Preliminary Analysis of Diesel-Degrading Bacteria Immobilized on Organic Carriers in Seawater

Liu Zhixiu; Xue Jianliang; Wu Yanan; Li Menglu; Sun Xiyu; Cui Hao; Cheng Lijie; Gao Yu; Xiao Xinfeng

(College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao 266590)

Preliminary Analysis of Diesel-Degrading Bacteria Immobilized on Organic Carriers in Seawater

Liu Zhixiu; Xue Jianliang; Wu Yanan; Li Menglu; Sun Xiyu; Cui Hao; Cheng Lijie; Gao Yu; Xiao Xinfeng

(College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao 266590)

Oil pollution in marine environment is becoming increasingly serious. Oil bioremediation by immobilization technology has been widely studied. However, the effect of bacteria immobilization was limited because of lack of nutrients. In this paper, the organic materials (corn straw, corn cob and corn leaf) were used as carriers. The diesel removal rate achieved by the immobilized bacteria was studied, and the effects of nutrients (e.g. nitrogen and phosphorus) released from organic carriers to the immobilized bacteria were analyzed. Test results indicated that a certain amount of nutrients was released from organic carriers. Additionally, the diesel removal rates achieved by different immobilized bacteria were all higher than those achieved by free bacteria. And, the diesel removal rates achieved by bacteria decreased in the following order: bacteria immobilized on corn straw (79%), bacteria immobilized on corn leaf (70%) and bacteria immobilized on corn cob (43%). These fndings indicated two aspects, viz.: the carriers porous structure and the nutrients, which were favorable to biodegradation. Finally, the changes in nitrogen and phosphorus contents that were released from different carriers during biodegradation were studied. The results showed that the rate of diesel biodegradation was fast in the initial phase because of the suffcient nutrients released from carriers. Meanwhile, in the fnal phase, the rate of diesel biodegradation was relatively slow because of few nutrients released from carriers.

oil pollution; microorganism immobilization; nutrition elements; marine environment

1 Introduction

With the rapid increase of petroleum demand, the environmental and ecological problems caused by oily wastewater formed in the course of oil exploitation and transportation have become increasingly serious. For example, the economic losses of marine fishery and tourism have been enormous, and can threaten the human health[1-2]. Therefore, much attention has been paid to clean up the pollution, especially the dispersed oil and dissolved oil, from the marine environments. Among the methods for remediation of oil pollution, the bioremediation method is more ideal than physical and chemical remediation methods, because of its good effect, cost-effectiveness and environmentally friendly nature. However, the effect of bioremediation is poor because the number of natural oil degradation bacteria in the polluted seawater is insignifcant. Some reports upon referring to the immobilization technology indicate that free bacteria are immobilized in a certain space area by physical or chemical means that can increase the biomass per unit volume and may maintain the bacteria activity[3-4]. Therefore, we considered that the first important factor is how to improve the amount of oil degradation bacteria and strengthen the bacteria activity[5-6].

However, to date, although the immobilization technology applied to marine environments is feasible, many key factors (e. g. nutrient) should be studied to optimize the remediation effect. Especially, many reports have focused on the development of synthetic carriers for immobilization. However, with the lack of enough nutrients, the degradation rate achieved by the immobilized bacteria also was not high. Chen[7]selectedpolyurethane-polyurea copolymer as the immobilization carrier, and the degrading efficiency was only 47.25% because of the inability to provide enough nutrients under a virtual marine condition. Thus, more reports have studied on how to solve the problem of insuffcient nutrients in synthetic carriers[8-9]. Wang[10]studied the oil-degrading bacteria immobilized into the expanded graphite with nitrogen element, and proved that the addition of nitrogen element could promote the growth of microorganisms and improve the degrading effciency. These researches all suggested that biodegrading efficiency of immobilized bacteria in synthetic carriers could be promoted by adding nutrient elements. However, He Liyuan[11]studied the differences of degrading efficiency between organic materials (rice straw, corn straw) and inorganic material (montmorillonite) serving as immobilized carriers, and found that the degrading efficiency of organic material was better than that of inorganic material used as the carrier. Furthermore, researchers also found that adding nutrient is another key factor to improve the degrading effciency[12-14].

Thus, to restore the oil-polluted marine environments, it is necessary to improve the degrading efficiency by increasing the amount of oil degradation bacteria and nutrients. In this paper, we selected organic materials (corn straw, corn cob and corn leaf) as carriers, studied biodegradation of diesel by means of free and immobilized diesel-degrading bacteria and analyzed the effects of nutrition elements (nitrogen and phosphorus) on bioremediation. The aim of the study is to propose a novel pathway of optimizing the immobilization technology in marine environments.

2 Experimental

2.1 Carrier materials

Three different organic materials (corn leaf, corn cob and corn straw) were selected as carriers in this study. Firstly, these materials were broken into similar pieces. Then, these pieces were immersed in distilled water for 5 h to 6 h to remove some sundry. Thirdly, these pieces were washed with alcohol and distilled water carefully two to three times. Fourthly, these pieces were dried at 105oC for 2 h. Afterwards, these pieces were used as the carriers to immobilize microorganisms (Figure 1).

Figure 1. Pictures of different carriers

2.2 Screening and identification of diesel-degrading bacteria

Many kinds of oil-degrading bacteria exist in natural seawater[15]. In this paper, the seawater samples were collected from the Tang Island Bay, Qingdao, Shandong Province, China. Firstly, a certain amount of seawater sample and 1 mL of sterile diesel were synchronously added into an autoclaved liquid mineral medium. Then, these samples were incubated at 30oC and under a rotary speed of 160 rpm for seven days. Afterwards, 10 mL of incubated sample was taken from the medium and was then added into the fresh medium to be subjected to the autoclaved reaction. The same steps were repeatedly operated until an adequate amount of petroleum degradation bacteria gathered. Then, the diluted bacteria liquid was coated on a plate, with the diesel fuel serving as the sole source of carbon with energy available to the developing colonies. After having been cultivated for five days, the colonies with different phenotypic characteristics were gathered and further purifed with the streak plate method. Different bacteria were isolated after repetition of this process for many times. The Tiangen bacterial genome DNA extraction kit was used to extract the genomic DNA from single strain, and the 16S rRNA genes were amplifed by polymerase chain reaction (PCR) using the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) primers.

The PCR products were sequenced in the Shanghai Sangon Biotechnology Company. We determined the species through sequencing results using the Basic Local Alignment Search Tool with the known 16S rRNA in GenBank.

2.3 Preparation of immobilized degrading bacteria

The immobilization technique was adopted from Gentili[16]. Up to 0.2 g of carriers were subsequently addedinto the flasks with 100 mL of immobilization medium and autoclaved at 121oC for 20 min. Then 10 mL of the prepared mixed diesel-degrading bacterial cultures were added into these flasks. Afterwards, these flasks were subject to incubation under a rotary speed of 150 rpm and at 30oC for five days. Finally, the immobilized dieseldegrading bacteria were obtained.

The immobilization medium was prepared by means of the following reagents (based on one liter of distilled water): 10 g of peptone, 4 g of beef extract and 5 g of NaCl. The pH of immobilization medium was adjusted at between 7.2—7.5.

2.4 Methods of analysis

2.4.1 Experimental design

The released quantities of nutrients into seawater were frst determined. The carriers were soaked in the artifcial seawater. The concentration of nitrogen and phosphorus was measured in the 2nd, 4th, 12thand 16thdays, subsequently.

A certain amount of different immobilized dieseldegrading bacteria and 1 mL of sterile diesel fuel were added into the fasks containing 100 mL of autoclaved artificial seawater. Then, all flasks were subjected to incubation under a rotary speed of 150 rpm and at 30oC in a rotary shaker. Afterwards, the total nitrogen (TN) content, total phosphorus (TP) content and residual diesel were measured in different time duration. The degradation effects of different immobilized bacteria and the changes in nitrogen and phosphorus contents were analyzed.

Meanwhile, the diesel removal ability of free-degrading bacteria was measured. The identifed average biomass of immobilized bacteria was 8.7×108colony forming units (cfu)/g. The average biomass of free bacteria was 8×108cfu/mL. To make the dosage of free bacteria comply with the amount of immobilized bacteria, the proportion of immobilized bacteria and free bacteria was set at 1 g per 1.088 mL. All groups were replicated in triplicate.

Artificial seawater was prepared using the following reagents (based on one liter of distilled water): 10 mL of diesel, 30 g of NaCl, 1 mL of CaCl2(0.01 mol/L), 1 mL of FeSO4(0.01 mol/L), 2 mL of MgSO4(0.03 mol/L). The pH of artifcial seawater was adjusted at between 7.2—7.5. The experiment used #0 diesel fuel. The number and relative content of homologues contained in #0 diesel are shown in Table 1[17].

Table 1 The composition of #0 diesel

2.4.2 Methods of analysis

(1) The analysis of TN in water was conducted according to the UV spectrophotometric method of alkaline potassium persulfate digestion[18].

(2) The analysis of TP in water was performed according to the ammonium molybdate spectrophotometric method[19].

(3) Determination of the residual diesel in artifcial seawater was performed using ultraviolet spectrophotometric method.

At first, 10 mL ofn-hexane were added to each flask containing the artificial seawater and the immobilized diesel-degrading bacteria. Then, the flask was shaken for 10 min. Afterwards, the liquid sample was placed in a separating funnel. Finally, the obtained extraction liquid was measured at 255 nm of wavelength. The concentration of residual diesel was calculated according to the following formula to obtain the diesel degradation rate.

in which,Y– the degradation effciency of diesel, %;

C0– the initial concentration of diesel, mL/L;

C1– the fnal concentration of diesel, mL/L .

3 Results and Discussion

3.1 Isolation and identification of diesel-degrading bacteria

Four pure bacteria were isolated from the seawater. According to the results of 16S rRNA sequence and the Basic Local Alignment Search, the isolated four bacteria were found in three different species, namely:Pseudomonassp.,Acinetobactersp.andRhodococcussp. The results are shown in Table 2.

Table 2 The strains of each sample (login)

All the three bacteria have already been reported in the process for utilization of petroleum hydrocarbons degradation[20-22]. However, there are significant differences in the chemical structure of target hydrocarbons.Acinetobactersp. isolated from soil can use long-chainn-paraffins as their carbon and energy source[23]. The effciency for removal of alkanes, aromatics, NSO (nitrogen, sulfur and oxygen-containing compounds) and asphaltenes byPseudomonassp.is equal to 48.9%, 41.2%, and 47.4%, respectively[24]. Quek[25]usedRhodococcussp. to biodegrade petroleum in marine environment, and free cells could biodegrade approximately 80% of the totaln-alkanes. Therefore, it is obvious that noticeable effect can be expected when the three bacteria work together in the course of degradation of petroleum hydrocarbons.

3.2 Amount of nitrogen and phosphorus released from different carriers

The amount of nitrogen and phosphorus in different immobilized carriers was released in artificial seawater (Figures 2 and 3). It can be seen from Figures 2 and 3 that a certain amount of nitrogen and phosphorus was released from different carriers. Upon taking the nitrogen and phosphorus released from corn leaf for example, the amount of nitrogen and phosphorus released can reach up to 18.8 mg/g and 0.224 mg/g in 16 days. Additionally, Figures 2 and 3 also indicate that the amount of nitrogen and phosphorus released was different between each carrier. Upon taking corn leaf and corn cob for example, the amount of nitrogen and phosphorus released from corn leaf was 16.28 mg/g and 0.1788 mg/g, respectively. And those released from corn cob were 8.432 mg/g and 0.0284 mg/g, respectively in 12 days. Obviously, the amount of nutrients released from corn leaf was greater than that released from corn cob. Moreover, the amount of released nitrogen decreased in the following order: corn leaf > corn straw > corn cob. The amount of released phosphorus decreased in the following order: corn straw > corn leaf > corn cob.

Figure 2 The amount of nitrogen released from different carriers

Figure 3 The amount of phosphorus released from different carriers

The factors influencing the release of nitrogen and phosphorus are different. The corn leaf, corn cob and corn straw contain different nutrient contents, and the water-soluble nutrient amount is different. The epidermalstructure of porosities, which will affect the water fowing in and out of the material, is different in each organic material. This structure also affects the rate of nutrients released into the water. The study will be unfolded in the follow-up research indicating that the amount of nitrogen and phosphorus released from corn leaf and corn straw would be signifcantly higher than that from corn cob.

3.3 Biodegradation of diesel by different dieseldegrading bacteria

The rate of diesel removed by different diesel-degrading bacteria showed an apparent difference as presented in Figure 4. At frst, the rates of diesel removed by different immobilized bacteria all were greater than that of free bacteria. For example, in 20 days, the rate of diesel removed by the immobilized diesel-degrading bacteria on corn straw reached up to 79%. Meanwhile, the rate of diesel removed by free bacteria was only 39%. Additionally, the diesel removal rates decreased in the following order: the immobilized bacteria on corn straw (79%) > the immobilized bacteria on corn leaf (70%) > the immobilized bacteria on corn cob (43%).

Figure 4 Biodegradation of diesel achieved by different diesel-degrading bacteria

According to Figures 2, 3 and 4, these results indicated two related aspects of interest, namely: the carriers’ more porous structure and the nutrients, which were favorable to biodegradation. Firstly, in the carriers, a significant amount of bacteria was supplied in a certain space full of porous structure. In contrast to free bacteria, the dieseldegrading bacteria can utilize nutrients released from these carriers. Moreover, the activity of these bacteria can be improved. Therefore, the diesel removal rate achieved by the immobilized bacteria was evidently better than that achieved by free bacteria.

Additionally, according to the concentration of TN and TP, the diesel removal rates achieved by the immobilized bacteria were obviously different between each other. In the initial phase, the nutrients released from carriers were high, and the speed of diesel biodegradation was fast. Moreover, in the final phase, the speed of diesel biodegradation almost slowed down. Some reports indicated that marine microbes can grow well at a nitrogen and phosphorus ratio of 16:1[26]. This study after analyzing TN and TP released from different carriers had disclosed that the ratio of TN and TP released from the corn straw and the corn leaf was 80:1. However, the ratio of TN and TP released from the corn cob was only 200:1. Meanwhile, the diesel removal rate achieved by bacteria immobilized on the corn cob was the lowest. Therefore, nutrient is the key factor of immobilization in order to improve the diesel biodegradation in marine environments.

Overall, the diesel removal rate achieved by the immobilized bacteria was evidently improved than that attained by free bacteria due to the porous structure and nutrient of carriers. Moreover, due to different amounts of nutrient released from different carriers, the diesel removal rates achieved by the immobilized bacteria were also different. This finding means that the phosphorus element is important for diesel-degrading bacteria.

3.4 Effects of nitrogen and phosphorus

In this paper, the nitrogen and phosphorus in artificial seawater were studied in the biodegradation reaction, with the results presented in Figures 5 and 6. As shown in Figures 5 and 6, the concentration of nitrogen element increased in seven days, and then slowly reduced. The concentration of phosphorus also increased in fve days. However, the phosphorus concentration obviously reduced after 10 days (especially, the phosphorus content changed during the biodegradation by the immobilized bacteria on corn straw and corn leaf used as carriers). This result was the same as that identified in the initial phase of the experiment, the amounts of nitrogen and phosphorus released from carriers were more. However, those nutrients used by immobilization bacteria wereless. In the late phase of experiments, the released amounts of nitrogen and phosphorus from carriers were less. Meanwhile those used by immobilization bacteria were more. Considering that the nutrients released from carriers were not suffcient, we suggested that suffcient nutrients (especially phosphorus) should be provided to enhance biodegradation in marine environments.

Figure 5 The change of nitrogen during the degradation process

Figure 6 The change of phosphorus during the degradation process

4 Conclusions

(1) A certain amount of nitrogen and phosphorus was released from different carriers. Obviously, the amount of nutrients released from the corn leaf was greater than that from the corn cob. Additionally, the amount of diesel removed by different diesel-degrading bacteria was evidently different. Moreover, the amount of diesel removed by different immobilized bacteria was always more than that removed by free bacteria. The diesel removal rate achieved by the bacteria decreased in the following order: the immobilized bacteria on corn straw (79%) > the immobilized bacteria on corn leaf (70%) > immobilized bacteria on corn cob (43%).

(2) The test results showed that in the initial phase, the speed of diesel biodegradation was rapid because of suffcient nutrients released from carriers. Meanwhile, in the fnal phase, the speed of diesel biodegradation almost slowed down because of less nutrients released from carriers.

Acknowledgments: This study was fnancially supported by the National Natural Science Foundation of China (Grant No. 51408347, 51474140), the Scientifc Research Fund Project of Introduced Talents (2014RCJJ015) and Young Teachers Taleuts Training Plan (BJRC20170502) of Shandong University of Science and Technology, and the Science and Technology Projects of Qingdao (Grant No. 15-9-1-58-jch and 15-9-1-32-jch).

[1] Chen Xiangfeng, Zang Hao, Wang Xia, et al. Metal-organic framework MIL-53(Al) as a solid-phase microextraction adsorbent for the determination of 16 polycyclic aromatic hydrocarbons in water samples by gas chromatographytandem mass spectrometry [J]. Analyst, 2012, 137(22): 5411-5419

[2] Zhang Wenrui, Tang Aidong, Xue Jianliang. Preparation of Cr-MnOx/cordierite and their properties for catalytic oxidation of 1,2-dichlorobenzene [J]. Spectroscopy and Spectral Analysis, 2016, 36(9): 3075-3082

[3] Stelting S A, Burns R G, Sunna A, et al. Survival in sterile soil and atrazine degradation ofpseudomonas sp.strain ADP immobilized on zeolite[J]. Bioremediation Journal, 2014, 18(4): 309-316

[4] Venkatsubramanian K. Immobilized Microbial Cells[M]. Washington: American Chemical Society, 1979

[5] Wang Xin, Wang Xuejiang, Liu Mian, et al. Adsorptionsynergic biodegradation of diesel oil in synthetic seawater by acclimated strains immobilized on multifunctionalmaterials[J]. Marine Pollution Bulletin, 2015, 92(1/2): 195-200

[6] El-Gendy, Nassar. Kinetic modeling of the bioremediation of diesel oil polluted seawater using pseudomonas aeruginosa NH1[J]. Energy Sources, Part A: Recovery, Utilization, and Environmental Effects, 2015, 37(11): 1147-1163

[7] Chen, C. C. The bioavailability of petroleum hydrocarbon contaminants in soil and seawater [D]. National Cheng Kung University, Taiwan, 2012

[8] Atlas R M, Bartha R. Degradation and mineralization of petroleum in sea water: limitation by nitrogen and phosphorus[J]. Biotechnology and Bioengineering, 1972, 14(3): 309-318

[9] Dua M, Singh A, Sethunathan N, et al. Biotechnology and bioremediation: successes and limitations [J]. Applied Microbiology and Biotechnology, 2002, 59(2-3): 143-152

[10] Wang Xin, Wang Xuejiang, Bu Yunjie, et al. Study on removing marine petroleum pollutants by floating immobilized microorganism[J]. Technology of Water Treatment, 2014, 40(09): 48-51 (in Chinese)

[11] He Liyuan, Guo Chunling, Dang Zhi. Different carrier fixed high efficient degradation bacteria removal in the study of oil water[C]. 2010 Academic Conference of Chinese Environmental Sciences Association, Shanghai China, 2010: 2775-2778 (in Chinese)

[12] Zhang Xiuxia, Qin LiJiao, Huang Congcong, et al. Study on the selection of immobilized carrier and its performance[J]. Chemical Industry and Engineering Progress, 2011, 30(12): 2781-2786 (in Chinese)

[13] Cao Xiaoqiang, Yan Bingqi, Wang Qian, et al. Adsorption of Cr(VI) from aqueous solutions on organic modified laponite[J]. Chemical Journal of Chinese Universities, 2017, 38(2): 173-181 (in Chinese)

[14] You Xiaofang, Xiao Xinfeng, Xue Jianliang, et al. Optimization and interactive effects of salinity, fertilizer and inoculums concentration on biodegradation in marine environment[J]. Journal of Engineering Research, 2017,5(1):23-33

[15] Uad I, Silva-Castro G A, Pozo C, et al. Biodegradative potential and characterization of bioemulsifers of marine bacteria isolated from samples of seawater, sediment and fuel extracted at 4000 m of depth (Prestige wreck) [J]. International Biodeterioration and Biodegradation, 2010, 64(6): 511-518

[16] Gentili A R, Cubitto M A, Ferrero M, et al. Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan fakes[J]. International Biodeterioration & Biodegradation, 2006, 57: 222-228

[17] Cai Zhiming, Zhang Junyong , Yang Kefeng, et al. Determination of composition of #0 diesel being sold at market by gas chromatography-mass spectrometry[J]. Journal of Tongji University, 2002,30(01):124-126 (in Chinese)

[18] HJ 636—2012: Water quality—Determination of total nitrogen—Alkaline potassium persulfate digestion UV spectrophotometric method[S]

[19] GB/T 11893—1989: Water quality—Determination of total phosphorus—Ammonium molybdate spectrophotometric method[S]

[20] Hanson K G, Nigam A, Kapadia M, et al. Bioremediation of Crude Oil Contamination withAcinetobacter sp. A3[J]. Current Microbiology, 1997, 35: 191-193

[21] Antai S P. Biodegradation of Bonny light crude oil byBacillus spandPseudomonas sp[J]. Waste Management, 1990, 10(1): 61-64

[22] Sharma S L, Pant A. Crude oil degradation by a marine actinomyceteRhodococcus sp[J]. Indian Journal of Marine Sciences, 2001, 30(3): 146-150

[23] Koma D, Hasumi F, Yamamoto E, et al. Biodegradation of long-chainn-paraffins from waste oil of car engine byAcinetobacter sp.[J]. Journal of Bioscience and Bioengineering, 2001, 91(1): 94-96

[24] Das K, Mukherjee A K. Crude petroleum-oil biodegradation effciency ofBacillus subtilisandPseudomonas aeruginosastrains isolated from a petroleum-oil contaminated soil from North-East India[J]. Bioresource Technology, 2007, 98: 1339-1345

[25] Quek E, Ting Y P, Tan H M. Rhodococcus sp. F92 immobilized on polyurethane foam shows ability to degrade various petroleum products[J]. Bioresource Technology, 2006, 97(1): 32-38

[26] Gundersen K, Heldal M, Norland S, et al. Elemental C, N, and P cell content of individual bacteria collected at the Bermuda Atlantic time-series study (BATS) site[J]. Limnology and Oceanography, 2002, 47(5): 1525-1530

date: 2016-12-15; Accepted date: 2017-03-20.

Xue Jianliang, E-mail: LL-1382@163. com; Gao Yu, E-mail: 13658675002@163.com; Xiao Xinfeng, xf.xiao@163.com.