Preparation of Modified Enteromorpha-Immobilized Microbial Agent and Research on Diesel Removal Performance

Yang Yuping; Li Nana; Duan Weichao; An Chenye; Xue Jianliang;Jiang Qing; Cheng Dongle; Shen Chanchan

(1.College of Safety and Environmental Engineering, Shandong University of Science and Technology, Qingdao 266590;2.Qingdao Oasis Environmental & Safety Technology Co., Ltd., Qingdao 266555;3.Centre for Technology in Water and Wastewater, School of Civil and Environmental Engineering, University of Technology Sydney, Sydney, NWS 2007, Australia;4.College of City and Architecture Engineering, Zaozhuang University, Zaozhuang 277160)

Abstract: Offshore oil pollution has caused serious impacts on plants and aquatic organisms in the marine ecosystem.Bioremediation of oil pollution by immobilized bacteria has aroused wide attention due to the high degradation rate compared with free bacteria.The properties of the carrier for immobilization play an important role in the oil degradation efficiency.In our study, a marine oil degrading bacteria Sp8 (Shewanella algae) was selected from sea water, and enteromorpha was used as carrier material for the immobilization of Sp8.In order to increase the hydrophobicity, sodium dodecylbenzene sulfonate (SDBS) was used as modifier to modify the surface of enteromorpha by the dipping method.Sodium alginate was used as the embedding carrier, and anhydrous calcium chloride was used as the cross‐linking agent to prepare the SDBS‐E immobilized microbial agent by the embedding method.Compared with the degradation rate achieved by free bacteria (78.87 ± 8.29%), the diesel removal rate accomplished by SDBS‐E immobilized microbial agent increased to 90.39 ± 1.24%.The analysis of diesel removal mechanism showed that the diesel removal pathway mainly included surface adsorption, internal uptake, and biodegradation.The diesel removal efficiency relied on surface adsorption in the early stage, and then depended on biodegradation in the later stage.The removal of diesel by SDBS‐E immobilized microbial agent conformed to the quasi‐first‐order degradation kinetic model.The results of software‐MOE suggested that enteromorpha‐immobilized microbial agent adsorbed diesel mainly through hydrogen bonds formed with diesel components.This study can provide a research basis and idea for the practical application of immobilization technology to remove petroleum from seawater in the future.

Key words: bioremediation; immobilization; diesel degradation; enteromorpha; modification

1 Introduction

In recent decades, offshore oil drilling and transportation have caused severe offshore oil spills, such as the Exxon Valdez oil spill and the Deepwater Horizon oil spill in the Gulf of Mexico.These accidents have caused serious impacts on plants and aquatic organisms in the marine ecosystem[1‐2].Physical methods (such as mechanical skimming and oil boom methods) and chemical methods(such as the application of dispersants and adsorbents)have been demonstrated to be feasible for the treatment of oil spills, but these methods usually suffer from the high‐cost and secondary pollution[3‐4].As a clean technology,bioremediation technology has the advantages of environmental safety, high economic efficiency and absence of secondary pollution[5‐7].Bioremediation has aroused wide interest in the treatment of organic pollutants, especially marine oil pollution.It is common practice to use free bacteria in the bioremediation of oil‐polluted sea area.However, practices have found that due to the harsh marine environment and lack of nutrients,the degradation rate of crude oil by simple free bacteriawas extremely low.The technology of microorganism immobilization can effectively overcome the above‐mentioned problems.Chen, et al.conducted diesel degradation experiments by immobilized cells under freshwater and seawater conditions to evaluate total petroleum hydrocarbon (TPH) degradability, and the results showed that the degradation rate could reach more than 80% under a phosphorous‐sufficient condition[8].Li, et al.used artificial inorganic shells to individually immobilizeBacillus cereusS-1, a hydrophilic petroleum hydrocarbon degrading bacterium, to endow the cell surface with required characteristics at the single‐cell level, achieving a 60% degradation rate of crude[9].More relevant studies demonstrated that the immobilized petroleum‐degrading bacteria were more adaptable to the environment than free bacteria, and showed better petroleum degradation effects[10‐12].

As for oil spill pollution, the immobilized microbial agent can overcome the difficulties encountered in the bioremediation of marine petroleum pollution to a certain extent, and it has become a promising research direction in marine petroleum pollution emergency treatment[13‐15].At present, although there have been massive studies on the treatment of marine oil pollution by immobilized microorganisms, there are still many problems in practical applications, such as insufficient adsorption sites for immobilized carriers[8].The key of microorganisms immobilization lies in the choice of carrier and immobilization method[10].For the immobilized carrier,the basic characteristics should be available, featuring low cost, reliable physical properties, and harmlessness to microorganisms, which can provide sufficient living space for microorganisms[16].Natural organic materials, such as agricultural product waste, due to their wide sources and low prices, are increasingly used as carrier material in the immobilization of microorganisms[6].But these materials usually suffer from small specific surface area, insufficient adsorption sites, and poor mass transfer performance.Therefore, natural organic materials are usually modified before being used as carriers[17‐18].Modification methods mainly include the acid‐base modification, the thermal modification, and the modifier modification.Li, et al.found that the degradation rate of crude oil achieved by immobilizedBacillus cereus S-1modified by sodium monododecyl benzene sulfonate could increase by more than 10%[9].Xu, et al.used high‐temperature carbonized corn stalks as the adsorption carrier for immobilization and the results showed that the diesel removal rate was greatly improved as compared with ordinary corn stalks[15].

In Qingdao, Shandong Province, the green tide of enteromorpha erupts every summer, and a large number of enteromorpha gathers to the shore, blocking the navigation channel, destroying the marine ecosystem,and seriously endangering the development of coastal fisheries and tourism[19‐20].The surplus enteromorpha is usually used in composting, feed production, scientific research and food processing[20‐21], but a large amount of enteromorpha is still piled directly on the beach every year, resulting in resource waste and environmental pollution.Enteromorpha, as a cheap and easily available raw material, can be used as immobilization material, but it may be ineffective for oil treatment if it is directly used as immobilized carriers due to the superhydrophilicity of enteromorpha[19].Therefore, it is of great significance to explore a modification method to increase the hydrophobicity of enteromorpha for better degradation performance.

This study proposes a new type of immobilized microbial remediation technology by using modified enteromorpha as the immobilized material, and investigates the remediation performance of diesel.The scopes are:(1) to find a suitable modifier and use it to modify enteromorpha; (2) to study the diesel degradation rate and degradation mechanism by the modified enteromorpha‐immobilized microbial agent; and (3) to simulate the diesel removal process using software‐MOE.

2 Materials and Methods

2.1 Chemicals

Sea water was collected from the Tangdao Bay (Qingdao,China).Diesel was obtained from PetroChina (Qingdao 151th Station).Enteromorpha was collected from the Yellow Sea area.It was washed with tap water several times to wash off the sediment and other impurities, and then washed with distilled water for 2 to 3 times to wash off the excess salts, and then it was naturally dried, andfinally stored in bags.

The minimal salt medium (pH=7.2‒7.5) was prepared to contain: 30 g/L of NaCl, 0.5 g/L of KH2PO4, 0.6 g/L of Na2HPO4, 5.0 g/L of (NH4)2SO4, 1.0 mL/L of CaCl2·FeSO4·MgSO4(1:1:2), and 2.0 g/L of yeast extract.The inorganic salt medium (pH=7.0‒7.2) was prepared to contain: 5 g/L of NaCl, 1.0 g/L of (NH4)2SO4, 0.25 g/L of MgSO4·7H2O, 2.0 g/L of NaNO3, 10 g/L of K2HPO4·3H2O, and 4.0 g/L of KH2PO4.

Tris‐HCl solution (pH≈7.4) contained 0.1 mol/L HCl and 1.2114 g/L of tris(hydroxymethyl)aminomethane.

2.2 Modification of enteromorpha

Different modifiers, including sodium dodecyl benzene sulfonate (SDBS), sodium oleate (SO), andn‐octyl triethoxy silane (TOS) were used to modify enteromorpha.The enteromorpha was modified by the method of dipping.A certain quantity of enteromorpha was dipped in a modifier solution for 24 hours.Then the enteromorpha was dried in an oven, ground, and bagged in ziplocks for later usage.

2.3 Preparation of immobilized bacteria

10 mL of bacterial solution and 0.75 g of modified enteromorpha were mixed into 50 mL of 4% sodium alginate solution, stirred adequately, and transferred into 3% anhydrous calcium chloride solution with a dropper to form a spherical immobilized bacterial agent.The prepared immobilized bacterial agent was stored in a polyethylene bottle containing 3% of anhydrous calcium chloride solution, placed in a refrigerator at 4 °C, and was subjected to cross‐linking for more than 24 hours.The immobilized bacterial agents were rinsed with normal saline and stored in distilled water before experiment.

2.4 Experimental set-up

After enrichment, separation, and identification,a highly efficient marine oil degrading bacterium named Sp8 was selected from seawater as previously described[22].Sp8 was immobilized by the embedding method using modified enteromorpha as the adsorption carrier to prepare immobilized bacterial agent.All batch experiments were carried out in conical flasks, which contained a minimal salt medium with 1% of diesel and 1% of petroleum degrading bacteria.These conical flasks were placed in a thermostatic oscillator for cultivation under the conditions covering a temperature of 29 ℃ and a rotational speed of 130 r/min.All the experiments were conducted in triplicate.

2.5 Analysis and calculations

2.5.1 Removal rate measurement

After being extracted withn‐hexane, the remaining diesel in the minimal salt medium was examined through UV spectrophotometry.The diesel removal rate is calculated as follows:

whereR(%) is the degradation rate of diesel,c0(mL/L)is the initial concentration of diesel, andc1(mL/L) is the final concentration of diesel.

2.5.2 Surface adsorption of diesel by immobilized bacteria

After the immobilized petroleum‐degrading bacteria were cultured in a minimal salt medium containing 1%of diesel for a period of time, the liquid medium was discarded.The residual immobilized petroleum‐degrading bacteria was washed with an inorganic salt medium once,then washed withn‐hexane (20 mL each time) twice, and finally washed with inorganic salt medium twice.These supernatants were collected and mixed, and were diluted to a certain multiple times.Then the amount of diesel adsorbed on the surface of immobilized microbial agent was measured by an UV spectrophotometer.

2.5.3 Internal uptake of diesel by immobilized bacteria

After degradation, the immobilized petroleum‐degrading bacteria were collected by centrifugation and washed twice with the inorganic salt medium.The immobilized microbial agent was transferred to a test tube containing 10 mL of Tris‐HCl solution, and after being shaken for 2 minutes was then crushed with a crusher for 20 minutes.After the immobilized microbial agent was crushed, 5 mL ofn-hexane were poured into the above test tube for extraction.The absorbance of organic layer was determined via UV spectrophotometry and then the amount of diesel adsorbed on the surface was calculated according to the standard curve equation of diesel.

2.6 Degradation kinetics

The degradation rate of diesel by immobilized microbial agent was measured every 12 hours, and the quasi‐zero‐order, quasi‐first‐order, and quasi‐second‐order degradation kinetic equations were used for fitting.Quasi‐zero‐order degradation kinetic equation:

Quasi‐first‐order degradation kinetic equation:

Quasi‐second‐order degradation kinetic equation:

wherek,k1, andk2represent the reaction rate constants.

3 Results and Discussion

3.1 Screening and identification of marine oil degrading bacteria

In our study, an efficient marine oil degrading strain Sp8 was isolated from the seawater of Tangdao Bay (Qingdao, China)as previously described[22].It can be seen that the selected Sp8 has a short rod shape (Figure 1).The BLAST comparison of Sp8 in GenBank revealed that the length of Sp8 gene was 1482 bits, and the gene coverage rate was 100%.The phylogenetic tree based on the 16S rRNA gene sequence proved a close relationship between Sp8 and theShewanella algae strain BPRIST022 with a similarity of 99% (Figure 2).

Figure 1 The SEM image of the selected marine oil degrading bacteria Sp8

3.2 Screening of modifiers and preparation of immobilized bacteria

3.2.1 Preparation and characterization of immobilized bacteria

The immobilized bacterial agents were light green, semi‐transparent microspheres (Figure 3).The SEM image showed the internal skeleton structure of the immobilized beads (Figure 4), which could provide the possibility for the attachment of highly‐efficient petroleum‐degrading bacteria.

3.2.2 Screening of modifiers

Three different modifiers, SDBS, SO, and TOS, were used to modify enteromorpha, and these modified enteromorpha and unmodified enteromorpha were used as carriers for the immobilization of Sp8.The diesel removal rates under different conditions were investigated, with the results shown in Figure 5.The removal rate achieved by the immobilized Sp8 with unmodified enteromorpha(E+Sp8) prepared with adsorption method (85.57±1.47%)was higher than that of free Sp8 (78.87 ± 8.29%),indicating the immobilization treatment promoted the removal of diesel.Meanwhile, different modification of enteromorpha greatly affected the diesel removal performance.Specifically, the embedding method treated immobilized bacterial agent prepared with enteromorphamodified with SDBS‐E showed a highest removal rate of diesel, reaching 90.39 ± 1.24 %, while the immobilized bacterial agent with SDBS‐E prepared by the adsorption method showed a low diesel removal rate (only 13.79± 5.58%).The immobilized bacterial agents prepared with enteromorpha modified with sodium oleate (SO‐E) showed poor oil degradation performance, as the diesel removal rates were lower than 10%.Besides, the diesel removal rate of the immobilized Sp8 prepared with TOS modified enteromorpha (TOS‐E) was lower than that of the immobilized Sp8 with unmodified enteromorpha (E+Sp8).It can be seen from Figure 5 that the immobilized microbial agents prepared by the two methods had little difference in the removal rate of diesel except for SDBS‐E+Sp8.Upon considering the practicality and efficiency of the two immobilization methods (the adsorption method and the embedding method), it was believed that the embedding method was more suitable for the implementation due to the puffy morphological characteristics of enteromorpha itself.In order to investigate the biological tolerance of Sp8 to the modifier, the growth curves of Sp8 with different doses of the modifiers, including SDBS and TOS, were determined by OD600values.It can be seen that the addition of a small amount of modifier would not reduce the activity of microorganisms (Figure 6).Considering that SDBS was more economical than TOS, SDBS was selected as the final modifier for enteromorpha.Given that the SDBS‐E+Sp8 prepared by the embedding method showed better performance on the removal of diesel,the petroleum‐degrading bacteria immobilized by the embedding method was utilized in our study.

Figure 2 Phylogenetic tree based on 16S rRNA gene sequence

Figure 3 The photo of modified enteromorpha-immobilized microbial agent

Figure 4 SEM image of profile of the immobilized bacterial agent prepared by the embedding method (SDBS-E+Sp8)

Figure 5 The diesel removal rate of bacteria Sp 8 immobilized by modified enteromorpha and different methods

3.3 Study on diesel degradation performance of SDBS-E+Sp8

3.3.1 The diesel removal rate achieved by SDBSE+Sp8

The diesel removal rates achieved by Sp8 and enteromorpha under different treatments are shown in Figure 7.It can be seen from Figure 7 that enteromorpha itself could adsorb diesel (18.35 ± 11.22%), and after treatment with modifier SDBS (SDBS‐E), the diesel adsorption capacity was significantly improved to 42.77%± 3.47%.The diesel removal rate of free Sp8 was 78.87%± 8.29%, however, the diesel removal rate achieved by Sp8 immobilized by unmodified enteromorpha (E+Sp8)was not increased due to deteriorated mass transfer.But after treatment by the modifier SDBS (SDBS‐E+Sp8),the diesel removal rate was significantly improved to90.39% ± 1.24 % from the original level of 78.87% ±8.29 %, indicating that SDBS improved the mass transfer of immobilized microbial agents, making it easier for diesel to enter microbial agents for internal ingestion or utilization by microorganisms.

Figure 6 Biological tolerance of Sp8 to SDB) and TOS

Figure 7 The diesel removal rate achieved by SDBS-E immobilized microbial agent

3.3.2 Mechanism of diesel removal by SDBS-E+Sp8

The diesel degradation kinetic curves achieved by SDBS‐E+Sp8 and Sp8 at room temperature are shown in Figure 8.It can be found that the removal of diesel by SDBS‐E+Sp8 and Sp8 both followed the first‐order degradation kinetic model.The diesel degradation rate constant achieved by SDBS‐E+Sp8 was 0.01045 d, which was significantly higher than that achieved by Sp8 (0.00479 d), indicating that the immobilization by enteromorpha modified with SDBS could facilitate the degradation of diesel.This might be due the capacity of sodium alginate and enteromorpha for adsorption of diesel components.Moreover, several studies had demonstrated that carrier material could provide the bacteria with a more suitable environment to carry out degradation[23‐24].

Figure 8 The diesel degradation kinetics curves achieved by by Sp8 and SDBS-E+Sp8

According to our previous studies, the removal of diesel by immobilized microbial agent includes three pathways, viz.: surface adsorption, internal uptake, and biodegradation[25‐26].It can be seen from Figure 9 that the removal of diesel by SDBS‐E+Sp8 relies on surface adsorption in the early stage and by biodegradation in the later stage.At the beginning, more than 90% of the diesel was adsorbed on the surface of SDBS‐E+Sp8 and did not enter the inside of the immobilized bacterial agent.Subsequently, diesel molecules passed through the surface and entered the interior of immobilized bacterial agent.In the meantime, the diesel was gradually degraded by the petroleum‐degrading bacteria Sp8.On the ninth day,11.79% ± 1.58% of diesel was adsorbed on the surface,and 11.79% ± 3.31% of diesel remained in the interior of SDBS‐E+Sp8 via internal uptake, and the residual diesel(76.42% ± 6.44%) was efficiently biodegraded by Sp8.

Figure 9 The diesel removal dynamics of SDBS-E+Sp8

3.4 Study on mechanism of diesel degradation by SDBS-E+Sp8

3.4.1 Molecular simulation

To better simulate the adsorption and binding of diesel on the surface and the inside of SDBS‐E immobilized microbial agent, organic components, which were present during degradation, were analyzed.In this study,enteromorpha, sodium alginate, oxidordeuctase and phthalic acid, andn‐hexadecane that was found in diesel components were selected as the research objects.The structure of phthalic acid, andn-hexadecane contained in diesel was acquired from https://www.chemicalbook.com/ProductIndex.aspx, the structure of biomacromolecule was acquired from the RCSB protein database.The MOE software was used to analyze the diesel removal mechanism of SDBS‐E immobilized microbial agent from the perspective of molecular simulation.In the simulation process, different binding sites were tested for connection,and the binding site with the strongest bond energy was retained, with the results shown in Figure 10.

The simulation results of enteromorpha combining with phthalic acid andn‐hexadecane molecules are shown in Figure 10a and Figure 10b, respectively.Figure 10a shows that the enteromorpha and phthalic acid molecules were connected by hydrogen bonds, of which H‐don bonds account for 41.8%, 11.3%, and 36.7%,respectively, and H‐acc bonds account for 34.9%,45.5%, 94.5%, 23.3%, and 34%, respectively.The bondlength of several hydrogen bonds is 1.16 Å, 2.82 Å,2.65 Å, 2.67 Å, 2.51 Å, 2.52 Å, 2.38 Å, and 2.89 Å,respectively.A large pocket in enteromorpha provided an entry site, through which phthalic acid approached the active site.Enteromorpha formed relatively weaker bonds withn‐hexadecane (Figure 10b).Figure 10c shows that the sodium alginate and phthalic acid molecules were connected by three hydrogen bonds with a bond length of 2.68 Å, 2.72 Å, and 2.89 Å,respectively.Figure 10d shows that the oxidordeuctase and phthalic acid molecules were connected by H‐don bonds (with a bond length of 1.45 Å), H‐acc bonds (with a bond length of 2.73 Å), and H‐acc bonds (with a bond length of 2.39 Å).These results suggested that SDBS‐E+Sp8 adsorbed diesel mainly through hydrogen bonds formed with diesel components.

Figure 10 The results of molecular simulation by MOE

3.4.2 The mechanism of diesel degradation by SDBSE+Sp8

The mechanism of diesel removal by SDBS‐E+Sp8 is shown in Figure 11.At the initial stage, diesel components were adsorbed onto the surface of the SDBS‐E+Sp8,crossed the pores on the surface, and entered the inside of microbial agent.Then diesel components were dispersed by SDBS into emulsified oil droplets for bioutilization.A part of diesel was attached to the skeleton of microbial agent, and the remainder was used by enzymes in the bacterium.The diesel components were continuously gathered and eventually degraded into CnHn, CO2, and H2O.

Figure 11 Diagram of the mechanism of diesel removal by SDBS-E+Sp8

4 Conclusions

In this paper, a highly efficient marine oil‐degrading bacteria was immobilized on modified enteromorpha.The selected modifier was SDBS, and the immobilized microbial agent (SDBS‐E+Sp8) was prepared by the embedding method.Based on our study results, we conclude the following:

(1) The immobilization of petroleum degrading bacteria Sp8 with modified enteromorpha could enhance the diesel removal rate.The diesel removal rate by the immobilized Sp8 with SDBS modified enteromorpha (SDBS‐E+Sp8)had been significantly improved to 90.39% ± 1.24 % from the original level of 78.87% ± 8.29 % of free bacteria.

(2) The mechanism of diesel removal by immobilized bacteria (SDBS‐E+Sp8) included three parts, viz.: surface adsorption, internal uptake, and biodegradation.The diesel removal process relied on surface adsorption in the early stage and biodegradation in the later stage.

(3) The degradation of diesel by SDBS‐E immobilized microbial agent conforms to the first-order degradation kinetic model.

(4) The results of MOE suggested that SDBS‐E+Sp8 adsorbed diesel mainly through hydrogen bonds formed with diesel components.The diesel components were continuously gathered by enteromorpha‐immobilizedmicrobial agent and were eventually degraded by enzyme(like oxidordeuctase) of bacteria into CnHn, CO2, and H2O.

Acknowledgements:This study was funded by the scientific research fund project of National Natural Science Foundation of China (Grant No.52070123), the Natural Science Foundation of Shandong Province (grant numbers ZR2020ME224,ZR2019PB031), the Project of Shandong Province Higher Educational Young Innovative Talent Introduction and Cultivation Team [Wastewater treatment and resource innovation team].