Journal of Water and Environment Technology, Vol.12, No.4, 2014 Biodegradation Potential of Organically Enriched Sediments under Sulfate- and Iron-Reducing Conditions as Revealed by the 16S rRNA Deep Sequencing

Tomoyuki HORI*, Makoto KIMURA*, Tomo AOYAGI*, Ronald R. NAVARRO*, Atsushi OGATA*, Akiyoshi SAKODA**, Yoko KATAYAMA***, Mitsuru TAKASAKI****

*Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan **Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan ***Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan ****Department of Food and Environmental Sciences, Faculty of Science and Engineering, Ishinomaki Senshu University, 1 Shinmito, Minamisakai, Ishinomaki, Miyagi 986-8580, Japan

ABSTRACT Organically enriched sediment has been found in water environments. The tsunami originating from the Great East Japan Earthquake in 2011 deposited large amount of sediment, thus providing evidence about its huge accumulation in coastal marine areas possibly due to human activities such as fish culture and marine product processing of industries. Here, degradation potential of organically enriched sediment deposited on a coastal site at Higashi-Matsushima, Miyagi, Japan was investigated under both sulfate- and iron-reducing conditions. Sediment slurry was prepared by mixing the sediment with artificial seawater. The effects of supplementation with sulfate and lepidocrocite (a crystalline Fe[III] oxide) on the structure and activity of the slurry microorganisms were examined by the combined physicochemical analyses and 16S rRNA deep sequencing. The sediment slurry was incubated for 5 days, during which the concentrations of TOC, sulfate, and ferrous iron remained at constant levels and the TG-DTA patterns did not change. The composition of dominant members of the microbial communities was stable, although the rare microbial populations slightly changed. The result in this study revealed that the organically enriched sediment was resistant to biodegradation under the sulfate- and iron-reducing conditions.

Keywords: Great East Japan Earthquake in 2011, microbial communities, organically enriched sediment, 16S rRNA deep sequencing

INTRODUCTION Organically enriched sediment has been found frequently on the sea floor below fish farms and/or close to coastal industrial areas (Kunihiro et al., 2011). The tsunami originating from the Great East Japan Earthquake in 2011 deposited a huge amount of sediment on the Pacific Coast in Tohoku district, Japan. This provided initial information that the organically enriched sediment has accumulated in the coastal marine areas. This may be attributed to human activities such as the fish culture and marine product processing of surrounding industries in the decades after the fish farming has been developed intensively (FAO, 1992). The environmental burden of the deposited sediment might pose serious problems. However, little is known about the detailed characteristics of the deposited sediment.

Address correspondence to Tomoyuki Hori, Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Email: [email protected] Received November 29, 2013, Accepted January 24, 2014. - 357 - Journal of Water and Environment Technology, Vol.12, No.4, 2014

The generative mechanism of organically enriched sediments at the bottom of the sea has been reported (Pearson and Rosenberg, 1978; Holmer and Kristensen, 1992); firstly excessive loading of organic matter on the sea-bottom sediment caused the depletion of dissolved oxygen that is used for degradation by aerobic microorganisms, and thereafter the organic matter recalcitrant to anaerobic decomposition accumulated as organically enriched sediments. There have been investigations focusing on aerobic degradation of organically enriched sediments (Wada et al., 2008; Kunihiro et al., 2011), yet the biodegradation potential especially under anaerobic conditions remains to be clarified. In-depth knowledge of the anaerobic degradation of organically enriched sediments is of importance to gain the future prospects for the on-site bioremediation.

Recently, the development of next-generation DNA sequencers has enabled researchers to obtain large-scale gene information directly from environmental genomic DNA. Among them, the 16S rRNA gene deep sequencing has provided precise insight into the composition of microbial communities including the population of rare microorganisms (Caporaso et al., 2011). Combining the deep sequencing with RNA-based examinations, the diversity and dynamics of the metabolically active microorganisms can be detected with a high sensitivity, thereby allowing the estimation of biological activities involved in the carbon and energy cycles in natural environments.

The reduction of sulfate and Fe(III) is the major electron sink for substrate oxidation under anaerobic conditions. On the sea floor sediment, sulfate is an important electron acceptor that is the driving force for the biotic carbon fluxes (Falkowski et al., 2008). Crystalline Fe(III) oxides such as lepidocrocite are one of the most abundant ferric iron minerals in sedimentary environments (Cornell and Schwertmann, 2003). In this study, we focused on the organically enriched sediment deposited by the tsunami originating from the Great East Japan Earthquake on a coastal site at Higashi-Matsushima, Miyagi, Japan. In order to clarify the biodegradation potential under both the sulfate- and iron-reducing conditions, the effects of supplementation with sulfate and lepidocrocite on the structure and activity of the sediment slurry microorganisms were investigated by a combination of physicochemical analyses and 16S rRNA deep sequencing.

MATERIALS AND METHODS Sampling site and proximate organic analysis of sediment Organically enriched sediment deposited on land by the tsunami originating from the Great East Japan Earthquake was collected in September 2011. The sampling site was located on a costal site at Higashi-Matsushima, Miyagi, Japan (38° 25′ N, 141° 14′ E). The collected samples were stored at 4°C prior to experimental use. The proximate analysis of organic components of the sediment was conducted in duplicate by gravimetric measurements with sequential extraction using different organic solvents and mineral acids (Watanabe et al., 1993). First, the lipid fraction was determined by Soxlet extraction of 1 g of the freeze-dried sediment using a benzene-ethanol (1:1) solution. The soluble polysaccharide was then extracted from the residue by distilled water under reflux. The hemicellulose fraction was determined after hydrolysis with 0.65 M HCl under reflux for 5 h. The cellulose fraction was obtained after further hydrolysis with 1.5 M H2SO4 at room temperature for 2.5 h and then with 0.42 M H2SO4 under reflux. Finally, lignin and ash contents were determined by heating the

- 358 - Journal of Water and Environment Technology, Vol.12, No.4, 2014 remaining residue at 600°C in a muffle furnace for 2 h. The percentage of each organic constituent was obtained from the decrease in mass of the freeze-dried samples after each extraction step.

Anaerobic incubation of sediment slurry Sediment slurry was prepared by mixing the sediment with artificial seawater (Daigo; Nihon Pharmaceutical, Tokyo, Japan) at a ratio of 1:3. Aliquots (20mL) of the homogenized slurry were placed anaerobically in 50-mL serum vials, which were then sealed with butyl rubber septa. The samples were pre-incubated in the dark at 25°C for more than 30 days in order to activate the slurry microorganisms. After pre-incubation, the headspace of each sample vial was flushed with N2. Two treatments were conducted: (i) addition of 20 mM sulfate as electron acceptor, and (ii) addition of 20 mM Fe(III) (in the form of lepidocrocite) as electron acceptor. Both treatments were run in triplicate with static incubation for 5 days at 25°C. No electron donor was supplied, that is, the indigenous organic reductants were solely utilized during incubation. Samples of the headspace gas, slurry water, and the solid-state sediment were removed at day 0, 2, and 5 from each vial of each set of the two treatments done in triplicate. Total CO2 in the headspace gases were analyzed by a gas chromatograph (GC-14B; Shimadzu, Kyoto, Japan) equipped with a packed column (ShinCarbon ST; Shinwa, Kyoto, Japan). Total organic carbon (TOC) was determined by the non-purgeable organic carbon (NPOC) method with a total organic carbon analyzer (TOC-L; Shimadzu, Kyoto, Japan) (Choi and Ng, 2008). In brief, the slurry waters were acidified to below pH 2 using HCl, sparged by N2 gas, and then analyzed 3 to 4 times by the TOC machine to produce a reliable data. The concentration of volatile fatty acids (VFAs) from the slurry waters was measured by a high-pressure liquid chromatograph (Alliance e26951, Waters, Tokyo, Japan) equipped with an RSpak KC-811 column (Shodex, Tokyo, Japan) and a photodiode array (2998, Waters, Tokyo, Japan). Ferrous iron was determined as described previously (Braunschweig et al., 2012). Briefly, 1 g of the sediment slurry samples was extracted for 5 days using 6 M HCl, and the extracted Fe(II) was determined using the ferrozine method. The sulfate concentration was determined by an ion chromatograph (DX-500; Dionex, Tokyo, Japan) equipped with an IonPac AS11 column (Dionex, Tokyo, Japan) and an ED40 electrochemical detector (Dionex, Tokyo, Japan). The thermogravimetry-differential thermal analysis (TG-DTA) pattern of the dried sediment was determined by using a simultaneous differential thermogravimetric analyzer (DTG-60, Shimadzu, Kyoto, Japan). The solid-state sediment samples were stored at –80°C for subsequent molecular analyses.

Extraction of total nucleic acids and the subsequent polymerase chain reaction (PCR) or reverse transcription (RT)-PCR Sediment samples were prepared from each vial of each set of the two treatments done in triplicate. Nucleic acids were extracted from the sediment samples using a direct lysis protocol involving bead beating (Hori et al., 2007). Total RNA and DNA were purified by digestion with DNase (RQ1; Promega) and RNase (Type II-A; Sigma-Aldrich, Tokyo, Japan), respectively. The mixture of the nucleic acids prepared from the triplicate samples was used as a template for PCR amplification. Polymerase chain reaction was performed with a high-fidelity DNA polymerase (Q5; NEB, Tokyo, Japan). Reverse transcription-PCR was carried out using a one-step RT-PCR system (Access Quick; Promega, Tokyo, Japan). The primer set 515f/806r was used to amplify the 16S

- 359 - Journal of Water and Environment Technology, Vol.12, No.4, 2014 rRNA genes and transcripts (Caporaso et al., 2012). Both the primers were modified to contain an illumina adapter region for deep sequencing with a MiSeq sequencer (Illumina, Tokyo, Japan), and the reverse primers were encoded with 6-bp barcodes for multiplex sequencing (Caporaso et al., 2012). The thermal profile of PCR was as follows: an initial denaturation at 98°C for 90 s, and then 25 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, and a final extension step at 72°C for 2 min. For RT-PCR of the 16S rRNA, reverse transcription was performed at 48°C for 45 min, while the thermal profile of the subsequent PCR consisted of a denaturation at 94°C for 3 min, and then 30 cycles, each including 30 s at 94°C, 45 s at 52°C, and 90 s at 72°C, which was followed by a final extension of 5 min at 72°C. The absence of the DNA contamination was confirmed by RT-PCR without reverse transcriptase.

Deep sequencing of the 16S rRNA genes and transcripts The PCR and RT-PCR products were purified firstly with AMPure XP kit (Beckman Coulter, Tokyo, Japan) and were subjected to the second purification with QIAquick gel extraction kit (QIAGEN, Tokyo, Japan) according to the manufacture’s instructions. The concentration of the purified DNA was determined spectrophotometrically with Quanti-iT PicoGreen dsDNA reagent and kit (Invitrogen, Tokyo, Japan) and a VersaFluor fluorometer (BIO-RAD, Tokyo, Japan). The copy number of the 16S rRNA gene fragments was measured by quantitative PCR with a GoTaq qPCR master mix (Promega, Tokyo, Japan) and a real-time PCR detection system (MyIQ2; BIO-RAD, Tokyo, Japan). Thereafter, an appropriate amount of the 16S rRNA gene segments (i.e., the barcode-encoded DNA libraries) was subjected to paired-end sequencing with a 300-cycle MiSeq reagent kit (Illumina, Tokyo, Japan) and a MiSeq sequencer (Illumina, Tokyo, Japan). Following the sequencing, the paired-end sequences with the scores of > Q30 were joined with the software ea-utils-1.1.2-301. Using the software Mothur version 1.31.2 (Schloss et al., 2009), the obtained sequences of the 16S rRNA genes and transcripts were aligned automatically, and therein the chimeric structures were detected and removed. More than 2,500 reads from each library were characterized phylogenetically using the software package QIIME version 1.6.0 (Caporaso et al., 2010).

RESULTS AND DISCUSSION Physicochemical parameters during the sediment slurry incubation From the proximate organic analysis, the deposited sediment consisted of 1.8% (average value of duplicate data) of lipid, 19.7% of soluble polysaccharide, 30.0% of insoluble polysaccharide (hemicelluloses), 12.3% of cellulose, 4.3% of lignin, and the remainder of ash, indicating that the sediment was organically enriched. Anoxic pre-incubated sediment slurry was supplemented with sulfate and lepidocrocite. The anaerobic incubation of the sediment slurry was implemented for 5 days that was enough to observe anaerobic respiration processes such as the sulfate and iron(III) reduction (Lueders and Friedrich, 2002; Hori et al., 2010). Carbon dioxide was produced immediately at the beginning of the incubation in both the sulfate and Fe(III) treatments (Fig. 1A and 1B), which might be caused by the degassing and establishment of the CO2-bicarbonate equilibrium. The CO2 concentration in the sulfate treatment remained at steady levels of 20.2 – 20.4 mM throughout the incubation, while that in the Fe(III)

- 360 - Journal of Water and Environment Technology, Vol.12, No.4, 2014 treatment decreased gradually from 21.4 mM at day 2 to 17.4 mM at day 5. The formation of siderite (FeCO3) in the Fe(III) treatment was considered to be a reason for the decrease in the CO2 concentration. The sulfate concentrations at day 0 under the sulfate and Fe(III) treatments were 34.2 mM and 15.4 mM, respectively (Fig. 1E and 1F), indicating that sulfate was originally present in the organically enriched sediment. The sulfate concentration in both treatments was nearly constant during the incubation; though a slightly decreasing trend at a rate of 0.27 – 0.38 mM/day was apparent. Ferrous iron, the product of the ferric iron reduction, did not increase in the sulfate and Fe(III) treatments, suggesting the almost non-occurrence of Fe(III) reduction (Fig. 1G and 1H). In both treatments, TOC was kept at approximately constant levels of 17.0 – 21.4 mg/L (Fig. 1C and 1D) and any VFAs were not detectable. These indicate that the main organic matter present in the sediment slurry were neither VFAs nor organic reductants coupled with the reduction of sulfate and lepidocrocite.

Further physicochemical characterization of the sediment samples in the two treatments was carried out using the TG-DTA analysis (Fig. 2). Significant difference was not observed in the TG-DTA patterns at the beginning (Fig. 2A) and at the end (Fig. 2B and 2C) of the incubation, which suggests that the chemical components of the sediment slurries were not changed by two treatments. All of the DTA patterns showed two endothermic peaks at around 80°C and 450°C. The former peak might be attributed to the dehydration process, and the latter one possibly resulted from the heat degradation of macromolecular compounds originally present in the organically enriched sediment. The residue of the TG fraction after the TG-DTA analysis is considered to be inorganic substances including silica and metal compounds. Note that the apparent residual inorganic fraction reflected by the TG-DTA graphs does not match the data obtained from the proximate organic analysis. This discrepancy may be due to the extraction of some metal compounds by the acid solutions, thus possibly leading to the overestimation of hemicelluloses and cellulose components during proximate analysis.

25 AB25 CD 20 20 15 15 mol/vial) μ

( 10 10 2

5 (mg/L) TOC 5 CO 0 0

40 EF2.5 GH 2.0 30 1.5

20 (mM) 1.0 2+ 10 Fe 0.5 Sulfate (mM) 0 0 025 025 025 025 Time (days)

Fig. 1 - Changes in physicochemical parameters during the anoxic incubation of the sediment slurries in treatments with sulfate (panels A, C, E, G), and Fe(III) (panels B, D, F, H); concentrations of CO2 (A, B), TOC (C, D), sulfate (E, F), and ferrous iron (G, H). The error bars represent the standard deviations of three replications.

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A B C

24 100

22 20 TG (mg) TG DTA (µV) DTA 20 −60

18 −140 0 200 400 600 0 200 400 600 0 200 400 600 Temperature (°C) Fig. 2 - Representative TG-DTA patterns of the sediment samples at day 0 (A), at day 5 in the sulfate treatment (B), and at day 5 in the Fe(III) treatment (C). The black lines indicate the TG curves, and gray lines indicate DTA curves.

Microbial community with and without metabolic activity in the sediment slurry The whole structure of microbial communities including the rare microbial populations with and without metabolic activity was phylogenetically characterized using deep sequencing of the 16S rRNA genes and transcripts (Fig. 3); (i) D0 (n = 25,152) and R0 (n = 2,556) libraries represented the DNA and RNA, respectively, at day 0, (ii) in the sulfate treatment, DS2 (n = 19,775) and DS5 (n = 21,813) libraries represented the DNAs at day 2 and 5, respectively, and RS2 (n = 5,760) and RS5 (n = 8,905) libraries represented the RNAs at day 2 and 5, respectively, (iii) in the Fe(III) treatment, DF2 (n = 19,584) and DF5 (n = 22,309) libraries represented the DNAs at day 2 and 5, respectively, and RF2 (n = 6,290) and RF5 (n = 11,579) libraries represented the RNAs at day 2 and 5, respectively. The composition of the major microbial populations was quite similar among the DNA libraries (Fig. 3A); the members affiliated with the class Deltaproteobacteria were the most predominant, followed by those affiliated with the phyla Chloroflexi and Bacteroidetes. The metabolically active microbial populations were also mostly stable among the RNA libraries (Fig. 3B), but differed considerably from the members detected in the DNA libraries. Almost all of the metabolically active microorganisms (i.e., > 70% of the total) were identified as belonging to the class Deltaproteobacteria as revealed by the composition of the RNA libraries. With a special focus on Deltaproteobacteria, its components were classified into family (a lower taxon) (Fig. 4A and 4B). The microbial members in RS5 and RF5 libraries with the top 5 highest relative abundance or increasing rate compared to day 0 were shown in Table 1. The composition of the deltaproteobacterial community in the RNA libraries was significantly distinct from that found in the DNA libraries, strongly suggesting that the selective metabolic activation of the specific microbial populations affiliated within the Deltaproteobacteria. In all RNA libraries, the members within the family Desulfobulbaceae that has been known as sulfate reducers was dominant, accounting for about half of the total. Other than the Desulfobulbaceae, the microbial members with high abundances in the RNA libraries were related to the known sulfate-reducing bacteria. It is tempting to speculate that, even though the decrease in sulfate in the two treatments was not obvious, sulfate reduction proceeded albeit weakly. Holmer and Kristensen (1992) reported the anaerobic degradation of organically enriched sediments by sulfate-reducing bacteria, coincided with the production of high levels of hydrogen sulfide. Additionally, the microbial members with high increasing rates in the RNA libraries were different between the sulfate and Fe(III) treatments (Table 1). However,

- 362 - Journal of Water and Environment Technology, Vol.12, No.4, 2014 these members represented low transcription levels (i.e., < 0.22% of the total), which indicate that their contribution to the carbon fluxes in the sediment slurries might be small.

AB 25,152 19,775 21,813 19,584 22,309 2,556 5,760 8,905 6,290 11,579 100 Gammaproteobacteria

80 Deltaproteobacteria 60

40

20 Relative abundance (%) abundance Relative Chloroflexi 0 D0 DS2 DS5 DF2 DF5 R0 RS2 RS5 RF2 RF5

Fig. 3 - Comparison of the 16S rRNA genes (A) and transcripts (B) obtained from day 0 (D0, R0) and from days 2 and 5 in the sulfate treatment (DS2, DS5, RS2, RS5) and from these days in the Fe(III) treatment (DF2, DF5, RF2, RF5). Relative proportions of the 16S rRNA gene sequences were affiliated phylogenetically. The numbers above the bars indicate the total numbers of reads sequenced in this study.

AB 100 Desulfobacteraceae

80 Pelobacteraceae Desulfuromonadaceae

60 other Desulfuromonadales

40 Desulfobulbaceae

20 Relative abundance(%)

0 D0 DS2 DS5 DF2 DF5 R0 RS2 RS5 RF2 RF5

Entotheonellaceae Entotheonellales (order) Syntrophorhabdaceae Syntrophobacteraceae Syntrophaceae Desulfobacteraceae Other Syntrophobacterales (family) Other Syntrophobacterales (order) JTB36 Sva0485 (order) Spirobacillales (order) Other SAR324 Other PB19 Other NKB15 NB1‐i MND4 JTB38 Other NB1‐j Other NB1‐j (order) Polyangiaceae OM27 Nannocystaceae Myxococcaceae Haliangiaceae Cystobacterineae Cystobacteraceae Myxococcales (order) Other Myxococcales MIZ46 (order) MBNT15 (order) IndB3‐24 (order) GW‐28 (order) GMD14H09 (order) FAC87 (order) Pelobacteraceae Geobacteraceae Desulfuromonadaceae Other Desulfuromonadales 2 Other Desulfuromonadales 1 Other Desulfurellales Desulfovibrionaceae Desulfomicrobiaceae Other Desulfovibrionales Nitrospinaceae Desulfobulbaceae Other Desulfobacterales 2 Other Desulfobacterales 1 DTB120 (order) Bdellovibrionaceae Bacteriovoracaceae Bdellovibrionales (order) BPC076 AF420338 Other Deltaproteobacteria Other Fig. 4 - Relative proportions of the 16S rRNA genes (A) and transcripts (B) that identified by family within the class Deltaproteobacteria.

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Table 1 - Relative abundances and increasing rates of the microbial members with the top 5 highest abundance and increasing rate in RS5 and RF5 libraries. Increasing rate Relative Taxon: Family (or higher affilication) Phylum or Class compared to abundance (%) day 0 (times) The RS5 library Members with the highest abundance Desulfobulbaceae Deltaproteobacteria 45.57 0.97 Pelobacteraceae Deltaproteobacteria 7.82 1.02 Desulfobacteraceae Deltaproteobacteria 6.09 1.33 Other Desulfuromonadales (order) Deltaproteobacteria 4.16 1.19 Desulfuromonadaceae Deltaproteobacteria 4.07 0.98

Members with the highest increasing rate Geobacteraceae Deltaproteobacteria 0.22 4.92 Acidobacteria (class) Acidobacteria 0.05 4.92 GMD14H09 (order) Deltaproteobacteria 0.05 4.92 Desulfobacterales (order) Deltaproteobacteria 0.04 3.99 Other Alphaproteobacteria Alphaproteobacteria 0.16 3.61

The RF5 library Members with the highest abundance Desulfobulbaceae Deltaproteobacteria 45.47 0.96 Pelobacteraceae Deltaproteobacteria 8.1 1.06 Desulfobacteraceae Deltaproteobacteria 6.62 1.45 Other Deltaproteobacteria Deltaproteobacteria 3.58 1.17 Desulfuromonadaceae Deltaproteobacteria 3.54 0.85

Members with the highest increasing rate Halomonadaceae Gammaproteobacteria 0.04 4.15 Other Acidobacteria (phylum) Acidobacteria 0.04 3.91 Desulfobacterales (order) Deltaproteobacteria 0.04 3.91 Brachyspirales (order) Spirochaetes 0.04 3.91 Planctomycetaceae Planctomycetes 0.03 3.42

CONCLUSIONS In this study, we investigated the effects of sulfate and lepidocrocite supplementation on the structure and activity of the slurry microorganisms using the combined physicochemical analyses and deep sequencing based on the 16S rRNA genes and transcripts. Although the physicochemical parameters (TOC, sulfate, and ferrous iron) and the TG-DTA pattern of the sediment slurries did not change during the anoxic incubation, the data from 16S rRNA deep sequencing demonstrated that the known sulfate-reducing bacteria were metabolically active. We hypothesize that sulfate reduction occurred slightly under both sulfate and Fe(III) treatments. The metabolic activity of sulfate reducers was detected by the highly sensitive genetic approach but apparently this was not involved in the carbon fluxes in the sediment slurries, since the sulfate and TOC concentrations were rather stable during the incubation for 5 days. The results in this study proved that the organically enriched sediment was resistant to biodegradation under sulfate- and iron-reducing conditions.

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REFERENCES Braunschweig J., Bosch J., Heister K., Kuebeck C. and Meckenstock R. U. (2012) Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology. J. Microbiol. Methods, 89(1), 41-48. Caporaso J. G., Kuczynski J., Stombaugh J., Bittinger K., Bushman F. D., Costello E. K., Fierer N., Peña A. G., Goodrich J. K., Gordon J. I., Huttley G. A., Kelley S. T., Knights D., Koenig J. E., Ley R. E., Lozupone C. A., McDonald D., Muegge B. D., Pirrung M., Reeder J., Sevinsky J. R., Turnbaugh P. J., Walters W. A., Widmann J., Yatsunenko T., Zaneveld J. and Knight R. (2010) QIIME allows analysis of high-throughput community sequencing data. Nat. Methods, 7(5), 335-336. Caporaso J. G., Lauber C. L., Walters W. A., Berg-Lyons D., Huntley J., Fierer N., Owens S. M., Betley J., Fraser L., Bauer M., Gormley N., Gilbert J. A., Smith G. and Knight R. (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J., 6(8), 1621-1624. Caporaso J. G., Lauber C. L., Walters W. A., Berg-Lyons D., Lozupone C. A., Turnbaugh P. J., Fierer N. and Knight R. (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. USA., 108(suppl 1), 4516-4522. Choi J. H. and Ng H. Y. (2008) Effect of membrane type and material on performance of a submerged membrane bioreactor. Chemosphere, 71(5), 853-859. Cornell R. M. and Schwertmann U. (2003) The iron oxides: Structure, properties, reactions, occurrences and uses. Wiley-VCH., Erlagsgesellschaft, Weinheim. FAO (1992) Aquaculture production, 1984-1990. FAO Fisheries Circular 815, 206. Rome, Italy. Falkowski P. G., Fenchel T. and Delong E. F. (2008) The microbial engines that drive Earth's biogeochemical cycles. Science, 320(5879), 1034-1039. Holmer M. and Kristensen E. (1992) Impact of marine fish cage farming on metabolism and sulphate reduction of under-lying sediments. Mar. Ecol. Prog. Ser., 80, 191-201. Hori T., Noll M., Igarashi Y., Friedrich M. W. and Conrad R. (2007) Identification of acetate-assimilating microorganisms under methanogenic conditions in anoxic rice field soil by comparative stable isotope probing of RNA. Appl. Environ. Microbiol., 73(1), 101-109. Hori T., Müller A., Igarashi Y., Conrad R. and Friedrich M. W. (2010) Identification of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing. ISME J., 4(2), 267-278. Kunihiro T., Takasu H., Miyazaki T., Uramoto Y., Kinoshita K., Yodnarasri S., Hama D., Wada M., Kogure K., Ohwada K. and Tsutsumi H. (2011) Increase in Alphaproteobacteria in association with a polychaete, Capitella sp. I, in the organically enriched sediment. ISME J., 5(11), 1818-1831. Lueders T. and Friedrich M. W. (2002) Effects of amendment with ferrihydrite and gypsum on the structure and activity of methanogenic populations in rice field soil. Appl. Environ. Microbiol., 68(5), 2484-2494. Pearson T. H. and Rosenberg R. (1978) Macrobenthic succession in relation to organic enrichment and pollution of the marine environment. Oceanogr. Mar. Biol. Annu. Rev., 16, 229-311. Schloss P. D., Westcott S. L., Ryabin T., Hall J. R., Hartmann M., Hollister E. B., Lesniewski R. A., Oakley B. B., Parks D. H., Robinson C. J., Sahl J. W., Stres B., Thallinger G. G., Van Horn D. J. and Weber C. F. (2009) Introducing mothur:

- 365 - Journal of Water and Environment Technology, Vol.12, No.4, 2014

open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol., 75(23), 7537-7541. Wada M., Zhang D., Do H. K., Nishimura M., Tsutsumi H. and Kogure K. (2008) Co-inoculation of Capitella sp. I with its synergistic bacteria enhances degradation of organic matter in organically enriched sediment below fish farms. Mar. Pollut. Bull., 57(1-5), 86-93. Watanabe A., Katoh K. and Kimura M. (1993) Effect of rice straw application on CH4 emission from paddy fields. Soil Sci. Plant Nutr., 39(4), 707-712.

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