Hydrocarbon Degradation and Bacterial Community Responses During Remediation of Sediment Artificially Contaminated with Heavy Oil
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Biocontrol Science, 2017, Vol. 22, No. 4, 187－203 Original Hydrocarbon Degradation and Bacterial Community Responses During Remediation of Sediment Artificially Contaminated with Heavy Oil SHARON N. NUÑAL1, SHEILA MAE S. SANTANDER-DE LEON2, WEI HONGYI3, ADEL AMER REGAL4, TAKESHI YOSHIKAWA3, SUGURU OKUNISHI3＊, 3 AND HIROTO MAEDA 1Institute of Fish Processing Technology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao, 5023 Iloilo, Philippines 2Institute of Marine Fisheries and Oceanology, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao, 5023 Iloilo, Philippines 3Education and Research Center for Marine Resources and Environment, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan 4National Institute of Oceanography and Fisheries, P.O. Box 182, Suez, Egypt Received 13 February, 2017/Accepted 23 June, 2017 Natural biodegradation of heavy oil in the marine environment can be accelerated by the addi- tion of nutrients or seeding of pre-selected microorganisms. In this study, a microcosm experi- ment was conducted to investigate the effects of inorganic nutrient supplementation（ biostimula- tion） and bacterial consortium amendment（ bioaugmentation） on the natural degradative processes of artificially contaminated sediment. Our results revealed that the addition of nutrients had greater effect on remediation than the addition of bacterial cells. Supplementation of inor- ganic nutrients promoted and sustained the growth of oil-degrading and heterotrophic bacteria throughout the experimental period. Highest reduction in the total petroleum hydrocarbons, and of their components, n-alkanes, polycylic aromatic hydrocarbons（ PAHs） and alkyl PAHs, were obtained in the biostimulated microcosms. Changes in the bacterial community were monitored by the PCR-DGGE（ polymerase chain reaction-denaturing gradient gel electrophoresis） method targeting the 16S rDNA gene. Results revealed different responses of the bacterial community to the addition of heavy oil and remediation agents. Shifts in the bacterial communities in the seawater were more dynamic than in the sediment. Results of this study showed that addition of remediation agents significantly enhanced the natural biodegradation of heavy oil in a sediment- seawater microcosm trial. Key words ： Microcosms / Hydrocarbon degradation / Bacterial community / Bioremediation. INTRODUCTION column while some settle at the bottom through the process of sedimentation. Once the oil is interspersed When oil is spilled in the sea, it undergoes changes in in the sediment at the bottom, it can penetrate beyond form through the passing of time brought about by the surface, making its removal difficult and can thus wave action, sunlight and inherent microbial community persist for a longer time（ Amadi et al., 1994; Colombo （Mackay and McAuliffe, 1988; Kingston, 2002）. Some et al., 2005; Reddy et al., 2002）. Natural catalytic components of the spilled oil disperse into the water processes by the microbial community in the seawater and sediment can, however, break down the hydrocar- ＊Corresponding author. Tel & Fax: +81-99-286-4133, E-mail : bons in the oil which can be completely mineralized into okunish（i a）fish.kagoshima-u.ac.jp CO2 and water（ Harayama et al., 1999）. In case of a 188 S. N. NUÑAL ET AL. tanker accident in the sea, the large oil influx causes an individually added. In the present study, the effective- imbalance in the carbon-nitrogen ratio which retards the ness of the bacterial consortium in degrading heavy oil growth of bacteria and subsequent utilization of the in the sediment, which presents more challenges in terms carbon sources（ Yakimov et al., 2007）. Furthermore, of accessibility to the oil as a substrate and to oxygen, the nutrients are also quickly assimilated by microorgan- was investigated. Furthermore, another bioremediation isms, thus depleting the nutrient reserves. This limitation approach, which is the addition of inorganic N and P in nutrient sources and dissolved oxygen, the toxicity of sources, was also explored. Temporal degradation of a high concentration of hydrocarbons and by-products the hydrocarbon components of heavy oil and growth of degradation to the bacterial community, and low of oil-degraders and other heterotrophic bacteria were numbers of the principal oil degraders in the sediment also measured. The effects of these remediation and seawater, can affect the rate and effectiveness of approaches to the natural succession of microbial natural biodegradation（ Boopathy, 2000）. communities were also determined by PCR-DGGE. Although natural biological remediation of an oil- Banding patterns were analyzed quantitatively by using polluted site happens without intervention, several tech- nonmetric multidimensional scaling（ NMDS） to describe niques aimed at enhancing the degradative capacities relationships of the microbial community in the different of the indigenous populations such as supplementation microcosms. Prominent bands were also excised and of nutrients or oxygen（ biostimulation） and addition of sequenced to determine their taxonomy. Generally this microbial cultures（ bioaugmentation） may be applied study is aimed at a better understanding of the degra- （Bento et al., 2005; Liang et al., 2009; McKew et al., dative processes and related shifts in the bacterial 2007; Simons et al., 2012; Thavasi et al., 2011; Tyagi et community structure, which can be helpful in the devel- al., 2010）. These human interventions are known to opment of more rational bioremediation strategies. impact the natural processes occurring within the different bacterial communities in the marine environment（ Tyagi MATERIALS AND METHODS et al., 2010; Vogel, 1996）. Thus, prior to the on site appli- cation of these bioremediation approaches, it is impor- Collection of seawater and sediment samples tant to determine microbial responses both at the meta- Sediment samples were collected from 2 locations in bolic and genetic levels. Yamagawa Bay, Kagoshima Japan（ Location 1: 31° The major bottleneck in studying microbial communi- 12'44. 48"N, 130°38'15.57"E; Location 2: 31°12'34.66"N ties is the fact that many environmental bacteria cannot 130°39'24.28"E） and were mixed in equal proportions. yet be cultured by conventional laboratory techniques Initial soil quality indices such as moisture（ gravimetric （Briones and Raskin, 2003; Ranjard et al., 2000）. Thus method）, grain size（ sieve analysis）, pH, Redox, total several advanced molecular methods have emerged to organic Carbon and total organic nitrogen（ CHNS provide more powerful tools to study bacterial commu- analyzer FLASH EA112, Thermo Fisher Scientific, nity dynamics by culture-independent techniques Massachusetts, USA）, and total organic Phosphorus （Pushpanathan et al., 2014; Yoshida et al., 2006）. One （Concentrated H2SO4 and dilute base extraction method; of the most widely employed DNA fingerprinting tech- Bowman, 1989） were measured. Results of the physico- niques for analyzing phylogenetic diversity is PCR-DGGE chemical characterization are presented in Table 1. （Muyzer et al., 1993）. PCR-DGGE allows profiling of the bacterial community through the banding patterns Preparation of bacterial consortium by which its diversity can be analyzed quantitatively. The bacterial consortium with an established oil- Furthermore, the intensity of bands provides a rough degrading capacity was prepared as we have previously estimate of the relative abundance of each species, and described elsewhere（ Nuñal et al., 2014）. The consor- the nucleotide sequencing of the excised bands tium is composed of strains related to: Pseudomonas provides a phylogenetic and taxonomic interpretation of aeruginosa, Marinobacter mobilis, Gaetbulibacter sp. the obtained amplicons. and Halomonas sp.（ DDBJ Acc. Nos. AB649102, In our previous study, we described the isolation, AB649110, AB649114, AB649101 respectively）. identification and characterization of bacterial strains with potential use in the bioremediation of oil pollution Microcosms Set-up （Nuñal et al., 2014）. From these strains a bacterial The sediment mixture was equilibrated at 26℃ for consortium was formulated and evaluated for its oil- seven days prior to the microcosm assembly. The equili- degrading capacities in an in vitro seawater experiment. brated sediments were then placed in 100 mL Erlenmeyer Results showed significant increase in the degradation flasks covered with perforated paper caps to allow of heavy oil in all treatments receiving the bacterial aerobic conditions. For each treatment, nine flasks were consortium compared to when the bacterial strains are prepared. Each flask contained 50 g sediment and 10 REMEDIATION OF OIL CONTAMINATED SEDIMENT 189 TABLE 1．Physico-chemical characteristics of the sediment eluents were combined, spiked with an internal stan- used in the bioremediation microcosms. dard（ pyrene-d10） and concentrated until 0.2 mL with Variables Values nitrogen stream. Moisture content（ %） 41.82% ± 0.88 Measurements of n-alkanes, polycylic aromatic hydro- carbons（ PAHs） and alkylated PAHs in the extracted Grain size（ sand, %） 81.9 ± 2.6 residual oil were conducted using a gas chromatograph Grain size（ silt/clay, %） 18.1 ± 2.6 （Agilent 6890; Agilent Technologies, USA） equipped pH 8.0 with a mass spectrometer（ Agilent 5973 MSD）. One Redox potentia（l mV） 105 ± 7.5 microliter of the sample was injected by an Agilent 7683B Total organic carbon（ %） 1.72 automatic sampler into