Exsitu assessment of potential free (FC) and immobilized cell (IC) bacteria used as engine oil (UEO) degraders in the degradation of UEO in liquid yeast extract minimal salt medium (YEMSM)

doi:10.21203/rs.3.rs-3313263/v1

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Exsitu assessment of potential free (FC) and immobilized cell (IC) bacteria used as engine oil (UEO) degraders in the degradation of UEO in liquid yeast extract minimal salt medium (YEMSM)

https://doi.org/10.21203/rs.3.rs-3313263/v1

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Used engine oil (UEO) contains potentially toxic substances that harmful humans and the ecosystem. Its dangerous pollution requires customized, efficient adsorptive bioremediation. This study investigates the local soil bacteria that can remove petrol-UEO (shell 15–40) by metabolic capability in UEO-YEMSM, previously isolated from UEO-polluted soil in KL, Malaysia. The biosorptive bioremediation capabilities of the free cell (FC) formula for UEO have been studied using pure Ochrobacterium intermedium LMG 3301 and mixed culture consortia of Ochrobacterium intermedium LMG 3301 plus Bacillus paramycoides MCCC1A04098 (BC), with an inoculation size of 3 × 10⁹ CFU/mL in 0.675% (v/v) UEO-YEMSM. To study the UEO adsorption bioremediation of IC systems and compare them to the same FC systems, pure and mixed bacteria cells were immobilized using alginate-attapulgite-calcium carbonate (AAC). On day 24, the IC systems exhibited the removal of nC9 to nC₁₇ with a 30% increase in TPH obtained with O.intermedium and the 18% by O. intermedium + B. paramycoides (BC) in UEO, as compared to the same FC systems. The AAC-IC of O. intermedium and the BC also removed nC₉-nC₂₉ to 93% and 98%, demonstrating the adsorptive-biosorptive bioremediation capacity of IC-systems with efficient mass transfers of experimental effectiveness factor (η) values of 1.04, and1.05, close to FC system η = 1. The enhanced degradation and bacterial growth in the AAC-IC systems indicated the high potential of AAC beads to be applied in the insitu bioremediation of UEO-polluted soils/water environments in KL, Malaysia.

Free cell (FC)

immobilized cell (IC)

sodium alginate-attapulgite-CaCO3 (AAC)

biosorption

adsorption

experimental effectiveness factor (η)

used engine oil (UEO)

Alginate immobilized cell (IC) bioremediation is an environmental technique that can be adapted with a variety of mixed matrices to treat used engine oil (UEO) polluted sites and that requires safe, novel, customized biological solutions (Elhamrouni et al., 2023).

This waste is a global problem (Pongsilp & Nimnoi, 2022), its discharge on the soil (previously/ recently) in garages has turned out to be a serious environmental threat (Patel & Shah, 2023). Because it accumulates and harms the soil ( Ghribi et al., 2019); it also harms a variety of sources: animal, vegetal, water, etc. (Diphare et al., 2013; Abdel-Shafy & Mansour, 2016; Ngozi et al., 2017; Haider et al., 2021).

The biological methods (e.g., free cell (FC), IC etc) are the most traditional, effective, and safe treatments towards the reduction or total removal of this waste danger. Several authors have studied the performance of these various systems in the removal of oil pollution (Vasilyeva et al., 2020; Partovinia et al., 2021; Pongsilp and Nimnoi, 2022; Soumeya et al., 2022). There are many non-biological methods as well for the treatment of oil pollution: chemical and physical (Gallegos Martínez et al., 2000). The remedy is very fast, but the cost, safety, efficacy, pollution, non-sustainableness, etc. (Bulai, et al., 2021; Ambaye et al., 2022), sometimes limit their applications.

Taking it all into account, as compared to the non-biological remedies, the biological microbial treatments are the most accepted choice for the ecological recovery of the oil-polluted sites (Varjani, 2017; Ławniczak et al., 2020). But, since oil-contaminated environments are challenging due to their low solubility, non-polarity, co-contamination with metals, slow biodegradation rate, etc. (Vidali, 2001; Liu et al., 2011; Ajiboye et al., 2020). In order to enhance the bioremediation of oil-polluted environments, the techniques of microorganism transfer and adsorption bioremediation methods should be applied (Mrozik and Piotrowska-Seget, 2010; Partovinia et al., 2018; Partovinia et al., 2021).

Partovinia et al. (2018) say that the most common way to work with microbes is to use the suspension culture stage of free cell (FC) inoculums. The activity and viability of potential microbes cannot always be completely assured by using the FC inoculum. Therefore, to increase bioremediation efficacy, various customized techniques of microorganism transfer to polluted areas have been investigated (Mrozik and Piotrowska-Seget, 2010; Partovinia et al., 2021). One of the more promising techniques in this respect is to use different carrier or support materials to produce wet solid coarse formulas (e.g., IC beads) or dry solid fine powder formulas (e.g., freeze/spray dried bacteria powder) of inoculums to maintain sufficient microbial activity for a long period of time, slow release, protection, even distribution, etc. (Banerjee & Ghoshal, 2013; Bayat et al., 2015; Estevinho et al., 2017; Partovinia et al., 2018; Abolhasani Soorki et al., 2019; Li et al., 2021). To which it increases the bioremediation efficiency and the tolerance to environmental perturbations, e.g., high pH, temperature, toxic oil CPDs, etc. (Cassidy et al., 1996; Hsieh et al., 2008; Zhang et al., 2008). Several offsite bioremediation techniques with novel characteristics and eco-friendly mixed carriers that support excellent bacteria viability and enhance UEO bioremediation have been reported elsewhere (Zhen-Yu et al., 2012; Hou et al., 2013; Partovinia & Rasekh, 2018; Li et al., 2021; Zhou, et al., 2021).

Besides that, so far, the soil oil-contaminated areas are rich with native microorganisms that can participate in bioremediation processes (Hossain et al., 2022). Many researchers isolated soil bacteria that can degrade UEO in pure cultures or in consortia, with various capabilities (Ibrahim, 2016; Hossain et al., 2022; O Jesubunmi et al., 2022; Javed et al., 2022; Muhammad & Adamu, 2022). The biodegradation capabilities of the native microorganisms in their same polluted sites are faster than those of the exotic microorganisms (Parach et al., 2017). Therefore, introducing the local efficient microbes that degrade UEO in the IC systems can be an effective solution to enhance the UEO exsitu/insitu bioremediation efficacy.

Bioremediation of oil pollution with FC/IC systems is not new (El-Borai et al., 2016; Farag et al., 2018; Umar et al., 2018), but alginate-attapulgite-calcium carbonate (AAC) IC is rarely used because it has only been used for diesel bioremediation (Wang et al., 2014). Also, the AAC-composite matrix can absorb UEO (Elhamrouni et al., 2023), and it is a material that is good for the environment. This makes it very interesting to compare the local efficient bacteria's ability to biodegrade AAC-IC with the same FC systems. In this work, the bioremediation metabolic capabilities of FC and AAC-IC systems for UEO were studied using a pure Ochrobacterium intermedium LMG 3301 and mixed culture consortia of Ochrobacterium intermedium LMG 3301 and Bacillus paramycoides MCCC1A04098 (BC), which had been previously isolated from UEO-polluted soil in KL, Malaysia (Elhamrouni et al., 2023). To study the adsorption bioremediation capability of IC systems, remove UEO, and compare them to the same FC systems, live pure and mixed bacteria cells were immobilized using AAC.

Maintaining cultures of UEO degraders

Murugan et al. (2017) kept Ochrobacterium intermedium LMG 3301/Bacillus paramycoides MCCC1A04098 in 50% (v/v) glycerol so that it could be kept for a long time. Following the procedures as described elsewhere (Elhamrouni et al., 2023). The culture cell suspension was grown over 24 h in nutrient broth (NB), at 32°C/150 rpm (Gupte and Sonawdekar, 2015). For short-term maintenance and daily requirements, the cells were routinely streaked on nutrient agar (NA) slants or plates (Gupte and Sonawdekar, 2015; Murugan et al., 2017).

The single (SC) and mixed (MC) inoculum preparation

The free cell (SC/MC)/immobilized cell (SC/MC) inoculum and immobilization process of AAC (%(w/v): Na-alginate (3.5), attapulgite (0.75), and calcium carbonate (5), were carried out and setup following the procedures as reported elsewhere (Ghazali et al., 2004; Wang et al., 2014), and performed according to Elhamrouni et al. (2023), with the inoculum size of 1x peptone water (PW) suspended cells in the gel making 6 x 10⁸ CFU/mL gel. So each 5 mL gel produced a total number of beads that entrapped the same amount of inoculum size as those added in the form of 0.670 mL suspended cells (4.5 x 10⁹ CFU/mL) in 20x diluted PW. The PW was diluted 20x to assume the equal relative influence of this nutrient in the comparisons of IC systems with FC systems. In these inoculum size equivalences, the inoculum size of 4.5 x 10⁹ CFU/mL, made in 20x diluted PW was considered the stardand inoculant size added per 100 mL UEO-YEMSM in the FC systems, while in the IC system, the total number of beads produced per 5 mL gel-entrapped bacteria was the equivalent inoculum size that was added per flask as to those in the FC systems. The beads were counted while adding to determine the number of beads that have to be taken periodically throughout Days 0–24, as equivalence to FC system 3-day volumetric cyclic sampling, that should not exceed 50% of the total number of beads/50 mL medium volume remaining on Day 24. The 0.670 mL of 20× diluted (PW) (4.5 × 10⁹ CFU/mL) inoculant, when diluted to 1 mL with normal saline, is equivalent to ≈ 3 x 10⁹ CFU/mL. At mean time, the total number of beads produced from 5 mL AAC also entraps the same concentration of inoculant (≈ 3 x 10⁹ CFU/5 mL gel).

The shake flask medium

The MSM of Zajic and Supplisson, (1972), with 0.005 g/L yeast extract (YE), was prepared according to optimized parameters obtained from shake flask experiment using RSM (pH 7.5, 35°C temperature, and 150 rpm agitation rate) (Non published data). The YEMSM and UEO were sterilized separately. Prio to sterilization (or autoclaving), the YEMSM was adjusted to pH 7.5, with 1M HCl / NaOH (Budde et al. 2010). After, the autoclaving (121°C/15 min), and the coolingup of the YEMSM to indoor temperature, an aliquote of filter sterilized UEO, with 0.45 µM pore size syringe microbial filter (PTFE, Sartorius) was added a septically per flask to attain the final concentration of 0.675%(v/v) UEO in 100 mL, YEMSM after the addition of inoculant. Then, the inoculated flasks with FC/IC were aseptically covered with aluminium foil and incubated (150 rpm/35°C) Day 0–24. The addition of inoculant sizes in the form of FC/IC was perfomed according to Elhamrouni et al. (2023). Each set of single/mixed inoculated flask of FC/IC cuture and their negative controls was prepared in triplicate.

Analytical methods

Spectrophotometric analysis of UEO residues

The UEO residue was monitored in the media /bead hexane extract using the spectrophotometry method at OD_500nm. The 5 mL broth sample/2 beads were taken per 3 days over the period Day 0–24. UEO in the gel beads was extracted using two steps. The 1st -step involved the dissolving of the two beads in 5 mL 0.2 M sodium citrate (Wang et al., 2014) and then the 2nd step was the extraction of homogenate with hexane ratio 1:1 (Darvishi et al., 2011). Step-1: 5 mL of 0.2 M sodium citrate was added to the two-gel beads in 10 mL Eppendorf tube to vol. 5mL (incubation 10 min at RT), then mixed using a vortex mixer. Step-2: 5 mL of hexane was added to the 5mL extract of sodium citrate and mixed correctly. Then following the same procedure as in step-2, 5 mL broth + 5 mL hexane was mixed with a vortex mixer to obtain the hexane broth extract. Subsequently the broth/bead homogenate hexane mixture was spun (5,000 rpm/20min). By the same steps, the hexane extractions of broth/bead control (without inoculants) were taken as a blank spectrophotometer reading to zero. The tubes were allowed to stand at room temperature (RT) for 10 min. Then 3 mL of the top upper phase was taken into a 10 mL Eppendorf tube and mixed well in a vortex mixer. The 2mL sample was poured into a quartz macro cuvette and used to measure UEO residue (OD_500nm) using a UV-Visible spectrophotometer (GENESYS 20, Thermo Spectronic, https://ww.thermofisher.com) against a blank (2mL broth/bead control hexane extract). This wave-length was chosen because it shows the highest absorbance peak for UEO at 500 nm in hexane. A reference curve was established for the 0 − 50,000 ng/µL UEO dissolved in hexane, to correlate the absorption readings with nanograms of UEO residues. To which the UEO degradation percentage (R_d) was calculated (Zhang et al., 2010, Deng et al., 2014) as below considering, D_Ab the UEO residue in “abiotic” treatment (control) and D_B UEO residue in “biotic” treatment (culture).

$${R}_{d}=\left[\frac{{D}_{Ab}-{D}_{B}}{{D}_{Ab}}\times 100\right]$$

Cell growth

The bacterial growth of FC/IC cultures in the media was determined using the 2, 3, 5-triphenyltetrazolium chloride (TTC) spectrophotometry method at OD_485nm (Moussa et al., 2013). The 2 mL sample broth/2 beads were taken periodically every 3 days throughout Day 0–24. The bacteria number in the medium was determined from the 2 mL culture broth while in the beads, 0.2 mol L^− 1 (filter sterilized pore size of the filter is 0.25 micrometers) sodium citrate cell extract of 2 AAC-beads (Wang et al., 2014; Elhamrouni et al., 2023). SC/MC standard calibration curve (0 to 3 × 10⁹ CFU/ mL) was prepared to correlate the OD_485nm tetrazolium formazan (TF) readings to bacteria CFU/mL.

GC-MS analysis of UEO residues

The hexane extraction of UEO residue broth samples was carried out as explained above (Section 2.4A). Then filtered through 0.45 µM microbial filter (PTFE, Sartorius) into 2-mL GC vials; the GC samples that were not analysed immediately were kept in a refrigerator at 4 ± 1°C. The GC-MS analysis of GC hexane sample extract was done as explained elsewhere (Ibrahim, 2016; Elhamrouni et al., 2023).

The UEO concentration $\left[{C}_{ni}\right]$ was determined from the standard curve (0–50, 000 ng/µL UEO of the same batch in hexane), the sample individual n-alkanes (n-C₉ to n-C₂₉) analysis peak areas (${PAC}_{ni}$) were converted into nanograms as shown in the equation below:

${C}_{ni}=\frac{{PAC}_{ni}}{\sum _{{n}_{i}=9}^{29}\left({PAC}_{ni}\right)}\times$ Concentration of TPH (ng/µL) in sample UEO standard

Where, ${\sum }_{{n}_{i}=9}^{29}\left({PAC}_{ni}\right)={PAC}_{9}+{PAC}_{10}+ {PAC}_{11}+\cdots \cdots {PAC}_{29}$. The peak area of fractions (n-C₉ to n-C₁₇; n-C₁₈ to n-C₂₉; n-C₉ to n-C₂₉) was combined to represent the amount of total UEO hydrocarbons (TPH) in those UEO fractions. Degradation percentage (R_d) of TPH in those fractions was calculated the same as above Section: 2.4A.

Microbial contamination

The absence or presence of microbial contamination was examined by checking shapes/sizes of the cells under an Olympus phase-contrast microscope (Olympus Corporation, Tokyo, Japan) with a magnification of 100 xs.

Equations

The slopes (d[ln(XV)]/dt) of the bacterial growth curves represent the specific growth rate (Oh et al., 2002), where specific growth µ_x (d^-1), growth (XV) CFU, and (t) is duration of treatment (d).

$${\mu }_{x}=\frac{d\left[ln\left(XV\right)\right]}{{d}_{t}}\cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots$$

The growth doubling time (t_d) for the individual or mixed culture can be determined following the Eq. 2(McNeil & Harvey, 2008, p.196):

$${t}_{d}=\frac{ln2}{k}\cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots$$

k is the individual or consortia culture growth rate (µ_x, d^-1) as determined from Eq. 1

For the UEO-%TPH degradation the slope (d[ln(R_di)]/(dt)) is degradation rate (µ_Rd,d^-1), as expressed in Eq. 3

$${\mu }_{Rd}=\frac{d\left[ln\left({R}_{di}\right)\right]}{{d}_{t}}\cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots$$

${R}_{di}=\left[\frac{{D}_{Abt}-{D}_{Bt}}{{D}_{Abt}}\times 100\right]$ , (D_Abt) is the abiotic (control) UEO concentration and (D_Bt) is biotic (culture) UEO concentration Day (t_i),

The biodegradation half-life times (t_1/2) was calculated based on the Eq. 4

$${t}_{1/2}=\frac{ln2}{k}\cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots$$

k the UEO-degradation rate (µ_Rd) (Eq. 3).

The ratio of the UEO-%TPH degradation rate with diffusion limitation (µ_Rd(IC)) to the UEO-%TPH degradation rate with no diffusion limitation (µ_Rd(FC)) represents the effectiveness factor (η) (Hsieh et al., 2008).

$$\eta =\frac{{{\mu }}_{\text{R}\text{d}}\left(\text{I}\text{C}\right)}{{{\mu }}_{\text{R}\text{d}}\left(\text{F}\text{C}\right)}\cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots \cdots$$

To determine the effect of free cell (FC) and immobilized cell (IC) culture on the biodegradation of the UEO in 0.675% (v/v) UEO-YEMSM. Four sets of experimental runs were performed. The 1st two sets were done on FC systems of single and mixed bacterial culture consortia; and the 2nd two sets on IC systems of the same single and mixed bacterial culture consortia. This was done in order to establish the effects of removal of UEO, and assuming the absence of AAC adsorption and/or diffusion resistance in the discrimination of the adsorptive AAC-IC bioremedy of UEO under similar conditions. The same FC/IC inoculum sizes were added in the form of free suspended cell / IC beads, to compare the biosorptive degradation. The 0.675% (v/v) UEO-YEMSM is equivalent to 0.05 g UEO per 100 mL MSM. Those FC culture conditions were important (for comparing) to determine the adsorptive-biosorptive role of AAC-IC systems in the bioremediation studies of UEO. AAC beads at a size of about ∅≈0.45cm were employed to encapsulate the FC, becoming an IC culture system.

Comparison of UEO biodegradation by free cell (FC)/ immobilized cell (IC) single culture ( O. intermedium ); and mixed FC/IC bacteria culture consortia ( O. intermedium + B. paramycoides)

Figure 1A demonstrates the time course of bacteria growth in the UEO batch remediation by single culture (O. intermedium) and the mixed bacterial consortia (O. intermedium strain + B. paramycoides) culture of FC systems in the bulk UEO-YEMSM. The measurements of the degradation were carried out by a spectrophotometer (OD _{500 nm}). As noted in Fig. 1A, during the UEO remediation days 0–24, the bacterial growth increased as the degradation of UEO increased in the interval days 0–15, reaching 28 x 10⁸CFU/mL and 32 x 10⁸CFU/mL, respectively. Then, between Days 16 and 24, the cell counts fall gradually with simultaneous marginal rises in UEO degradation until Day 24. However, throughout Day 0–24, both systems exhibited almost the same degradability ability for UEO. This early rise in bacterial counts indicates the quick adaptability of O. intermedium and O. intermedium strain + B. paramycoides to the UEO. The reason for the rise in UEO degradability is thought to be due to better bacterium growth during that period. Better growth can lead to better UEO degradation, as growth density and inoculum size of potential oil degraders are significant factors that can affect the degradation of hydrocarbon pollutants (Manohar et al., 2001; Thapa et al., 2012).

Figure 1B shows the time course of bacteria growth during the UEO batch remediation by single culture (O. intermedium) and mixed bacteria culture consortia (O. intermedium + B. paramycoides) IC systems in the bulk UEO-MSM. As observed in Fig. 1B during remediation by the IC (i.e., single culture /mixed culture), the cell leakage (CL) started within the 1st 3 days during the period Day 0–24. Then, afterwards, it continued increasing linearly as the degradation of UEO increased until Day 24. These bacterial counts in the media are thought to be due to the release of growing cells from the beads into the media (Trelles & Rivero, 2013; Nunal et al., 2014; Wang et al., 2014). The evidence of this result is the fall of bacterial counts in the same FC systems following Days 15–24 (Fig. 1A), at the same time in the IC systems in the same interval (Fig. 1B), the bacteria count increased, suggesting that those rises are due to the released cells growing in the beads into the medium.

Figure 1C demonstrates the time course of bacteria growth and the UEO degradation in gel beads by single culture (O. intermedium) and mixed bacterial consortia (O. intermedium + B. paramycoides) IC systems. During remediation of the single and mixed AAC-IC systems, the results show that the bacterial cell numbers started rising within the first three days in the beads. Then, the numbers continued increasing until Day 18. Between Days 18 and 24, the single IC numbers simultaneously remained nearly constant, and the mixed AAC-IC counts continued to rise until Day 24. However, during Days 0–24, as shown in Fig. 1C, the UEO degradability by AAC-single and mixed IC started to rise within Day 3 of treatment and continued to increase steadily until Day 24. Generally, in partially open/closed microbial biological systems, the co-metabolism and the dynamic conditions improve the microbial populations (Nzila, 2018). As explained before, during immobilization (Elhamrouni et al., 2023), it is expected that the addition of fresh peptone water to the inoculum formula promoted the growth and mineralization of UEO as a sole carbon energy source. The peptone-water base medium is normally used to test the ability of organisms to utilize HC sources (Holt et al., 1994). In this particular case, it seems its presence in the beads was well applied to the utilization of UEO. The growth-promoting substrates initiate and increase the HC degradation rate (Nzila, 2018). For example, Ambrosoli et al. (2005) found that the bacteria present in the inoculum could not utilize the test PAHs to support growth but could degrade them when growth-promoting substrates (glucose and acetate) were provided. Therefore, the increased growth of mixed cultures might be attributed to the peptone water and the increased incorporation of C-source (UEO) into biomass. The inoculum comprising two or more microbial cultures has increased synergistic effects and a more powerful degradation potential than the individual culture strain in the utilization of used engine oil (UEO) (Kuppusamy et al., 2016; Ibrahim, 2016).

Figure 1 The growth and biodegradation of UEO by single (O. intermedium)/mixed bacterial consortia (O. intermedium + B. paramycoides) of free cell systems (FC systems)/immobilized cell systems (IC systems) in UEO-MSM. A) Growth and %TPH-D of UEO by single free cell (S/FC) and mixed free cell (M/FC). B) The growth and %TPH-D of UEO by single immobilized ^(IC) cell of O. intermedium and mixed immobilized ^(IC) cell bacterial consortia. S/CL: Single/Cell leakage; M/CL: Mixed/ Cell leakage. C) The growth and %TPH-D of UEO by single immobilized ^(IC) cell, and mixed immobilized ^(IC) cell consortia in the beads. S/IC: Single/Immobilized cell; M/IC: Mixed/Immobilized cell.

This result indicates an increased rate of UEO degradation by mixed consortia (O. intermedium + B. paramycoides) AAC-IC compared to its single counterpart (O. intermedium).However, because the biodegradation of UEO by AAC immobilized cells can be associated with the entrapped cells in the beads and the released cells (CL) in the bulk medium, In this study, the progress of UEO degradation was measured in both the beads and medium, and the combined effect was represented in Fig. 2B. Figure 2B shows that the equilibrium point of the mixed IC combined effect due to the entrapped and released bacterial consortia in the bulk UEO-MSM was also established before its individual IC system. This indicates that, regardless of AAC adsorption, the mixed bacterial corsortia entrapped in the beads and released in the bulk UEO-YEMSM were more efficient in the removal of UEO. In this case, the effect of this quick equilibria is most likely expected to be due to the synergistic degradation potential of microbial consortia (Kuppusamy et al., 2016; Ibrahim, 2016), which tends to be mostly related to the enhanced biological activity and to a lesser extent associated with the adsorptive properties of the AAC matrix.

Tables 1a, b show the comparison of FC and AAC-IC culture growth and GC-MS analysis of TPH-degradation parameters in UEO-MSM Day 24. Table 1a shows that single and mixed FC systems featured bacterial

No. of 21.72 ± 0.94 x 10⁸ CFU/mL and 24.20 ± 1.90 x10⁸ CFU/mL. The specific growth rates of 0.060 and 0.063 per day (Fig. 3A) with UEO TPH degradation up to 90.75% and 93.03% (Table 1b). At meantime the single and mixed IC systems recorded bacterial numbers of 27.97 ± 0.46 x 10⁸ CFU/mL, and 32.26 ± 1.86 x 10⁸ CFU/mL, and specific growth rates of 0.125 and 0.127 per day (Fig. 3A).

Table 1

a: The comparison of free cell (FC) and AAC-immobilized cell (IC) culture growth parameters in the bulk UEO-MSM Day 24
Parameters	FC systems⁽¹⁾		IC systems⁽¹⁾
	Single(S)⁽²⁾/Mixed(M)⁽³⁾, FC		Single(S)/Mixed(M), IC
	(S/FC)	(M/FC)	(S/IC)	(M/IC)
Growth (x 10⁸ CFU/mL)	21.72 ± 0.94^C	24.20 ± 1.90^C	27.97 ± 0.46^B	32.26 ± 1.86^A
Growth rate (µ_x)(day^− 1)	0.060	0.063	0.125	0.127
Doubling time (t_d)(day)	11.55	11.00	5.54	5.45

Values are the mean ± standard deviation (n = 3).

Within a row (A–C), significant differences (p < 0.050) are indicated, as measured by the Duncan DMRT Test. and UEO-TPH degradation of up to 93.28% and 98.42% (Table 1b) respectively. This growth kinetics data (Table 1a) shows that after 24 days of treatment, the IC systems increased the specific growth rate (Eq. 1) by two folds (0.125–0.127 day^− 1) as compared to their FC systems (0.060–0.063 day^− 1). Besides that, the AAC-IC systems also decreased the generation time (Eq. 2) to around 48% (5.54 days) and 50% (5.45 day) in single and mixed AAC-IC bacterial consortia as compared to their single (11.55 days) and mixed bacterial consortia (11.00 days) FC systems. The highest and lowest values of growth rates and generation time in the bulk media were recorded with IC systems.

The growth and UEO degradation kinetic, under two criteria of single (S) and mixed (M) cultures, are free cells and immobilized cells in the bulk UEO-MSM from days 0–24.

Figures 3A, B compares the kinetic growth of FC and their IC. The cases include single and mixed and free cell (FC) O. intermedium, FC O. intermedium + B. paramycoides. And, immobilized cell (IC) O. intermedium, and immobilized cell O. intermedium + B. paramycoides.

As noted in Fig. 3A, B the mixed of FC/IC systems showed the best growth than single cuture in agreement with Nzila (2018). Figure 3B shows that the IC systems attained slightly increased specific growth than their FC systems.

Table 1

b: The GC-MS, analysis comparison of TPH-degradation parameters by free cell (FC) and immobilized cell (IC) culture in the bulk UEO-MSM Day 24
Parameters	FC systems		IC systems
	Single(S)⁽²⁾/Mixed(M)⁽³⁾, FC		Single(S)/Mixed(M), IC
	(S/FC)	(M/FC)	(S/IC)	(M/IC)
Control (C₀) (mg/mL)	88.475	88.475	82.73	82.74
TPH-residue (mg/mL)	8.188	6.167	5.564	1.31
TPH% degradation	90.75	93.03	93.28	98.42

1) Table 1a, b indicates parameters measured in the UEO-MSM; 2) individual culture of O. intermedium; 3) mixed consortia of O. intermedium + B. paramycoides. indicating that with the use of the IC system, the specific growth rate increased and the generation time decreased.

Figure 3C compares the kinetics of UEO degradation by FC and their IC. The IC systems established increased degradation rates from Day 0 to 24 as compared to their FC systems, demonstrating the impact of adsorption due to attapulgite in the AAC- IC systems (Elhamrouni et al., 2023). Table 2 summarizes the spectrophotometer (OD-500nm) measured kinetic parameters of UEO biodegradation by FC and IC systems, Day 0–24. As noted in Table 2, the mixed bacterial consortia of FC and IC, recorded relative increased UEO degradation rates of 0.23 day^-1 and 0.24 day^{-1 as} compared to the individual FC and IC cultures that recorded 0.22 day^-1 and 0.23 day-1, respectively. Those slight increased UEO degradation rates by FC/IC mixed bacterial consortia compared to individual FC/IC, are most likely attributed to the synergistic bioeffect of mixed bacterial consortia, and more/least to biosorption in FC mixed bacterial consortia, and biosorption and adsorption in IC mixed bacterial consortia (Elhamrouni et al., 2023). Chih-Wen & Hwai-Shen (2011) also observed that around 90% oil removal was achieved in diesel oil or crude oil when working with Rhodococcuse rythropolis strain NTU-1 bioremedy, 24% was biodegradation, and 63% biosorption in suspended cell aerobic batch culture. As related to our case, it seems that the cutures were more degradative and least biosorptive, which might be due to the ability of those microbes to produce biosurfactants, increasing the availability of UEO in YEMSM to their microbial biodegraders.

The degradation rates of UEO were shown to be higher when exposed to individual and mixed bacterial IC cultures, resulting in a drop of 3.50% and 4.92% in the half-time degradation of UEO (measured in days), compared to the corresponding FC systems. The experimental effectiveness factor (η) values achieved by the individual and mixed bacterial IC cultures were 1.04 and 1.05, respectively. These values closely resemble those observed in similar FC systems, where η = 1. The obtained outcome suggests that the mass transfers in the AAC-IC systems are highly efficient. The reduced structural resistance to the flow of UEO in the beads may be attributed to the characteristics of AAC-IC beads before and after the ion exchange process, such as the internal and peripheral porosity of the beads (Wang et al., 2014; Elhamrouni et al., 2023). This effect is likely unrelated to the protective structure of the larger diameter beads (∅≈0.45cm). The mass transfer and biodegradation performance of alginate beads are negatively affected by their large diameter, as it increases the resistance of the structural matrix (Dursun & Aksu, 2002; Dursun & Tepe, 2005). However, the present study suggests that this impact is not significant when using large diameter beads (∅≈0.45cm), likely due to the unique characteristics of AAC beads (Wang et al., 2014; Elhamrouni et al., 2023).

The production of biosurfactant can also be another reason for that impact that decreased the resistance to the flow of this viscous substrate. The oil degraders produce biosurfactants (Franzetti et al., 2010; Su et al., 2011; Phulpot et al., 2021), to which they probably homogenized the UEO in the bulk YEMSM. Bezza et al. (2015) and Karlapudie et al. (2018) reported that the genus of Ochrobacterium and Bacillus produce biosurfactants. Nehal & Singh (2022) found that Bacillus paramycoides produces biosurfactant, which forms the bacteria consortia in this study. Therefore, enhancing the diffusion of UEO inside the AAC-IC systems; besides that, O. intermedium and B. paramycoides are facultative anaerobic. The anaerobic degradation of UEO in the beads is expected to release intermittent gas bubbles, which probably led to the increased internal/peripheral porosity of AAC-IC beads.

The living microorganisms, growth and gas evolution lead to significant mechanical disruption of beads (Dursun& Tepe, 2005); but because AAC-IC systems are known to be of strong consistency, durability, and more porosity (Wang et al., 2014), they remained intact over Day 0–24. Indicating that the anaerobic gases produced mostly opened the internal pores in the AAC-IC bead layers without the breaking of matrix, therefore contributing positively in this respect.

Degradation of UEO by FC systems and AAC-IC systems

The GC-MS analysis degradation of UEO by FC systems and AAC-IC systems

Figures 4, 5, and 6 demonstrate the GC-MS analysis of UEO. As observed in Figs. 4B, C, and 5B, C the decrease of the area under the curve (10–28 min) in biotic treatments as compared to the same abiotic controls (Fig. 4A/ 5A), it indicates the biodegradation of UEO. The more decreased patterns of the area under the curve (AUC) in Figs. 4C / 5C, demonstrate here that the mixed bacteria of free cell (FC)/immobilized cell (IC) cultures were more efficient in removing UEO, than their FC/IC pure cultures of O. intermedium (Fig. 4B and 5B). As a significant result, this effect is believed to be due to the "synergistic effect" of "mixed culture" on the degradation of UEO.

Table 2

The effect of FC systems and IC systems (Bead size, ∅≈0.45cm)^(A) on the UEO-%TPH degradation rate (µ_Rd(d^-1)), degradation half life tme (t_1/2(d^-1)) and the experimental effective factor(η).
Culture system	µ_Rd(d^− 1)	t_1/2(d^− 1)	η
a) FC system
Pure⁽¹⁾	0.22	3.14	1.00
Mixed consortia⁽²⁾	0.23	3.05	1.00
b) IC system
Pure⁽¹⁾	0.23	3.03	1.04
Mixed consortia⁽²⁾	0.24	2.90	1.05

A) The gel bead size was determined based on the wate displacement (Sun & Griffiths, 2000; Elhamrouni et al., 2023).

1) Table 2 indicates individual culture of O. intermedium; 2) Mixed consortia of O. intermedium plus B. paramycoides.

A- The GC-MS analysis profile of HC-Cpds (C₉-C₂₉) degradation of UEO by FC systems

Figure 6 shows the GC-MS analysis of the HC-Cpds (C₉-C₂₉) degradation of UEO by FC O. intermedium and O. intermedium + B. paramycoides. The red bars in Figs. 6A-D / 7 A-D, are negative controls (C_n-Abiotic control (µg/mL) to which no biodegradation occurred for those C_n-fractions, while the green, white and blue bars are biodegradation results [(C_n-Abiotic control) – (C_n-Biotic residue)] of O. intermedium and O. intermedium + B. paramycoides as expressed in µg/mL.

As noted in Fig. 6A, O. intermedium and O. intermedium + B. paramycoides exhibited various degradation abilities for the HC-Cpds (C₁₁-C₁₇). The C₉ and C₁₀ were not detected during the GC-MS analysis profile of C₉-C₂₉ hence, the zero value is due to non-detection. Figure 6A shows the zero degradation of C₁₅ with O. intermedium, and C₁₂, C₁₃ and C₁₅ (red arrows) with O. intermedium + B. paramycoides, as their C_n residue concentrations increased higher than their negative control following the GC-MS analysis of (C₉-C₂₉). These offshoots of C₁₅ with O. intermedium, and C₁₂, C₁₃ and C₁₅ (red arrows) with O. intermedium + B. paramycoides, above their abiotic control were expected due to catabolic repression and HC metabolites resulting from the increased degradation of HC-Cpds fractions ≥ C₁₇ in the range of nC₁₇-nC₂₉. The evidence that corroborates this effect was attributed to the increased degradation to almost completion for the HC-Cpds C₁₇ (Fig. 6A) and nC₁₈ to nC₂₉ (Fig. 6B).

These results agree well with Cerqueira et al. (2011), who reported on the catabolic repression of HC-Cpd, which cannot be degraded in the presence of another metabolite Cpd acting as a repressor to the synthesis of enzymes involved in metabolic pathways. However, as observed in Fig. 6C, the O. intermedium, and O. intermedium + B. paramycoides enabled the TPH-UEO% degradation of 61% and 75% in the HC-Cpds range of C₁₁-C₁₇. Meanwhile, Fig. 6D shows the same formulas also featured the TPH-UEO% degradation of 91% with the total bacteria counts of approximately 21.72 ± 0.94 x 10⁸ CFU/mL, and 93% with 24.2 ± 1.9 x 10⁸CFU/Ml in HC-Cpds range of C₉-C₂₉. The losses caused by O. intermedium or O. intermedium + B. paramycoides during the incubation Day 0–24, were mostly within the HC-Cpds of nC₁₈ - C₂₉, to which they represent the highest percentage of HC-Cpds in UEO, GC n-Alkane (C₉-C₂₉) analysis.

Due to the high percentage of n-C₁₈ to n-C₂₉ in UEO, the TPH degradation % of UEO remarkably increased as the degradation% of n-Alkane fraction nC_i (i > 17) increased. The decreased degradation was caused by n-C₁₁ to n-C₁₇ as explained above. It was indicated that these bacteria formula were least efficient to utilize some of the HC-Cpds in that n-alkane chain length. Zhang et al. (2011) reported that the increased degradation efficiency of HC-Cpds in oil decreased as alkane chain length increases, which is in contrast to our results that exhibited high degradation % TPH in nC_i (i > C₁₇). Several studies on the degradation of polyaromatic hydrocarbons by bacteria demonstrated a tendency toward an inverse relationship between the biodegradation rates and the number of aromatic rings and molecular mass (Molina et al., 2009; Vitte et al., 2011). As explained above, the increased principal consumption of those HC-Cpds in the range ≥ C₁₇ might be the reason that led to the non-degradation of C₁₂, C₁₃ and C₁₅. This effect is thought to be caused by the increased ability of O. intermedium to degrade, aliphatic HCs in UEO (Mahendhran et al., 2018).

As noted in Fig. 6D, the reduction of 91%, TPH-UEO % was achieved under this experimental condition. Additionally, it was observed that, in the Fig. 6D, the bacteria consortia cause increase in TPH-UEO removal up to ≈ 2.19% (93%) than O. intermedium (91%). The slight rise in TPH-UEO removal associated with bacteria consortia might be attributed to the synergistic combined bioeffect resulting from the individual organisms in this consortium. The inoculum co-cultures usually have least/more increased synergistic effects, and more powerful degradation potential than their mono cultures in the utilization of used engine oil (UEO) (Kuppusamy et al., 2016; Ibrahim, 2016; El-Aziz et al. (2021). Gargouri et al.(2011), also observed an increase in degradation performance of HC-Cpds, when working with the mixed cultures. The result of the TPH-UEO removal efficiency by individual and mixed bacteria consortium was close to each other. This result indicates that, the O. intermedium was contributing so much; with least/more effect relating to B. paramycoides as its surface is hydrophillic. The O. intermedium is hydrophobic surface bacterium (Mahendhran et al., 2018; Alipour Asadabadai). Bacterial surface properties are essential to the effective biodegradation of hydrophobic hydrocarbon substrates and their adhesion mechanisms (Zhang et al., 2015). Ron & Rosenberg (2014) found that adherence of hydrophobic pollutants to bacterial cells is mainly related to hydrophobic fimbriae, fibrils, outer-membrane proteins and lipids, as well as certain small molecules present in cell surfaces such as gramicidin S and prodigiosin. Bacillus spp, are weak hydrophobic surface petroleum hydrocarbon degrading bacterium (Wang et al., 2019), to which they cannot directly adhere to oil substrate to facilitate their uptake. Individual otherwise mixed microbial consortia with the abilities to degrade petrol oil have been reported elsewhere previously. For example, the same authors Gaur et al. (2021) reported on the consumption of TPH-UEO % up to 74.35 ± 0.037% by Pseudomonas aeruginosa gi|KP 16392| in 5% (v/v) UEO-MSM. Phulpoto et al. (2021) reported 31.10 ± 0.08% to 40.50 ± 0.11% UEO biodegradation with Ochrobactrum sp. S1MM and Bacillus nealsonii S2MT.

The current study used O. intermedium to achieve the extra increase of 22% removal of TPH-UEO in 0.675% (v/v) UEO-YEMSM than Pseudomonas aeruginosa gi|KP 16392| and 2.93 times reduction than Ochrobactrum sp. S1MM. Phulpoto et al. (2021), also observed as similar to our results increased biodegradation efficiency of UEO with consortia comprising of two (i.e., Ochrobactrum sp. S1MM + Bacillus nealsonii S2MT), and/or more bacterial cultures. The reason for these variations in these degradability abilities is due to the variations in medium condition (e.g., oxygen, pH, temperature, UEO concentration, UEO composition etc.) and the microorganism growth and formulas (Bhattacharya et al., 2015; Larik et al., 2016; Phulpoto et al., 2021; Premnath et al., 2021).

Generally, waste lubricating oils have highly variable compositions to which it depends on the extent of combustion process during its functioning (Bhattacharya et al., 2015). Another reason for this variation could be also due to the genetic makeup, since different genetic makeup can lead to different biodegradability abilities of UEO. The bacteria surface properties. e.g., hydrophobic petroleum hydrocarbon degrading bacterium can be more effective to the biodegradation of hydrophobic hydrocarbon substrates (Zhang et al., 2015).

B- The GC-MS analysis profile of HC-Cpds (C9-C29) degradation of UEO by IC systems

Figure 7 (A-D) shows the GC-MS analysis of HC-Cpds (C₉-C₂₉) degradation of UEO by AAC-immobilized O. intermedium and O. intermedium + B. paramycoides. As, seen from the results in the Fig. 7A, O. intermedium and

O. intermedium + B. paramycoides exhibited various degradation abilities for the HC-Cpds (C₁₁-C₁₇). The C₁₀ was not detected during the GC-MS analysis profile of C₉-C₂₉, and so the zero value is due to non-detection. In the same Fig. 7A, the reason of the zero degradation for C₁₃, C₁₄, C₁₅ and C₁₆ with O. intermedium (Red arrows) as related to the results in Fig. 7B, can be explained basically as the same as explained above in FC systems.

However, as noted in Fig. 7C, it can be thought that O. intermedium and O. intermedium + B. paramycoides allowed the removal of 87% and 89% TPH-UEO in the carbon range nC₉-nC₁₇. The closeness in the result of the removal of TPH-UEO% (nC₉-nC₁₇), by O. intermedium, and O. intermedium + B. paramycoides was probably caused by the adsorption of beads, and/or to some extent the degradation of HC-Cpds, of C₁₇ by O. intermedium and nC₁₃-nC₁₇ by O. intermedium + B. paramycoides in the carbon range of nC₉-nC₁₇. In the same carbon range nC₉-nC₁₇, the calculated removal of TPH- UEO% by AAC-immobilized cell systems (Fig. 7C), as compared to their counter FC systems (Fig. 6C) was 30% high in O. intermedium immobilized cells and at meantime the AAC- immobilized O. intermedium + B. paramycoides system allowed only 18.6% increased removal in TPH-UEO% (nC9-nC17), than their counter FC systems (Fig. 6C).

The increased removal of TPH-UEO% by IC systems than FC systems in nC₉-nC₁₇ range indicates the increased adsorptive bioremediation efficiency, and it can also be due to the low viscosity of the HC-Cpds in that particular n-alkane (C₉-C₁₇), which it caused the easy flow of those HC-Cpds through pores inside the beads.

Generally, it is more difficult for sorbent materials to absorb highly viscous oil ((Hoang) Anh et al., 2021). An important outcome shows that AAC beads will selectively increase the removal of the n-alkane range (C₉-C₁₇), to which it was less efficiently degraded by FC system as explained before (Fig. 6A). The hydrocarbon fraction by carbon range C₉-C₁₇ is thought to include HC-Cpds of Kerosine blend (Sayed et al., 2021), are light and less viscous, as compared to heavy petrol HC-Cpds. However, those comparisons as made in the carbon range of nC₁₈-nC₂₉ for the systems in Fig. 7C vs Fig. 6C resulted in 1% and 3% increased removal of UEO by AAC-IC systems as compared to their counter FC systems. This result shows that, the removal of TPH-UEO % were almost close to each other in Fig. 7C vs Fig. 6C. Thus the observed removals of nC₁₈-nC₂₉ of UEO Day 24 are attributed mainly to bacterial digestion rather than adsorption. As an important out come, this result indicates that the both systems are more efficient to remove the carbon range nC₁₈-nC₂₉ to almost completion. The result is mostly subjected to digestion by O. intermedium or O. intermedium + B. paramycoides of nC₁₈ - C₂₉, in UEO, GC n-Alkane (C₉-C₂₉) analysis. Besides that, since those were the magnitude of influence of AAC-IC O. intermedium or O. intermedium + B. paramycoides on the partial loss of HC-Cpds in the range nC₉-nC₁₇ and nC₁₈-nC₂₉. It is also important to report on this impact on the HC range nC₉-nC₂₉ as related to the growth in these AAC-IC bioremedies.

These bacteria No. are high than those attained by their the same FC systems to which they permitted the bacteria concentrations of 21.72 ± 0.94 x 10⁸ CFU/mL, and 24.2 ± 1.9 x 10⁸ CFU/mL, with O. intermedium and O. intermedium + B. paramycoides Day 24.

Wang et al. (2014) also recorded high total bacteria No. of up 10⁸-10¹³ CFU/mL AAC geL, when working with the Pseudomonas sp. DG17 AAC-IC in the bioremediation of diesel. The increased TPH-UEO % removal is associated with increased total bacteria No, in the media with immobilized AAC-IC. This outcome might be attributed to the improved conditions in the beads that led to better bacteria growth and protection (Banerjee & Ghoshal, 2011; Bayat et al., 2015).

From the results in the Fig. 7D, it can be said that, the AAC-IC O. intermedium enabled the TPH-UEO% reduction of 93%, with total bacterial No. of 28 ± 0.46 x 10⁸ CFU/mL; and, the AAC-immobilized O. intermedium + B. paramycoides permitted the losses to almost completion with only 2% remaining, with total bacterial No. of 32.26 ± 1.86 x 10⁸ CFU/mL.

The bacteria protection can lead to prolonged bioremediation and bacteria growth. The innoculum sizes and increased No. of potential bacteria are significant inputs in oil bioremediation processes (Thapa et al., 2012), and they can be a good indicator to the increased bioremediation (Prescott et al., 2002, p. 20–38, 96–132, 1012–1018). The increased No. of microbial oil biodegraders are normally achieved on the expense of C-sources incorporated into biomass.

Beside the effect of increased bacteria No. in the AAC-IC consortia on the removal of TPH-UEO %, this rise in TPH-UEO removal associated with AAC-IC bacteria consortia can be attributed to the genetic variations of those individual bacteria in their degradative abilities and the growth on the HC-Cpds during bioremediation. Genetic diversity in bacterial oil biodegrading population lead to syntrophic processes to which it promotes their mutualistic interaction and boosts their degradative ability to awide variety of HC-Cpds. Syntrophic process is a mutualistic interaction (Sieber et al., 2012) in which individual of microbial consortium participates in the biodegradation of the complex oil contaminant efficiently.

Gargouri et al. (2011) found that high degradation performance of HC-Cpds, range n-alkanes (C₁₀–C₃₅) was attained when working with hydrocarbon-rich industrial wastewater effluents and mixed cultures. Phulpoto et al. (2021), Iyobosa et al. (2021) & El-Aziz et al. (2021) also reported that the mixed cultures are powerful tools for biodegradation. This conclusion agrees well with our results, of the bioefficiency of O. intermedium + B. paramycoides, as explained above. The novelty of this result is the combining of the adsorption and the prolongation of the biodegradation efficiency of UEO, by the AAC-IC formulas in liquid medium. This finding will be important to further cheap insitu applications of bioremedy technique to oil polluted water sites that requires expensive non sustainable physical and/or chemical approaches, lasting shorter. The adsorption and the prolongation of the biodegradation efficiency of UEO by AAC biocatalyst here is good as it will reduce the cost of bioremediation applications, and the total dependence on abiotic expensive physical and/or chemical methods.

The individual cultures were also found to be competent in some cases, for example Throne-Holst et al. (2007) found that the genus Acinetobacter had the ability to degrade a broad range of the n-alkanes C₁₀–C₄₀. Several inherent oil-degrading bacteria with the ability to degrade n-alkanes HC-Cpds has also been reported by other investigators elsewhere previously i.e., Baldwin et al. (2009); Mavrodi et al. (2003); and Whyte et al. (1999) etc. Rhodococcus sp. strain Q15 (C₁₂–C₃₂) (Whyte et al., 1999); Pseudomonas putida G7 (naphthalene) (Mavrodi et al., 2003), and Pseudomonas aeruginosa JI104 (toluene) (Baldwin et al., 2009). Thus, the success of any bioremediation process is the potential ability of the individual/mixed microorganisms to remove the pollutants (Varjani & Upasani, 2013) to almost completetion. In this study, the high removal of TPH-UEO% by individual/mixed AAC-immobilized cell in the range 93–98% indicates most likely the maximum efficiency of these systems to remove TPH UEO% in the liquid medium. This result allows us to suggest that these AAC immobilized cell bio remedies will be efficient to remove petroleum hydrocarbon products and fractions by carbon ranges of nC₉-nC₂₉, i.e., Kerosine, heating oils, etc. (Sayed et al., 2021) in the marine environment. Wang et al. (2014) also observed the removal of petro hydrocarbon ranging from 33.56% ± 3.84–56.82% ± 3.26% after 20 days, when working with the immobilization of Pseudomonas sp. DG17 onto the AAC. These, low removal of the TPH% as compared to our study can be mainly attributable to the type of petrol product used, concentration and the bacteria used. The bacteria differ in their petrol hydrocarbon degradative bioefficiencies based on formula i.e., single or mixed bacterial cultures, otherwise biosurfactants and/or liopolytic enzyme producing cultures (Phulpoto et al., 2021; Larik et al., 2016).

To test the adsoptive bioremedy of the IC system to remove UEO, individual (O. intermedium) and mixed bacterial consortia (BC) (O. intermedium plus B. paramycoides) were immobilized using AAC and compared to the similar FC systems. The growth and degradation of UEO by AAC-IC culture were followed in a shake flask (0.675% (v/v) UEO-YEMSM, agitation: 150 rpm, day: 0–24). The results showed that the IC cultures promoted exponential bacteria growth and the UEO removal on days 0–24. And, with reduced growth generation time and experimental effectiveness factor (η) values of 1.04 and 1.05 close to the FC systems, = 1. The beads remained intact throughout treatment. The AAC-IC systems removed the nC₉-nC₁₇ Cpds in UEO by adsorption and digestion, with a 30% TPH-UEO% rise by individual AAC-IC and 18% by BC-AAC-IC compared to the same FC system. The FC/IC systems efficiently digested nC₁₈ -nC₂₉ in UEO. The individual AAC-IC enabled the TPH-UEO% reduction of 93%, with a bacterial number of 28 ± 0.46 x 10⁸ CFU/mL, and the BC the loss of 98% TPH-UEO, with 32.26 ± 1.86 x 10⁸ CFU/mL in nC₉-nC₂₉. These findings clearly demonstrate the efficient capacity of the IC-BC system in terms of adsorption and biodegradation.

Acknowledgements

The researchers would like to express their gratitude to the Faculty of Forestry and Environment at the Universiti Putra Malaysia for their assistance in supplying this study with the equipment and supplies that were required in order to successfully finish this research.

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No competing interests reported.

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Exsitu assessment of potential free (FC) and immobilized cell (IC) bacteria used as engine oil (UEO) degraders in the degradation of UEO in liquid yeast extract minimal salt medium (YEMSM)

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Maintaining cultures of UEO degraders

The single (SC) and mixed (MC) inoculum preparation

The shake flask medium

Analytical methods

Spectrophotometric analysis of UEO residues

Cell growth

GC-MS analysis of UEO residues

Microbial contamination

Equations

Results and discussion

Degradation of UEO by FC systems and AAC-IC systems

A- The GC-MS analysis profile of HC-Cpds (C₉-C₂₉) degradation of UEO by FC systems

B- The GC-MS analysis profile of HC-Cpds (C9-C29) degradation of UEO by IC systems

Conclusion

Declarations

Acknowledgements

References

Additional Declarations

Status:

Version 1

Exsitu assessment of potential free (FC) and immobilized cell (IC) bacteria used as engine oil (UEO) degraders in the degradation of UEO in liquid yeast extract minimal salt medium (YEMSM)

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Maintaining cultures of UEO degraders

The single (SC) and mixed (MC) inoculum preparation

The shake flask medium

Analytical methods

Spectrophotometric analysis of UEO residues

Cell growth

GC-MS analysis of UEO residues

Microbial contamination

Equations

Results and discussion

Degradation of UEO by FC systems and AAC-IC systems

A- The GC-MS analysis profile of HC-Cpds (C9-C29) degradation of UEO by FC systems

B- The GC-MS analysis profile of HC-Cpds (C9-C29) degradation of UEO by IC systems

Conclusion

Declarations

Acknowledgements

References

Additional Declarations

Status:

Version 1

A- The GC-MS analysis profile of HC-Cpds (C₉-C₂₉) degradation of UEO by FC systems