explored. The percentage of crude oil biodegradation by biosurfactant sounds fast. This may be because that the microorganisms in the oil sludge have the ability of using the remaining crude oil as a source of carbon and energy. In sum, degradation of crude oil was reached as 22%.
Optimization of bacterial growth in order to maximize biosurfactant production
Single factor test3
Impact of pH: To increase the amount of biosurfactant production by the Kocuria rosea ABR6 during the selective condition, five parameters need to be optimized. According to the results, the highest emulsification was related to pH 9 and around 80% (Fig. 3A) respectively, emulsification of the isolated bacteria in pH 7, 8, 9, and 10 were 70%, 75%, 80%, and 65%. Impact of carbon sources: The most production of biosurfactant resulted to be 80% and was related to olive oil about 80%, also fermentation of glucose, tributyrin, crude oil by isolated strain were 40%, 53%, 75% (Fig. 3B). Impact of nitrogen sources: In order to find the best nitrogen sources, numbers indicated yeast extract with 75% was at the best nitrogen source. Other sources include, peptone, NH4NO3, NaNO3, and tryptone were 70%, 73%, 52%, and 55%. (Fig. 3C). Impact of agitation: results show the best agitation was 120 rpm, the rest of the results of agitation under 80 rpm, 100 rpm, 120 rpm, and 140 rpm were, 75%, 78%, 83%, and 80% (Fig. 3D).
Response surface analysis
The response surface analysis results are showed in Fig. 4. The highest growth was related to 72 h after incubation; hence, the highest emulsification was around 96 percentages. According to graph after 96 h, isolated strain showed the most biosurfactant production. It shows, Kocuria rosea had maximum biosurfactant production in stationary phase. According to Fig. 4, pH 9 provided the best condition for biosurfactant production in this case study. Based on the emulsification activity around 97.38, aeration speed optimally reported to be 120 rpm. In order to find the highest level of biosurfactant production by Kocuria rosea ABR6, a two-stage experiment was designed. In the first stage, 40 tests were performed and after that 15 tests. Variable factors in these tests were pH, aeration speed and incubation. Based on these results, if the conditions are pH 9, aeration speed 120 rpm and incubation 72 h, the production of biosurfactant will be 100%.
The effect of different factors in three dimensions
Statistical analysis and analysis of variance (ANOVA) in the second stage of optimizing biosurfactant production by Kocuria rosea ABR6 was proceed. Figure 5 shows, the statistical analysis and analysis of variance (ANOVA) that for each variable, the corresponding correlation coefficient is P-value and F-value. Based on regression analysis and based on the evidences, it was found that pH and incubation time were positive factors and the P-value was less than 0.05. In general, the model was significant and significantly affected the production of biosurfactants. Figure 6 illustrated actual and predicted amounts of biosurfactant production found to be in linear form. Figure 7 shows, the effect of pH, agitation rate and incubation time factors on producing biosurfactant and emulsification index. As the slope of the invoice line increases, the effect of that factor on the response will be steeper, as well as, the Smoother line slope shows the effect of that factor on response was less. In this experiment, the representative factor, pH had a steep line slope and other factors like incubation time and agitation rate had slower line slope, therefore, they have less effect on the production of biosurfactants. Also, the production equation of 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.
$$ {\text{Y }} = 93/71 - 71/0{\text{A}} + 26/{\text{3 B }} - 77/{\text{8C}} - 29/{\text{4AB}} + 48/{\text{7 A}}^{{2}} $$
Y = Biosurfactant production; A = pH; B = Incubation time; C = Agitation rate.