A novel characterization of alginate-attapulgite-calcium carbonate (AAC) gel adsorption in bacterial biodegradation of used engine oil (UEO)

Abstract Biodegradation of used engine oil (UEO) that pollutes the environment is attracting increasing attention. The aim of this study was to characterize the adsorption of used engine oil (UEO) and the immobilization of UEO biodegraders in Ochrobacterium intermedium LMG 3301 and Ochrobacterium intermedium LMG 3301 plus Bacillus paramycoides MCCC1A04098 (BC) using alginate-attapulgite-calcium carbonate (AAC). Twenty AAC beads were tested separately in 20 mL of soil water extract (SWE) and hexane containing 1% UEO for their adsorptive capacities of UEO in 50-mL capped flasks. The results showed that the UEO removal efficiency (%RE) was bead-number dependent, recording a maximum of 83 ± 0.32% in SWE, and 11.50 ± 0.52% in hexane correlating with the highest number of AAC-granules (20 beads). The 1st order kinetics in SWE showed a UEO adsorption rate, K1 (h−1), of 0.11–0.15 h−1 correlating with 20 AAC-granules and 1, 2, 3 and 4% (w/v) UEO. The intra-particle diffusion kinetic model suggested that intra-diffusion was the main mode of mass transfer in AAC granules. The microscopic study and 2,3,5-triphenyltetrazolium chloride (TTC) positive stain showed that bacteria grew well in AAC. This result demonstrates that this matrix can be applied in the formulation of novel adsorptive granular formulas for bioremediation of UEO.


Introduction
Surface and underground water are increasingly polluted with used engine oil (UEO) owing to the rapid global rise in the use of petroleum fuel and oil-based lubricants [1,2].Specifically, the use of engine oil has increased as a result of the rising use of vehicles and automobiles [3].Normal and accidental spillages of oil products into natural environments (air, land and water) have drawn critical public attention [4], since waste oil is considered one of the most dangerous urban wastes.Oil contaminants include high percentages of polycyclic aromatic hydrocarbons (PAHs), aromatic and aliphatic hydrocarbons, and nitrogen, sulfur and heavy metals (Zn, Pb, Cr and Fe) [5].PAHs are toxic, mutagenic and carcinogenic [6].Toxic heavy metals in UEO get dissolved in water and can move in soil, eventually polluting surface and/or groundwater, representing a health hazard in the food chain that can build up to toxic levels.
During the past years, more research on bioremediation has focused on the bioremediation of large-area oil spills caused by crude oil, diesel and petrol such as UEO disposals at car workshops.Hazards related to the continuous accumulation of UEO contamination remain a serious cause of concern [7].Such contamination needs a quick intervention to reduce the relevant risks.Many research efforts have been aimed at minimizing the environmental consequences of oil spills chemically, physically and naturally [7].Among all methods, Ghribi et al. [7] mentioned that bioremediation is considered the best choice to date due to its advantages over approaches using synthetic chemicals which are costly and limited in their efficiency [8].Studies have shown that the use of formulas with indigenous single/mixed bacteria consortia is efficient [9].These microbes present a high potential for the formulation of efficient free suspended cells, freeze dried cells and adsorptive immobilized cell beads.Bacteria entrapment using a blended composite matrix (e.g.alginate-attapulgite-calcium carbonate) increases porosity, mass transfers, and strength of produced beads [10].These results can support better bacterial growth of immobilized cell formulas, with protection from harsh mechanical damages in the liquid and/or solid medium.An optimal combination ratio of composite alginate-attapulgite-calcium carbonate has been reported previously by the same authors [10] in the immobilized cell bioremediation of diesel.Diesel has some chemical properties similar to those found in used engine oil.Therefore, this matrix can be used in the immobilized cell bioremediation of UEO.
This study was aimed at investigating sodium alginate-attapulgite-calcium carbonate (AAC) as a matrix for UEO sorption and/or the immobilization of oil-degrading bacteria.

Isolation
UEO-degrading bacteria were isolated from oil-polluted soil by enrichment [11].Ten grams of UEO-contaminated soil taken from a composite sample (Table 1) was added aseptically to 100 mL sterilized 1% UEO-enriched minimal salt medium (EMSM) in a 250-mL Erlenmeyer flask without baffles and incubated on a rotary shaker at 32 °C/150 rpm for one week.Subsequently, 1 mL of microbial enriched spent medium was transferred into freshly prepared 99 mL, 1.01%UEO-EMSM in a 250 mL Erlenmeyer flask and incubated under the same condition (32 °C, 150 rpm, for a week).The same procedure was repeated for the third enrichment culture medium.Then, 100 µL of inoculum appropriate dilution was made from the last sub-culture Erlenmeyer flask and was evenly added to 1% UEO-MSM agar plates; the cultures were incubated at 32 °C for 2 to 5 days.Pure bacterial colonies were selected based on their ability to grow well, colony shape, color and microscopic inspection of cell morphology; then they were streaked onto another NA plate.

Single (SC)/mixed (MC) culture inoculum preparation
A loopful of revived culture bacteria (O.intermedium LMG 3301/B.paramycoides MCCC1A04098) grown on nutrient agar (NA) at 32 °C/24h was transferred in 100 mL of sterilized nutrient broth (NB) in a 250-mL Erlenmeyer flask.The culture was then incubated at 32 °C/24h on a shaker (Protech) at 150 rpm.Subsequently, 50 mL culture broth made up to 1 OD 600 nm (3 × 10 9 cfu mL −1 ) was transferred into a 50 mL sterilized Eppendorf tube and then centrifuged at 10,000 rpm/10 min, 4 °C (Hettich, Universal, 320) [14].The pellet was washed three times with normal saline and then was resuspended in 5 mL of peptone water.The resulting inoculant was used as a seed culture in the free or immobilized cell formulas.The bacterial consortia of O. intermedium LMG 3301 + B. paramycoides MCCC1A04098 inoculum was prepared separately following the same procedures, and then grown according to the principle of equal quantity and cell concentration in NB.The appropriate volume of harvested washed cells from 50 mL culture was resuspended with 5 mL peptone water as the bacterial consortia (BC) inoculant seed in the free cell or AAC immobilized cell experiment.

Spectrophotometric analysis of UEO residues
Several 2 mL samples were taken from each batch flask in 15 mL Eppendorf tubes throughout a period of 0-12 h.To monitor the UEO in each 2 mL sample per 2 h, the sample was mixed with hexane at a v/v ratio of 1:2 using a vortex mixer.Subsequently, the tubes were allowed to stand at room temperature (RT) for 10 min.Then 3 mL of the top upper phase was taken Before the sampling at each ueo-contaminated soil sampling point, the top surface soil was removed using a sterile garden trowel [19].then, three separate soil samples of 100 g each were scooped at a depth of 15 cm from three different sampling points at each workshop.b each three individual soil samples collected from the same garage site point were mixed together in one polyethylene bag, hence making two composite samples per site; the sample bags were kept in a fridge at 4 °c until the next day for isolation by enrichment [11].
into a 10 mL Eppendorf tube and mixed well in a vortex mixer [15].
The sample was poured into a quartz macro cuvette and used to measure UEO residue (OD 500 nm ) using a UV-Visible spectrophotometer (GENESYS 20, Thermo Spectronic, https://ww.thermofisher.com)against a blank of 5 mL soil water extract/hexane.This wavelength was chosen because it shows the highest absorbance peak for UEO at 500 nm in hexane.The measurements were performed in duplicate, and a reference curve was obtained for the 0-50,000 ng µL −1 UEO standards in hexane.According to Moses et al. [16], the adsorption capacity ( q e ) measured in µg kg −1 and removal efficiency (RE) were calculated using Eqs.
where C 0 and C e are the initial UEO concentration (at t = 0) and the UEO concentration at equilibrium (ngµL −1 ) at any other time points, respectively; V is the solution volume (mL) and w (g) is the weight of adsorbent.

Bacterial immobilization process
In this study, the immobilization of cells was carried out by using a gel medium with the following components dissolved in water measured at %(w/v): Na-alginate (3.5), attapulgite (0.75), and calcium carbonate (5).The rest of the procedure was performed according to Wang et al. [10].The procedure was performed by taking 100 mL of the sodium alginate-attapulgite-CaCO 3 gel (AAC) in a 0.25 L scourge bottle and septically adding an appropriate inoculum volume (≈670 µL)) to attain an inoculum size of 2 × 10 8 cfu mL −1 gel and hardening in CaCl 2 .5H 2 O (Figure 1).The gel bead size was determined based on the water displacement method [17].To test the cell leakage, 100 mL of sterilized 1% UEO-MSM (in a 250 mL Erlenmeyer flask) was loaded with AAC-immobilized cell beads.A total of 74 beads from SC and 60 beads from MC produced from the 5 mL, ACC-cell mixture entrapping, 3 × l0 9 cfu mL −1 of SC (O.intermedium LMG 3301 strain) or MC (O.intermedium LMG 3301 strain + B. paramycoides MCCC1A04098 strain) were added in separate flasks, and incubated at 32°C (150 rpm/24 days).

Cell growth
SC/MC bacteria count (BC) in the media and beads were determined using the 2,3,5-triphenyltetrazolium chlroride (TTC) spectrophotometry method at OD 485nm [18].The released bacteria count (BC) in the medium was determined from the 2 mL culture broth withdrawn periodically every 3rd day until the 24th day.In the meantime, the growth of the cells immobilized in the beads was measured from the 2 mL, 0.2 mol L −1 (filter sterilized pore size of the filter is 0.25 micrometers) sodium citrate cell extract of 2 AAC-beads [10].The samples were diluted with sterile normal saline before TTC assays.SC/MC culture standard calibration curve was prepared in a range from 0 to 3 × 10 9 cfu mL −1 (exponential growth phase) to correlate the OD 485nm tetrazolium formazan (TF) readings to BC (cfu mL −1 ).

Immobilized cell scanning electron microscopy (SEM)
To view the immobilized bacteria growth in AAC, SEM micrographs of AAC were taken with a JSM-5410LV scanning electron microscope (JEOL).The AAC immobilized bacteria were activated in NB using a shaker (PROTECH) set at 32 °C, 150 rpm/24h, according to the protocol at the Microscopy Unit of the Institute of Bioscience, UPM, Malaysia.The immobilized cell SEM samples were prepared by 'fixation' with 4% (v/v) glutaraldehyde, followed by 1% aqueous Osmium tetroxide.Then, the samples were dehydrated by sequential acetone gradients (35%/10 min; 50%/10 min; 75% overnight at 4 °C; 95/10 min; 100%/15 min, three times), and desiccation with a critical point dryer (HCP-2, Hitachi, Tokyo, Japan).After that, the samples were coated in an ion coater instrument (IB-3, Eiko, Japan) in an argon atmosphere.The micrographs were captured with SEM (JEOL).

GC-MS analysis of UEO residues
To perform the GC-MS analysis, the extraction of UEO from the spent medium was done with hexane.Following the centrifugation (5,000 rpm/20 min) of the 5 mL sample plus 5 mL hexane, the mixture was allowed to stand for phase separation, and the top phase was collected.The sample was then filtered through a 0.45 µm microbial filter pore size (PTFE, Sartorius) into 2 mL GC vials.GC samples that were not analysed immediately were kept in a refrigerator at 4 ± 1°C.For analysis by GC [19], the HCs-Cpd of n-C 12 -C 29 in the sample extract were separated using GC-2010, ZEBRON ZB-5ms column (30 M × 0.25 mm I.D × 0.25 µm film thickness) and detected with MS-detector.The GC conditions were as follows: injector temperature at 300°C; detector temperature at 280°C.The carrier gas (nitrogen) flow rate was 1.0 mL/ min, and the auto injected sample volume during analysis was 1 μL.The oven temperature was programmed in the range of 40°C-300°C, which allowed a temperature profile of 40°C held for 2 min; then ramped by 15°C/min to 300°C, and finally held for 10 min.

TTC test of activated immobilized cell gel beads
The viability of immobilized cells in the activated gel beads was examined using the modified TTC stain technique [18].The sample was washed 2-3 times in activated AAC gel beads in sterile normal saline.The beads were aseptically placed in 5 mL sterilized Eppendorf tubes, and then submerged with TTC stain.They were then incubated in a hot water bath (MEMMERT S/N: 820078) set at 32 °C/2h.The red bead color observed with naked eyes indicated a positive result.

Preparation of the adsorbent and soil water extract
The granular adsorbent was prepared as described above, without inoculum (AAC), and without the attapulgite and inoculum in the case of alginate-calcium carbonate (AC).Soil water extract was prepared following the partially modified method of soil extract agar [20].To prepare 1,500 mL of soil extract, 750 g of soil was weighed correctly and added in 2 L Scourge bottle containing magnet rod.Then, the distilled water (DW) was added to turn the total volume of scourge bottle content to around 1350 mL, and the content suspended, using a cold magnet mixer.After that, to sterilize the scourge bottle constituents, the bottle cup was placed rightly.Then the bottle was placed in autoclave, and steam sterilized for 1 hour at 121°C.Following, the removal of the scourge bottle from the autoclave and the cooling to the RT.The sterilized soil extract was poured in a filter setup (utilizing a large Buchner funnel, no. 5 Whatman filter paper, and air suction pump setup) and iltered into a Buchner flask.After, the filtration the final volume of the filtered extract was turned to a total volume of 1.5 liter with sterile distilled water, and kept in the fridge until further uses.

Batch sorption
The adsorption of UEO by AAC/AC gel beads was investigated by batch experiments.In each experiment, 20 mL of 200 mg/L NaN 3 soil water extract or hexane solvent containing 1% UEO was transferred into capped flasks (vol.50 mL).Then a number of AAC/AC gel beads (0, 4, 8, 12, 16 and 20) were added separately to each flask and shaken at 150 rpm, 32 °C/24 h.The 24-h batch was chosen here, as the time point to which the system was already in equilibrium (C e ).The zero gel bead solutions were taken as control.At 24 h, the samples in soil water extract or hexane were taken to measure the residual UEO (see above).

Adsorption linear kinetic models
The pseudo 1st-order kinetics model can be rewritten as a logarithmic equation in terms of the variables as defined in Eqs. ( 1) and ( 2) and rate constant ( k I ) as: Moreover, Salifu et al. [21] has graphically determined k I using the slope of the plot of ln( ) q q e t − versus t .Following the linear kinetic model, the intra-particle diffusion kinetic model was developed by Weber and Morris [22] as described in Eq. ( 4).The aim of the intra-particle diffusion is to determine the intra-particle diffusion rate constant ( K id ) measured in (mgg −1 h 1/2 ) and the constant C (mgg −1 ) which is proportional to the boundary layer thickness [23]: If intra-particle diffusion is involved in the adsorption process, the plot of q t vs t 1 2 / will be linear.At / , the diffusion rate is only due to intra-particle diffusion.In the case some other mechanisms are involved besides the intra-particle diffusion, then C ≠ 0 [24].In the experiments for the effects of initial UEO concentration on the adsorption of attapulgite, the initial concentrations of UEO (1, 2, 3 and 4%) were added separately into each Eppendorf tube containing 20 mL of 200 mg/L NaN 3 soil water extract and 20 AAC-gel beads.The sample was then shaken with a shaker set at 150 rpm and temperature 25 °C; at 12 h the remaining UEO in the sample was measured.

Adsorption kinetics of UEO
The adsorption of UEO in 20 mL soil water extract was studied at various initial UEO concentrations of 0-4% (0, 1, 2, 3, and 4%) in 50 mL Eppendorf tubes, each shaken at 150 rpm in an incubator shaker at 25 °C.In a batch kinetic adsorption experiment [25] (Shi et al. 2014), 20 gel beads (AAC) were added per tube as an adsorbent, and were taken separately over periods of 0 h, 2 h, 4 h, 6 h, 8 h, and 12 h.At the end of each batch point time, a 2 mL sample was taken into a 15 mL Eppendorf tube, while the UEO residue was extracted with 4 mL of hexane, and then measured as described above.The UEO uptake at the time points, q t (mgkg −1 ), was calculated by Eq. ( 2), and the data obtained were fitted to the models (Eqs.( 3) and ( 4)).

Adsorption capacity of AAC gel beads for UEO
In this study, two formulas of sodium alginate beads (AAC and AC) were tested in soil water extract and hexane solvent containing UEO. UEO removal by the sorbent materials (AAC/AC) was verified based on the various numbers of beads (0, 4, 8, 12, 16 and 20) added to the 20 mL of 200 mg/L NaN 3 soil water extract or hexane solvent containing 1% UEO (w/v) in capped flasks (50 mL).The adsorbent was prepared as discussed in Sec.3.6.

Statistical analysis
The collected data were analysed with SPSS software (version 17; SPSS Inc, Chicago, IL, USA) and one-way analysis of variance (ANOVA) for the % RE of UEO.When ANOVA detected significant (p < 0.05) differences, Duncan's multiple range test (DMRT) was used for mean comparisons [26].

Growth dynamics of free suspended Ochrobacterium intermedium, Bacillus paramycoides and their mixed consortia (BC) in shake flask system
The growth potentials of the individual and BC on UEO are shown in Figure 2.After an initial count of 3 × 10 7 cfu/mL on Day 0, the population of the individual culture O. intermedium increased exponentially to 27.5 ± 1.9 × 10 8 cfu/mL on Day 15 but declined steeply to 20.27 ± 1.6 × 10 8 cfu/mL on Day 21.B. paramycoides strain exhibited the slowest simultaneous growth pattern.Its highest value of 17.5 ± 0.51 × 10 8 cfu/mL was on Day 12 and significantly declined to 11.42 ± 0.76 × 10 8 cfu/mL on Day 24.Growth of the mixed culture followed almost the same growth patterns of O. intermedium in the first 6 days (0-6 days).Then, its cell counts continued rising slowly from Day 6 up to 28.57 ± 0.53 × 10 8 cfu/mL on Day 12, and decreased gradually from 27.09 ± 1.70 × 10 8 cfu/mL on Day 15 to finally 19.07 ± 0.68 × 10 8 cfu/mL on Day 24.In all graphs  the establishment periods of <3 days were represented as an increase from Day 0 to Day 3.

Degradation of UEO by SI: O. intermedium, SII: B. paramycoides and their mixed consortia (SI + SII)
In Figure 3, the decreased GC-chromatogram peak area patterns in the culture condition as compared to the control indicate the degradation of UEO. Figure 3(D) shows that in the mixed bacteria consortia, the decrease of the GC-chromatogram peak areas was mostly throughout the GC-MS analysis base line (blue arrows) with one particular area peaking (red arrow).

Removal efficiency of UEO by AAC/AC formulated gel beads
First, we studied the removal efficiency (RE) depending on the number of gel beads (sorbent) for the UEO in the polar and non-polar solvents.Soil water extract was chosen here as the polar solvent because it is considered as one of the important phases in the heterogenous soil system phases (i.e.solid, liquid and gas) in which the mass transfers occur, and in which the adsorptive efficiency of Alginate-Attapulgite-Calcium Carbonate bead sites can be well exposed, to remove UEO and characterized to predict their efficiency in the soil water environment of ionic and nonionic interactions.Hexane was chosen here as a homogenous system in which the UEO dissolves, and in which it is expected that most likely this system discloses the Alginate-Attapulgite-Calcium Carbonate nonionic properties in the removal of UEO.This system was chosen mainly to disclose the Alginate-Attapulgite-Calcium Carbonate beads' inert ionic properties to remove UEO.
The formulated gel bead (AAC/AC) removal efficiency was tested in the respective solvents.The AAC beads exhibited high UEO removal efficiency.They were able to adsorb more UEO in both solvent conditions (Figure 4a and 4b).
Figure 4(a) shows that in the polar solvent, when the number of gel beads increased from 4 to 20 granules, RE increased up to 83 ± 0.32%.The same behavior was also observed with AAC in the non-polar solvent (hexane) (Figure 4b), but with a lower percentage of RE (11.50 ± 0.52%) than that in the polar solvent (Figure 4a).In these observations, it is also important to mention that in the non-polar system (hexane), although the AAC's UEO RE percentage was better than the AC's, this sorbent exhibited high mean deviations among replicates (Figure 4b).

Effect of UEO concentration on the adsorption capacity of AAC gel beads in soil water extract
Figure 5(A) shows the adsorption capacity ( q t ) of AAC beads over 12 h calculated every two hours at initial UEO concentrations of 1, 2, 3, and 4% (g/L).The adsorption capacity increased as the UEO percentage concentration increased.A linear fitting curve was plotted for each concentration to determine the maximum adsorption capacity over 12 h.In Figure 5(B), it can be seen that q e increased linearly with increasing UEO concentration in the soil water extract.respectively.Table 2 summarizes the zero and first-order adsorption kinetics parameters of the UEO and the intra-particle diffusion kinetics model parameters of AAC, under the different concentrations of UEO (1 to 4%).When the UEO concentration was varied in the range from 1% to 3%, the UEO adsorption rate, K 1 (h −1 ), also increased in the range of 0.11 to 0.15 h −1 .At 4% UEO, it decreased to 0.13 h −1 as compared to 0.14 h −1 and 0.15 h −1 attained in 2% and 3% UEO soil water extracts, respectively (Adam et al. 2015).

Kinetics of UEO adsorption by AAC
However, as related to the conditions in Table 2, the 1st order kinetics results also show that the adsorption half-life, t 1/2 (h), of AAC decreased as the UEO concentrations varied in the range from 1% to 3% in the soil water extract (6.30, 4.95, and 4.62 h, respectively) and subsequently increased to 5.33 h as the UEO concentration reached 4%.In this study, the intra-particle diffusion kinetic model was also used to resolve the characteristics of intra-particle diffusion.

AAC diameter characterization
By using the water displacement method [17], the AAC bead diameter (Ø) was determined as approximately 0.45 cm (Figure 7A).It is also interesting to mention here that, after washing the activated AAC gel beads three times in sterile normal saline and their incubation     with TTC stain, the AAC immobilized gel beads revealed the spots at which the bacteria densely populated the beads and growing as indicated by increased dark red color spots inside the beads (Figure 7C).The water displacement method was used for the determination of AAC bead diameter (Ø = D b ) (Figure 7B and 7C), followed by TTC staining of 24 h grown AAC immobilized single (S: O. intermedium LMG 3301) bacterium and mixed (O.intermedium LMG 3301LMG 3301+ B. paramycoides MCCC1A04098) bacteria in NB.The control beads (Figure 7D) were without cells and incubated in NB.The dark color shown in Figure 7(C) suggests that the bacteria are densely populated at these spots.This observation is reported in this work and to the best of the authors' knowledge, it has not been mentioned prior to this work.Figure 8(A) and 8(B) shows the SEM micrographs of the immobilized cells [18].

Cell leakage (CL) from the AAC immobilized cell system
Figure 9 shows the time course of cell leakage, from the SC (O.intermedium LMG 3301) and MC (O.intermedium LMG 3301 strain + B. paramycoides MCCC1A04098) AAC-ICS.During the 0-24 days' interval, the cell leakage (CL) started within 3 days, and then continued, linearly increasing as the immobilized cell growth increased until Day 24.On Day 24, the SC and MC immobilized cell systems (ICS) in the medium enabled cell leakage counts of 27.97 ± 0.46 × 10 8 cfu mL −1 and 32.26 ± 1.86 × 10 8 cfu mL −1 , with the beads still remaining intact.

Discussion
In Figure 2, the growth of O. intermedium LMG 3301LMG 3301 + B. paramycoides MCCC1A04098, and their mixed culture in 1% UEO-EMSM, indicates most likely the degradative ability of these screened potential bacteria culture to utilize UEO as a carbon source.In Figure 2, the closeness of O. intermedium counts to those of bacteria consortia can be attributed to the dominance of O. intermedium in these mixed bacterial cultures.However, since all the pure and mixed cultures grew well on UEO-EMSM (Figure 2), as well as the degradation of UEO occurring (Figure 3) at the expense of those cell growth.As noted from the chromatogram peak areas in Figure 3(B)-(D), as explained above the decrease in peak areas as compared to the control chromatogram (Figure 3A) indicated the biodegradation of UEO.In Figure 3(D), the chromatogram peak area which mostly decreased to the base line confirms that most of the varieties of HCs-Cpds in UEO were fully decomposed with just a few of them remaining as can be inferred from the sharp peak shown in Figure 3(D) (red arrow).
However, because the O. intermedium and O. intermedium plus B. paramycoides formulas were relatively efficient in the utilization of UEO, these bacterial formulas were taken as potential bacteria models in this study of the characterization of Alginate-Attapulgite-Calcium carbonate as a matrix for the immobilized cell bioremedy formula to further study UEO biodegradation in other future studies.
The adsorptive methods for bioremediation of crude oil and diesel pollution have been studied previously [10,27].The novel characterization of formulated adsorptive composite carrier materials of AAC for the bioremediation of UEO pollution has not been reported, with the exception of the bioremediation of diesel [10].This report demonstrates for the first time the characterized adsorptive capacities of Alginate-Attapulgite-Calcium carbonate (AAC) gel for UEO removal from a soil water extract (polar-system model) and hexane (non-polar system model).In a previous study [10], as mentioned above, this composite matrix was applied in the bioremediation of diesel.
The present report shows the difference between the percentage of RE for the same adsorbent (AAC) in polar and non-polar solvents (Figure 4A and 4B).We observed that the adsorption sites on the AAC in hexane were not well exposed, compared to the soil water extract.We further explored the adsorption mechanisms involved in this process.As attapulgite behaves as an adsorbent and chemi-sorbent simultaneously in SWE systems, it is expected that the adsorptive capacity increases based on the study of Galan [28].The incorporation of attapulgite in the alginate blend can increase the sorption capacity for free UEO.Moreover, this increase can enhance the removal efficiencies of UEO in aqueous media via the cationic adsorption of the charged oil residues [29].This work emphasizes adsorptive biodegradation, therefore, the concentration of attapulgite was fixed at 0.75 (% w/v) to avoid the likely precipitation of attapulgite, which may be toxic to the immobilized cells in gel beads [10].Mousa et al. [30] found that clay is biocompatible in a formula when it remains stable without precipitation.In this study no precipitants were observed during AAC gel synthesis, indicating that most likely the non-toxicity may result from the use of AAC in the entrapment of bioagents.
On the other hand, when a low percentage of RE (Figure 4B) was observed, the increased standard deviations of the RE percentage may be attributed to the non-ionic interaction.This result suggests that the hydrophobic interaction is not the only factor contributing to the stability of binding and the increased adsorption of UEO to this hydrogel-based sorbent.In Figure 4(A), the increased UEO RE percentage in the soil water extract and the adsorption stability might be attributed to the enhanced ionic and non-ionic interactions.These interactions probably involved various ionic, physical forces (π-π interaction, van der Waals, etc.) and hydrophobic interactions [31].Alginate is an anionic polysaccharide hydrogel and the HCl treated AAC beads [10] contained cationic attapulgite [32].Therefore, the adsorption stability of AAC beads in SWE toward UEO might be mostly attributed to the combined ionic interaction resulting from alginate and attapulgite [33].
The characterized adsorptive affinity properties of AAC to UEO demonstrate that this material can be used in the development of efficient biocatalysts, which can effectively sorb and biodegrade hydrocarbon pollutants in a multiphase system e.g. in oil polluted water.The high-performance sorption would lead to increased mass transfer efficiency in the AAC bead system.In Figure 5(B), the linear UEO concentration-dependent adsorption with a constant AAC bead number demonstrates that the AAC-gel beads (adsorbent mass) are constant.Hence, the determined amount of active adsorption sites on the constant number of AAC-gel beads, as demonstrated by the concentration of UEO in soil water extract, shows that when UEO per unit volume of soil water extract increases.The ratio of the UEO-HC fractions to the available adsorption sites also increased and more UEO fractions in the soil water extract could be adsorbed on AAC beads, resulting in an increase in the q e .This result indicates that the 0-4% UEO (w/v) studied in these batch wise sorbent systems with a constant mass of adsorbent (No. of beads) were not in a range that had reached saturation.Hence, more adsorption sites were still available to engage more UEO when the UEO adsorptions were investigated until 4% UEO (w/v).
Figure 6 and Table 1 indicate that when the percentage of UEO is 4%, the adsorption rate capacity of AAC decreases.The reason behind the decrease in sorption capacity at 4% UEO can be explained by the accumulation of UEO on active sites which results in a decrease in the total surface area available for the attachment of UEO molecules.This result demonstrates that the maximum sorption rate capacity of AAC is at 3% UEO.The toxicity of oil contamination on the microbial community has been established to be at >3% oil [34].Consequently, the UEO concentration-dependent adsorption rate is good because it will reduce the toxicity of the dynamic UEO concentration gradient on the released cells in the medium and the immobilized cells in AAC beads at various concentrations of UEO in the soil water extract.The low adsorption rate (0.11 h −1 ) at 1% UEO seemed to be due to the low UEO concentration in the soil water extract (Sun & Yang, 2003) [35].
The decrease in the total available surface area for the attachment of UEO, as explained above, can also be demonstrated based on the adsorption half-life values.At a low concentration of UEO, the sorbent remains active before it equilibrates and becomes partially inert.For example, in 1% UEO soil water extract, the adsorption half-life of AAC was 6.30 h, while in 4% UEO soil water extract, AAC exhibited a half-life of 5.33 h.These differences in the adsorption half-life values of AAC can be useful in the dynamic control of UEO loads in AAC biocatalysts and in enhancing microbial cell stability.This enhancement allows a prolonged process operation of degradation.On the other hand, mass transfer is one of the limiting factors in developing effective immobilized cell systems [36].The intra-particle diffusion model at 1% UEO (Table 2) suggests that the only mechanism involved in mass transfer is the intra-particle diffusion.The reason for this mechanism might be the unclogged pores at the low UEO percentage, and the free flow of UEO because of the increased porosity of AAC beads.Wang et al. [10] found that the addition of fibrous clay mineral attapulgite (clay) and CaCO 3 in alginate-based blends increases the strength and forms many pore paths, making honey-comb-like structures in the AAC carrier.
In contrast, between 2-4% UEO concentration it seems that the in/out flow of UEO in beads is controlled by intra-particle diffusion and other unknown inter and/ or intra dependent properties of the AAC gel beads.Under these circumstances, the effect of UEO can be resolved by using multi-linearity correlations [37].The involvement of such attapulgite properties is caused by the presence of micropores and channels in attapulgite (or palygorskite) together with the fine particle size.The fibrous/ribbon like structures of palygorskite increase the surface area for the adsorbate contact and attachment [28].Nevertheless, although the biocompatibility of attapulgite without microorganisms was initially investigated during synthesis, because this work focused mainly on the development of a matrix for immobilized cell adsorptive bioremedy, the biocompatibility of AAC immobilized cells in the beads was further examined using SEM and TTC stain.The SEM micrographs and TTC positive stain technique demonstrate that this matrix can support excellent bacterial growth.
However, the dark spots (Figure 7C) in the TTC-stained AAC activated gel beads with immobilized cells indicate the heterogeneous mass flow from the outside toward the inside.This flow might be caused by a gradient of respiratory activity and nutrients throughout the immobilized biomass-phase confinements [38].This behavior could be attributed to the heterogeneously crosslinked calcium alginate networks during the entrapment of the bacterial cells [39].
A significant result can be seen in using the TTC stain technique that allows a quick, simple and novel economical approach for monitoring and imaging the viability of AAC immobilized-cell whole-bead system without causing cross sectioning.The result characterizes the AAC material as a novel matrix of supplementary attributes and biocompatibility for the formulas that will protect, provide a growth matrix and attain combined mixed effects for expediting the biodegradation rate of UEO.
In Figure 9, the linearity in the cell leakage without the breakage of beads is thought to be due to the slow release of growing cells from the beads into the media [10,40,41].Evidence in support of this is shown by the rise in growth of SIC/MIC in the beads associated with the increased cell leakage &have an advantage that will permit the combined effect of both free cell (FCS) and the ICS removal of UEO in the liquid and/or solid media (soil).These outcomes upgrade the AAC-ICS as a system of excellent mass transfers, to a system that allows a slow release of actively growing cells during bioremediation in the remediation by both FCS and adsorptive ICS.These overall observations suggest that the AAC matrix could efficiently support excellent bacterial growth with good physical strength and mass transfer attributes.It is suggested as having potential in designing prolonged bioremediation processes in liquid and/or solid media (soil) involving both FCS and ICS.

Conclusions
In this study, the properties of AAC in removing UEO to support cell growth were characterized.The results of the AAC bead UEO removal efficiency (% RE) showed that the UEO RE percentage was bead-number-dependent with elevating % RE (83 ± 0.32%) in soil water extract and hexane (11.50 ± 0.52%) associated with the highest number of AAC-granules.The SEM imaging and TTC staining assay showed that the AAC can support good bacterial growth by showing that this carrier can be applied in the formulation of granular adsorptive UEO bioremedies.

Figure 2 .
Figure 2. growth profiles of the individual (Si: O. intermedium; Sii: B. paramycoides), and mixed bacteria strains (Si + Sii) on enriched mineral salt medium (emSm) supplemented with 1% ueo (at ph 7.0, 32 °c temperature, and 150 rpm agitation rate).Data points represent the mean of three replicate flasks and error bars that represent standard deviation were removed for clarity.

Figure 6 (
Figure6(A) and 6(B) shows the analytical first-order kinetics graph of UEO adsorption by AAC, and its intra-particle diffusion graphical kinetics model,

Figure 6 .
Figure 6.First-order kinetics (a) fitted modeling graph of ueo adsorption and intra-particle diffusion; (B) fitted plots of ueo into aac.

Figure 5 .
Figure 5. Kinetics of ueo adsorption and removal at 12 h: (a) Zero-order kinetics of the amount of ueo adsorbed per unit mass of aac, (B) adsorption capacity (q e ) of ueo by aac beads at equilibrium 12 h.

Figure 7 .Figure 9 .
Figure 7. measurements of the diameter of aac beads (a, B, and D), and the spots on the beads where the bacteria are densely located and growing (c).

Table 1 .
number of samples and their sampling point locations in Selangor.

Table 2 .
model for fitted data kinetics parameters of aac-ueo adsorption studies.