Exploring the Degradation Potential of Halomonas Bacteria from Oil-contaminated Marine Environment

Wu Yanan; Liu Yingying; Xue Jianliang; Shi Ke; Gao Yu; Xiao Xiaolong

(1. College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao 266590; 2. Sinopec Petroleum Engineering Corporation, Dongying, Shandong, 257026)

Abstract: The degradation potential of a diesel-degrading bacteria (HDMP1) isolated from the oil-contaminated marine environment was explored systematically by analyzing the environmental conditions and synergistic action with other diesel-degrading bacteria. The result indicated that HDMP1 was conf irmed as a strain of Halomonas by the method of strain identification. And, HDMP1 showed good diesel degradation performance with a diesel degradation rate of up to 79.59% after 7 days. By analyzing the effect of environmental conditions on HDMP1, the best carbon and nitrogen sources were found to be lactose and peptone, respectively, at a pH value of 7.5 and a salinity of 4 g/(100 mL). Additionally, the synergistic effect of HDMP1 combined with other diesel-degrading bacteria was analyzed by orthogonal experimental design. The inocula of HDMP1, HDMP2, HDMP3 and HDMB3 were optimized, with the best results equating to 0.4%, 0.1%, 0.4% and 0.9% in 100 mL of MSM, respectively, while the degradation rate of diesel was identified to be 73.5% within 5 days in the presence of optimum inocula.

Key words: diesel-degrading bacteria; synergistic effect; marine environment

1 Introduction

At present, many studies focus on repairing oilcontaminated areas. Unexpected oil spill accidents often occur in the course of exploration, transporting and ref ining process of oil[1-4]. After the oil spill, there will be many chain reactions that will result in the death of a large number of organisms in the surrounding environment, and change in the community structure causing the destruction of the ecological balance[5]. Some technologies were studied and developed to control the pollution-affected area. Among the many available methods, bioremediation has been proven to be eco-friendly for the removal of oil from the oil-contaminated areas.

Diesel-degrading bacteria are significant for oil biodegradation in oil-contaminated areas. Some researchers have conf irmed that diesel-degrading bacteria are widely found in the natural environment. At present, many kinds of diesel-degrading bacteria have been reported for the effective remediation of oil-contaminated areas, such asHalomonas,BacillusandPseudomonas[6-8]. But, the biodegradability is still limited by environmental conditions such as nutrients, salinity, pH value, and even the presence of other bacteria[5,9]. However, because of the complex mixture of various components, different dieseldegrading bacteria show remarkably different degradation performance. The present study is aimed at enhancement in the degradation rate of diesel by construction of a complex bacteria system which involves symbiotic and synergistic effects of diesel-degrading bacteria[10]. The synergistic effect of different nutrient sources, pH value and salinity, and other diesel-degrading bacteria have a great impact on the diesel-degrading bacteria’s metabolism and the degradation rate of diesel. However, few reports have been working on these problems in the marine environment.

In order to systematically solve the above problems, an efficient diesel-degrading bacteriumHalomonaswas isolated from the oil-contaminated marine environment, and its degradation potential was explored by analyzing the environmental conditions such as nutrient, pH value and salinity. Additionally, degradation performance of the diesel-degrading bacteria combined with other diesel-degrading bacteria in the biodegradation process was analyzed. Through a series of experiments, the potential of the diesel-degrading bacteria was explored during bioremediation of oil-contaminated marine environment.

2 Experimental

2.1 Chemicals

All chemicals and reagents used in the present study were analytically pure reagents. Diesel, used as a target substance for biodegradation, was provided by the PetroChina Corporation (the 151st Station in Qingdao).

2.2 Medium and culture conditions

Mineral salt medium (MSM) contained: 0.6 g/L of Na2HPO4, 0.5 g/L of KH2PO4, 3 g/L of NH4NO3, 2 g/L of yeast extract, 30 g/L of NaCl, and the trace element composition consisted of: 1.11×10-3g/L of CaCl2; 3.04×10-3g/L of FeSO4; and 7.20×10-3g/L of MgSO4, with the pH value equating to 7.2―7.5.

Artificial seawater (AS) contained: 0.6 g/L of Na2HPO4, 0.5 g/L of KH2PO4, 30 g/L of NaCl, and the trace element composition consisted of: 1.11×10-3g/L of CaCl2; 3.04×10-3g/L of FeSO4; and 7.20×10-3g/L of MgSO4.

The condition of culture incubation: the culture was incubated at 30 °C, under a rotary speed of 140 r/min. All mediums were sterilized at 120±2 °C for 20 min before inoculation.

2.3 Isolation, enrichment and screening of bacteria

The samples were collected from a Ferry Terminal in Huangdao District, Qingdao, Shandong province, China. The diesel-degrading bacteria were obtained by isolation, which showed the highest degradation rate of diesel. Four diesel-degrading bacteria were isolated, and were labeled as HDMP1, HDMP2, HDMP3 and HDMB1, respectively.5 mL of diesel-degrading bacteria were put into a 500 mL flask containing 250 mL of MSM on a sterile operating platform, and then 5 mL of diesel was added. The flask was cultured for 3 days. 5 mL of inoculum from the flask were added into a new 500 mL flask containing 250 mL of MSM to repeat the above steps. In order to obtain high concentrations of diesel-degrading bacteria, these materials were subject to three successive cycles of enrichments in the Erlenmeyer f lasks.

To determine the degradation performance of each dieseldegrading bacterium, 1 mL of diesel-degrading bacteria was placed with 100 mL of MSM and blended with 1% (volume fraction) diesel, and the culture flasks were incubated in a shaker (140 r/min, 30 °C) for 5 days.

2.4 Determination of degradation rate of diesel

The diesel concentration was determined by measuring the absorbancy at 250 nm using ultraviolet spectrophotometry, and the method used to analyze the degradation rate of diesel has been described previously[11-12]. According to the equation, the degradation rate of diesel was calculated as follows:

whereYis the degradation rate of diesel (%), C0is the initial concentration of diesel (mL/L) and C1is the f inal concentration of diesel (mL/L).

2.5 Optimization of degradation performance of diesel-degrading bacteria

In order to provide higher degradation performance of the diesel-degrading bacteria, batch experiments and optimization of parameters were performed simultaneously. The parameters contained the carbon sources (glucose, sucrose, lactose, starch and urea), the nitrogen sources (potassium nitrate, ammonium nitrate, peptone, urea, ammonium chloride), the pH value (6.0―8.0), and the salinity (1―5 g/(100 mL)). The degradation rates of diesel were determined after 5 days.

2.6 Synergistic effect of HDMP1 combined with other diesel-degrading bacteria

The synergistic effect of HDMP1, HDMP2, HDMP3, and HDMB1 was studied by orthogonal test. Simultaneously, a complex bacteria system was obtained. The factors and levels of the orthogonal test are shown in Table 1. According to Table 1, different inoculums of dieseldegrading bacteria were inoculated into 100 mL of MSM and supplemented with 1% of diesel. The degradation rate was determined after 5 days.

Table 1 The factors and levels of orthogonal test

3 Results and Discussion

3.1 Screening of diesel-degrading bacteria

Firstly, to study the diesel bioavailability of dieseldegrading bacteria, the degradation rate of diesel was determined after 7 days. The performance of dieseldegrading bacteria for degrading diesel is shown in Figure 1, indicating that HDMP1 had the highest rate for degradation of diesel (79.59%). Based on the above experimental data analysis, HDMP1 was used as dominant species in the further experiments.

Additionally, based on the 16S rDNA gene sequences coupled with using the Gen Bank BLAST tool, the 16S rRNA gene sequences of HDMP1, HDMP2, HDMP3 and HDMB1 strains were automatically aligned to reference sequences of the generaHalomonas,Halomonas,Pseudomonas,andPlanomicrobiumobtained from the Gen Bank (https://blast.ncbi.nlm.nih.gov/Blast.cgi), respectively. A phylogenetic tree was constructed (Figure 2) based on the neighbor-joining method using the software MEGA version 7.0. The complete 16S rDNA gene sequence of the six strains existed in the GenBank database, and the accession number covered AY204638.1 (HDMP1), AB917466.1 (HDMP2), KJ735915.1 (HDMP3), and NR_042259.1 (HDMB1). AlthoughHalomonashas resulted in a significant degradation of oil[6], there are few related studies on the restoration of oil-contaminated seawater byHalomonas. The present study shows thatHalomonashas better degradation performance and can be applied to the microbial enhanced oil recovery (MEOR).Pseudomonasis a kind of common diesel-degrading bacteria, and the performance ofPseudomonasfor degrading dieselhas been studied[13-16].Planomicrobiumis considered as a kind of the biosurfactant-producing bacteria which is halotolerant at a moderate concentration of NaCl[17]. Based on the analysis of the test results, those highly efficient bacteria were used in the succeeding experiments.

Figure 1 The degradation rate of diesel-degrading bacteria

The scale bar indicates substitutions per nucleotide. Reference strain organisms are included and sequence accession numbers are given in parentheses. Bootstrap values from 1000 replicates.

Figure 2 Phylogenetic tree based on 16S rDNA gene sequence showing the phylogenetic position of HDMP1

3.2 The degradation performance optimization of HDMP1

Some researchers focus on how to promote the degradation rate of diesel by optimization. The present study was also aimed at enhancement in the degradation rate of diesel by selection and optimization of parameters. To determine the optimal parameters of HDMP1, the degradation efficiency of HDMP1 was evaluated with different combinations of carbon sources, nitrogen sources, pH value, and salinity conditions.

As shown in Figs. 3 and 4: (1) the sequence of the degradation rate of HDMP1 under different carbon sources was as follows: lactose (LA) ＞ starch (ST) ＞ sucrose (SU) ＞ urea (UR) ＞ glucose (GL) ＞ absence of carbon sources (WCS); (2) the sequence of the degradation rate of HDMP1 under different nitrogen sources was as follows: peptone (PE) ＞ ammonium nitrate (AN) ＞ ammonium chloride (AC) ＞ absence of nitrogen sources (WNS) ＞ urea (UR) ＞ potassium nitrate (PN). The degradation rate of diesel was about 80% when lactose was used as the carbon source. Uniformly, the degradation rate of diesel was about 80% when lactose was used as the nitrogen source.

Figure 3 The degradation efficiency of HDMP1 with different carbon sources

Figure 4 The degradation efficiency of HDMP1 with different nitrogen sources

The diesel-degrading bacteria need different nutrients, such as carbon sources, nitrogen sources, etc.[18-20]The carbon source is used mainly to provide the energy required for the bacteria’s vital activities in the growth and metabolism environment. Different carbon sources may affect the morphological structure of biofuels and the microbial communities of biof locs in recent researches[21]. In this study, the test result indicated that lactose promoted the degradability of HDMP1. Furthermore, peptone was found to be the best nitrogen source for diesel-degrading bacteria. Peptone is rich in organic nitrogen compounds, and also contains some vitamins and carbohydrates, which can provide the growth factors in the metabolic process[22-23].

Figure 5 Effect of different pH value on the degradation performance of HDMP1

Figure 6 Effect of different salinity on the degradation performance of HDMP1

As shown in Figure 5, the degradation rate of HDMP1 was determined at different pH value (6.0―8.0). The degradation rate of diesel was up to 52.82% when the pH value was 7.5. The degradation rate decreased sharply at a pH value of below 7. As shown in Figure 6, the best salinity value was found to be 4 g/(100 mL), and the degradation rate of diesel oil reached 64.52%. When the salinity was less than 4 g/(100 mL), the degradation rate of HDMP1 decreased with a decreasing salinity. When the salinity was greater than 4 g/(100 mL), the degradation rate of HDMP1 decreased with an increasing salinity. During the experiments, the optimum condition of carbon source and nitrogen source covered lactose and peptone, respectively, while the pH value and salinity were equal to 7.5 and 4 g/(100 mL), respectively. It could be concluded from Figure 7 that the degradation rate of HDMP1 reached about 90% after 7 days.

Figure 7 The degradation rate of HDMP1 to diesel

Many researchers have studiedthe growth and influence of different values of pH and salinity onHalomonas[24-25]. In this study, the similar effective ranges of pH value and salinity were also obtained.

3.3 Synergistic effect of HDMP1 and other dieseldegrading bacteria

The synergistic effect of HDMP1 combined with other diesel-degrading bacteria was further analyzed by orthogonal experiment. As shown in Table 2,ref lects the influence of various factors on the diesel degradation process. R characterizes the effect level of the impact factor on the degradation rate of diesel.

The degradation capacity of HDMB1 was lower than HDMP1, HDMP2 and HDMP3 (Table 3). The factors’ Sig. were greater than 0.05 (Table 3), which indicated that there was no significant difference between the inoculums of the corresponding factors.

Table 2 Visual analysis of orthogonal test

The biodegradability of oil was significantly promoted by a complex bacteria system[16,26]. In order to enhance the synergistic effect of the complex bacteria system, the quantity of inoculation was adopted to comply with the condition of maximumK. The inoculums of HDMP1, HDMP2, HDMP3, and HDMB3 were optimized and found to be 0.4%, 0.1%, 0.4%, and 0.9%, respectively. The effect curve is a graphic depiction of the visual analysis chart for an orthogonal experiment. As shown in

Figure 8 The effect curve of the diesel degradation rate

Figure 8, it revealed that the mean values of degradation rate of each factor were at level 1―4. The degradation rate of diesel was up to 73.5% within 5 days in the complex bacteria system.

Table 3 Test of between-subjects effects (Dependent: degradation rate)

4 Conclusions

In this study, four diesel-degrading bacteria were selected, which includedHalomonas,Halomonas,PseudomonasandPlanomicrobium,respectively.The degradability of diesel-degrading bacteria was evaluated by analyzing parameters and synergistic effect with those of other diesel-degrading bacteria, with the results shown below:

1) HDMP1 was conf irmed to be a strain ofHalomonasby the method of strain identified. And, the degradation rate of HDMP1 was up to 79.59% after 7 days.

2) The degradability of HDMP1 was explored by analyzing the influence of parameters including different carbon sources, nitrogen sources, pH value and salinity. The optimum condition of appropriate carbon source and nitrogen source covered lactose and peptone, respectively. The pH value and salinity were found to be 7.5 and 4 g/(100 mL), respectively.

3) The synergistic effect of HDMP1 combined with other diesel-degrading bacteria was analyzed. A complex bacteria system (HDMP1+HDMP2+HDMP3+HDMB1) was obtained by orthogonal experimental design. The inoculums of HDMP1, HDMP2, HDMP3, and HDMB3 were optimized, and the best results were 0.4%, 0.1%, 0.4%, and 0.9% (in 100 mL MSM), respectively.

Acknowledgements:This study was financially supported by the Scientific Research Fund Project of National Natural Science Foundation of China (Grant No. 51408347), the Open Research Fund Program of Shandong Provincial Key Laboratory of Oilfield Produced Water Treatment and Environmental Pollution Control (Sinopec Petroleum Engineering Corporation) (No.201801), the SDUST Young Teachers Teaching Talent Training Plan (BJRC20170502), the SDUST Graduate Tutor Guidance Ability Promotion Project (KDYC17023), and the Sinopec Petroleum Engineering Corporation Key Scientific and Technological Project (JP15036).