The relation between the denitrification rate and quinone compositions of the microorganisms in the water treatment apparatus for paddy drainage was evaluated. Q-8 content, the highest ubiquinone, had the strongest correlation coefficient between the denitrification rate (DNR) and the detected ubiquinone. MK-7 had the strongest correlation with DNR among the detected menaquinones. From the results of principal component analysis for DNR using quinone content as the variables, the first principal components were Q-8 and Q-9, and the second principal component was MK-8. Q-8, Q-9, MK-7 and MK-8 containing bacteria strongly contribute to DNR in the water treatment apparatus and around the paddy environment
Journal of Water and Environment Technology, Vol. 8, No.4, 2010 Address correspondence to Koji Hamada, Laboratory of Water Environment Conservation, National Institute for Rural Engineering of Japan, Email: hamada34@affrc.go.jp Received July 31, 2010, Accepted October 21, 2010. - 421 - Evaluation of the Characteristics of Microorganisms that Contribute to Denitrification in the Paddy Drainage Treatment Apparatus by Quinone Composition Measurement Koji HAMADA*, Asa MIURA**, Masafumi FUJITA***, Tadayoshi HITOMI*, Tomijiro KUBOTA*, Eisaku SHIRATANI* *Laboratory of Water Environment Conservation, National Institute for Rural Engineering, 2-1-6, Kan’nondai, Tsukuba, Ibaraki 305-8609, Japan **Faculty of Education and Regional Studies, University of Fukui, 3-9-1, Bunkyo, Fukui 910-8507, Japan ***Department of Urban and Civil Engineering, Ibaraki University, 4-12-1, Nakanarusawa, Hitachi, Ibaraki 316-8511, Japan ABSTRACT The relation between the denitrification rate and quinone compositions of the microorganisms in the water treatment apparatus for paddy drainage was evaluated. Q-8 content, the highest ubiquinone, had the strongest correlation coefficient between the denitrification rate (DNR) and the detected ubiquinone. MK-7 had the strongest correlation with DNR among the detected menaquinones. From the results of principal component analysis for DNR using quinone content as the variables, the first principal components were Q-8 and Q-9, and the second principal component was MK-8. Q-8, Q-9, MK-7 and MK-8 containing bacteria strongly contribute to DNR in the water treatment apparatus and around the paddy environment. Keywords: denitrification, paddy drainage, quinone composition, water treatment. INTRODUCTION Any excess nitrogen runoff from paddies causes eutrophication in downstream water environments. Nitrogen concentrations in the drainage must be reduced for water environment conservation. Charcoal is expected to function as a media for water treatment in a natural environment, and it was used in a water treatment apparatus for surface drainage from paddy fields (Miura et al., 2009). Porous charcoal is an absorbent, microorganism’s attachment media. Microorganisms around the charcoal have nitrogen removal activities and it contributes high nitrogen removal activities for a significantly long time. Biological nitrogen removal activities of microorganisms depend on the kinds of microorganisms in there. Therefore, information on the microorganism community structure or an alternate indicator is very important for understanding the microbial activities. Biologically activated carbon systems are widely used in the treatment of drinking water. Many reports have been published on the biologically activated carbon systems. Burlingame et al. (1986) and Stewart et al. (1990) reported that the microorganism community structures of influent water and that on the surface of activated carbon are not significantly different; that microorganisms on the activated carbon originate from the influent water, and no specific microorganisms dominate the activated carbon surface when compared to the influent water. Therefore, the microorganism community Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 422 - structure within the apparatus in this study is considered to be affected by the effluent from the paddy and the paddy environment. Many methods to evaluate microorganism community structures exist based on biological, biochemical and chemical analyses. Quinone profiles based on quinone composition in microorganisms provide important information on the community structure of the microorganism (Hiraishi, 1989). Isoprenoid quinones are used as coenzymes in the electron transport chain for respiration of microorganisms. The major type of quinone contained in each microorganism depends on the features of the microorganism (Collins’ and Jones, 1981). For example, obligate anaerobic bacteria use only menaquinones, and the major quinones of other species show similar tendencies. The quinone analysis is based on only chemical analysis and does not require any biological and biochemical knowledge or skills. Thus, the quinone profiling has high affinity with analysts. Moreover, quinone composition used in quinone profiling can achieve a qualitative evaluation of the biomass and biological community structure in one analysis. In this study, quinone compositions of microorganisms on the charcoal chips and the denitrification rates (DNRs) by the microorganisms attaching charcoal chips were analyzed. Charcoal chips were experimentally used in the water treatment apparatus for the paddy surface drainage. Correlations between DNRs and the microorganism community structure were evaluated. MATERIALS AND METHODS Water treatment apparatus for drainage from paddy field Fig. 1 shows the water treatment apparatus. The apparatus was 300 mm long, 300 mm wide and 150 mm high (inside dimensions), and was made of transparent acrylic plates (10 mm thick). The apparatus had three independent reacting spaces. Each space was separated by partitions with holes (100 mm wide and 280 mm height) on one side as shown in Fig. 1b. Holes on the partition were netted with nylon (NB90, φ155 µm, Sogo Laboratory Glass Works Co., Ltd., Japan). Each space was filled with 50, 70 and 90% (v/v) charcoal in order of the flow direction. Influent water flowed in a zigzag pattern through the apparatus. 蓋 本体 300 150 3 0 0 (a) Experimental paddy (b) Water treatment apparatus Fig. 1 - Outline of the experimental field and the water treatment apparatus 水路堰板 流 入 流出 装置 (透明) メータ 水 田 畦(コンクリ) 水田への 流入 3,000 3,000 Padd y Influent Effluent Weir Influent to the paddy Water treatment apparatus Filled with charcoal chips Sampling from 3 reacting space - Upstream side area - Middle area - Downstream side area Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 423 - Paddy field The characteristics of the paddy field and the planting were as follows; 3-m-square lysimeter (Fig. 1a), the inlet point was in the middle, the outlet point was at the corner, surface soil type was sandy loam, no fertilizer was added in 2007, the crop plant was Akita-Komachi Rice, and the planting rate was 0.30 m (ridge pitch) and 0.15 m (planting pitch). The paddling and transplanting were conducted on June 6 and June 8, and the inundation began on June 8 in 2007. A weir was at the outlet point, the height of which was fixed during the observation period. The water treatment apparatus was set just in front of the weir, and water levels in the paddy field and the apparatus remained approximately constant during the observation period. A mid-summer drainage was not carried out. Harvesting was carried out in late October (after the last batch experiment). Charcoal chips The chemical and physical characteristics of charcoal chips mainly depend on the tree type and the carbonized temperature. The charcoal chips used in this study were made from thinned Japanese cedar (Sugi in Japanese), and carbonized at 1,050°C in order to produce charcoal that is good at removing recalcitrant dissolved organic matter from water (Miura et al., 2007). Evaluation of denitrification activity of microorganisms Batch experiments for the evaluation of DNR, and a quinone analysis for the evaluation of the microorganism community structure were conducted once a month; July 19 - 20, August 6 - 7, September 2 - 3 and October 9 - 11 in 2007. Charcoal tips were collected from the upstream, the middle and the downstream reacting spaces as shown in Fig. 1b. Three experiments using each three charcoal were conducted at a time, and a total of twelve experiments were conducted in this research. Charcoal chips were collected from the experimental apparatus. The denitrification potential of microorganisms on the charcoal chips was evaluated by a batch experiment. Charcoal chips (approximately 100 g-wet and 15 g-dry) and medium water (300 mL) were put into a flask together, and purged with N 2 gas to keep the anoxic condition. All experiments were conducted at temperatures under 20 degrees Celsius. Sample water was periodically collected, and the nitrate concentration was analyzed after filtration with the membrane filter (pore size 0.2 μm, Whatman, USA). Nitrate concentrations were analyzed by ion chromatography (DX-320, Dionex Corp., USA). Dissolved oxygen concentrations and pH levels were measured by the Galvanic DO sensor (DO-24, TOA DKK, Japan) and the pH sensor (WM-50EG, TOA DKK, Japan) at the beginning and the end of the batch experiment. The medium water contained C 6 H 12 O 6 : 113.3 mg (40 mg-C), KNO 3 : 101.1 mg (10 mg-N), NH 4 Cl: 10.7 mg, KH 2 PO 4 : 1.82 mg, Na 2 HPO 4 ·12H 2 O: 7.17 mg, MgSO 4 ·7H 2 O: 9.0 mg, CaCl 2 ·2H 2 O: 1.4 mg, KCl: 3.6 mg, EDTA: 0.3 mg, FeCl 3 ·6H 2 O: 45 μg, H 3 BO 3 : 4.5 μg, CuSO 4 ·5H 2 O: 0.9 μg, KI: 0.9 μg, MnCl 2 ·4H 2 O: 3.6 μg, Na 2 MoO 4 ·2H 2 O: 1.8 μg, ZnSO 4 ·7H 2 O: 3.6 μg, CoCl 2 ·6H 2 O: 4.5 μg per liter. The medium water has a buffering function up to pH 7.2. Microorganism community structures Quinones were extracted from the charcoal chips in the apparatus according to the Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 424 - 0 25 50 75 100 1.0 1.5 1.5 1.6 1.7 1.8 3.2 3.3 6.2 9.3 11.0 12.0 Denitrificatin rate [g-N･ μ g-quinone -1 ･d -1 ] Quinon content [%] MK-11(H2) MK-11 MK-9(H2) MK-10 MK-9(H2) MK-9 MK-8(H2) MK-8 MK-7(H2) MK-7 MK-6 Q-10 Q-9 Q-8 Fig.2 - Comparison between DNRs and the quinone contents following procedures; 1) quinone mixture was extracted with a chloroform/methanol solution (2:1 v/v), 2) quinone mixture was dried with a rotary evaporator, 3) quinone mixture was extracted from the dried residue with hexane, 4) quinone mixture was adsorbed onto a Sep-Pak plus Silica column (Waters Corp), and 5) menaquinones were extracted with a diethyl ether/hexane solution (2:98 v/v), and ubiquinones were extracted with a diethyl ether/hexane solution (10:90 v/v) from the column. Ubiquinones and menaquinones were quantitatively determined by reversed phase HPLC ( SCL-10AVP , Shimadzu, Japan) with ZORBAX ODS column ( 4.6φ× 250 mm, Aglent Technologies, USA) and photodiode array detector (SPD-M10A VP, Shimadzu, Japan). RESULTS Evaluation of denitrification rates (DNR) The observed DNRs in the batch experiments were 1.0 - 12.0 g-N/μg/d (average: 4.5 g-N/μg/d, Fig. 2). These values were calculated by the changes of nitrate-nitrogen concentration during the initial hour of the batch experiments, by the suspended charcoal chips volume, and by the quinone composition extracted from the charcoal chips. In addition, DO concentrations and pH levels in the reactors were under the detection limit and ranged from 7.1 - 7.3. Quinone compositions of the microorganisms The quinone compositions are compared with DNRs in Fig. 2. Three ubiquinones (Q-8, Q-9 and Q-10) and eleven menaquinones (MK-6, MK-7, MK-7(H 2 ), MK-8, MK-8(H 2 ), MK-9, MK-9(H 2 ), MK-10, MK-9(H 6 ), MK-11 and MK-11(H 2 )) were isolated and identified. In this study, the typical proportion of ubiquinones was Q-8 > Q-10 > Q-9. This proportion is similar to that reported for the activated sludge in a municipal wastewater treatment plant (Hiraishi et al., 1996). The major menaquinones were always MK-6, MK-7, MK-8 and MK-8(H 2 ). These menaquinones are also the major ones in activated sludge (Hiraishi et al., 1996). The molar ratio of menaquinone to ubiquinone (MK/Q) is used as an indicator of the respiratory state such as the balance between anaerobic and aerobic respiration. The Denitrificatin rate [g-N/μg-quinone/d] Quinone content [%] Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 425 - Table 1 - Correlation coefficients (CC) between DNR and the quinone contents CC Q-8 0.58 Q-9 0.43 Q-10 0.03 MK-6 -0.70 MK-7 0.86 MK-7(H 2 ) 0.17 MK-8 -0.67 MK-8(H 2 ) 0.53 MK-9 0.05 MK-9(H 2 ) -0.29 MK-10 0.17 MK-9(H 6 ) -0.32 MK-11 -0.68 MK-11(H 2 ) -0.56 Ubiqinone 0.50 Menaquinone 0.50 MK/Q ratio was 1.41 - 3.38 (average: 2.36). This value is reasonable when compared to reported values. The MK/Q molar ratio in farmland is reported around 2.4 (calculated by authors from the figure in Hiraishi et al., 2003). Hiraishi et al. (2003) also reported MK/Q ratios in several other environments; aquatic environments were 0.14 - 0.54, wastewater environments 0.5 - 1.0, soil environments 2.5 - 3.4. The MK/Q molar ratio in the apparatus was closest to the soil environment. DISCUSSION Correlation between denitrification rate and quinone compositions Table 1 lists the correlation coefficients for DNRs and the contents of quinones. The correlations for the denitrification rates and each quinone were calculated by linear correlations. As for ubiquinones, Q-8 content, the highest ubiquinone, had the strongest correlation coefficient. DNR tended to increase with an increase in the Q-8 content, and the denitrification rate was independent of Q-10 with the second highest ubiquinone content. As for menaquinones, MK-7 had the strongest correlation with DNR. In addition, the content of MK-6, MK−8 and MK-11 were negatively correlated to DNR. Quinones related to DNR Principal component analysis (PCA) for DNR was carried out using the fourteen identified quinones, three ubiquinones and eleven menaquinones as variables. Fig. 3 shows a graphic representation of the PCA analysis of DNR. The X-axis is the first principal component (PC1) and Y-axis is the second principal component (PC2). The diameters of the circles indicate DNRs. The plots were randomly scattered, and it is difficult to find clear trends, but the three highest DNRs (9.3, 11.0, 12.0 g-N/μg-quinone/d) were plotted in the low PC1 area. Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 426 - -3 3 -4 4 PC1 PC2 : 10 mg-N/ μ g-quinone/d Fig.3 - Results of PCA for DNR Table 2 - Factor loading of PC1 and PC2 PC1 PC2 Q-8 -0.37 -0.18 Q-9 -0.32 -0.12 Q-10 0.10 0.25 MK-6 0.23 0.00 MK-7 -0.27 0.14 MK-7(H 2 ) 0.23 0.25 MK-8 0.30 -0.34 MK-8(H 2 ) -0.31 0.13 MK-9 -0.11 -0.44 MK-9(H 2 ) 0.23 0.29 MK-10 0.12 0.51 MK-9(H 6 ) 0.34 -0.06 MK-11 0.30 -0.29 MK-11(H 2 ) 0.32 -0.23 The contribution ratio of PC1 and PC2 were 43.8% and 18.6%, respectively, and the accumulated contribution ratio for PC1 and PC2 was 62.3%. Table 2 shows the factor loading of PC1 and PC2. As for PC1, Q-8, Q-9, MK-9(H 2 ) and MK-11(H 2 ) indicated higher factor loading values. Content of these quinones were 5.1 - 15.7% for Q-8, 3.6 - 13.7% for Q-9, less than 2% for MK-9(H 6 ), and less than 1% for MK-11(H 2 ). The contributions of MK-9(H 2 ) and MK-11(H 2 ) were neglected, because the contents of MK-9(H 6 ) and MK-11(H 2 ) were very small. PC1 was considered to be mainly Q-8 and Q-9. As for PC2, MK-8, MK-9 and MK-10 indicated higher factor loading values. MK-8 content was 7.2 - 17.7%, MK-9 content was 2.2 - 2.7%, and MK-10 content was 0.4 - 1.3%. PC2 was considered to be mainly MK-8, because MK-9 and MK-10 contents were relatively small. From the results of PCA, Q-8, Q-9, MK-7 and MK-8 appear to be very important factors in DNR in the water treatment apparatus. CONCLUSION The relation between denitrification rates and characteristics of microorganisms were Journal of Water and Environment Technology, Vol. 8, No.4, 2010 - 427 - evaluated. The microorganisms were collected from the charcoal chips in the water treatment apparatus for paddy surface drainage. Characteristics of Microorganisms were evaluated by the quinone compositions of microorganisms. From the results, we conclude that Q-8, Q-9, MK-7 and MK-8 containing bacteria appear to strongly contribute to denitrification in the environment surrounding the paddies. Q-8 especially appears to be the key factor for denitrification in the paddies in addition to the apparatus for paddy surface drainage treatment. This information is an important indicator of the stabilization of denitrification activity in the water treatment apparatus and will help with the water treatment apparatus improvement. Furthermore, the types of quinone related to denitrification rates in the apparatus were thought to be similar to those in the surface of paddy soil, because microorganisms in the apparatus were thought to have originated from the surface of paddy soil. Thus, Q-8, Q-9, MK-7 and MK-8 containing bacteria strongly contribute to denitrification rate in the paddies. Q-8 especially appears to be the key factor for denitrification in the paddy. Quinone composition provides information for the evaluation of the biological potential for denitrification in paddies. The arrangement of environmental factors of paddy, focusing on the Q-8 and other highly-contained-quinones, will lead to the finding of paddy characters accomplishing high denitrification activity. REFERENCE Burlingame G. A., Suffet I. H. and Pipes W. O. (1986). Predominant bacterial genera in granular activated carbon water treatment systems, Canadian Journal of Microbiology, 32, 226-230. Collins’ M. D. and Jones D. (1981). Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications, Microbiological Reviews, 45(2), 316-354. Hiraishi A. (1989). In Recent Advances in Microbial Ecology (Hattori T., Ishida Y., Maruyama Y., Morita R.Y. and Uchida A. ed.), Japan Scientific Societies Press, Tokyo, 663 (In Japanese). Hiraishi A., Ueda Y., Ishihara J. and Mori T. (1996). Comparative lipoquinone analysis of influent sewage and activated sludge by high-performance liquid chromatography and photodiode array detection, The Journal of General and Applied Microbiology, 42, 457-469. Hiraishi A., Iwasaki M., Kawagishi T., Yoshida N., Narihiro T. and Kato K. (2003). Signification of lipoquinons as quantitative biomarkers of bacterial populations in the environment, Microbes and Environments, 18(2), 89-93. Miura A., Shiratani E., Hitomi T., Yoshinaga I., Takaki K. and Hamada K. (2007). Removal of recalcitrant dissolved organic matter using carbonized recycled sugi wood, Journal of Japan Society on Water Environment, 30(1), 39-44. (in Japanese with English abstract) Miura A., Shiratani E., Hamada K., Hitomi T., Yoshinaga I. and Kubota T. (2009). Removal of nutrients, organic matter and heavy metals from paddy field drainage by charcoal, Journal of Water and Environment Technology, 7(1), 9-17. Stewart M. H., Wolfe R. L. and Means E. G. (1990). Assessment of the bacteriological activity associated with granular activated carbon treatment of drinking water, Applied and Environmental Microbiology, 56, 3822-3829. . the first principal components were Q -8 and Q-9, and the second principal component was MK -8. Q -8, Q-9, MK-7 and MK -8 containing bacteria strongly contribute. Vol. 8, No.4, 2010 - 425 - Table 1 - Correlation coefficients (CC) between DNR and the quinone contents CC Q -8 0. 58 Q-9 0.43 Q-10 0.03 MK-6 -0.70 MK-7 0 .86