Biocontrol Science, 2016, Vol. 21, No. 1, 29－36

Original Isolation and Characterization of a Purple Non-Sulfur Photosynthetic Bacterium Rhodopseudomonas faecalis Strain A from Swine Sewage Wastewater

HONGYI WEI1, SUGURU OKUNISHI2＊, TAKESHI YOSHIKAWA2, YUTO KAMEI3, AND HIROTO MAEDA2

1The United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-8580, Japan 2Education and Research Center for Marine Resources and Environment, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan 3Department of Applied Biochemistry and Food Science, Faculty of Agriculture, Saga University, 1 Honjo, Saga, Japan

Received 21 May, 2015/Accepted 25 September, 2015

A purple non-sulfur photosynthetic bacterium（ PNSB）, PSB Strain A was isolated from swine sewage wastewater. Phylogenetic analysis revealed that PSB Strain A was most closely related to Rhodopseudomonas faecalis. Growth of the isolate under anaerobic-light conditions with a variety of carbon sources was investigated. Both PSB Strain A and the standard strain showed good growth with acetic acid, propionic acid, and n-butyric acid at a concentration of 20 mM. At the high concentration of 200 mM, PSB Strain A showed better growth in pyruvate, acetate, propionate, succinate and malate. By applying PSB Strain A to treat swine sewage wastewater, the concentration of VFAs, which were acetic acid and propionic acid, decreased from 158.0 mM to 120.2±2.9 mM, and 14.9 mM to 9.3±0.9 mM, respectively, after 216-h incubation. After 330-h incubation, the concentrations of TOC and ammonia nitrogen dropped from 4508.0 mg/L to 3104.0±451.5 mg/L, and 629.7 mg/L to 424.1±7.4 mg/L, respectively. The isolated PSB Strain A showed almost the same efficiency compared with the standard strain on the removal of VFAs and TOC. The results suggest the possibility of using the isolated strain to treat swine sewage wastewater.

Key words ： Purple non-sulfur photosynthetic bacterium / Rhodopseudomonas / 16S rDNA / Swine sewage wastewater.

INTRODUCTION Kitamura et al., 1980）. Removal of VFAs and ammonia has also been reported by using PNSB（ Lee et al., The majority of purple non-sulfur bacteria are species 1992; Zhan et al., 2013）. In addition, one advantage of of the genus Rhodopseudomonas. They have a complete using these bacteria is that various types of bacterial tricarboxylic acid cycle（ TCA cycle） which allows them biomass could be produced during the process. The to grow anaerobically in the light or aerobically in the biomass produced could then be used in the diet for dark with different carbon sources and electron donors aquaculture, animal feed stock supplements and biofer- （Pfennig et al., 1974; Kitamura et al., 1984）. Since the tilizers（ Kobayashi and Kurata, 1978; Noparatnaraporn 1970s, the bioremediation of wastewater with a high et al., 1983）. concentration of organic matter has been investigated Swine wastewater contains a large amount of organic by applying PNSB（ Kobayashi and Tchan, 1973; matter and a high concentration of ammonia, which is toxic to fish and other aquatic organisms（ Obaja et al., ＊Corresponding author. Tel & Fax: +81-99-286-4133, E-mail : 2003）. Directly discharging such wastewater into the okunish（i a）fish.kagoshima-u.ac.jp environment might cause eutrophication and present a 30 S. OKUNISHI ET AL. potential risk to public health（ Bernet et al., 2000）. the liquid cultures by using the DNeasy Plant Mini Kit Many strategies have been developed to treat this kind （Qiagen, Hilden, Germany）. The extracted DNA was of wastewater, such as gravity sedimentation, precipita- used for PCR amplification of 16S ribosomal RNA tion, and activated sludge（ Martinez et al., 1995; Liao genes（ 16S rDNA） with a universal primer set et al., 1993; Guo et al., 2013）. PrSSU.1F（ 5’-AGAGTTTGATCATGGCTCAG-3’） and In this study, a PNSB Strain was isolated from swine PrSSU.1R（ 5’-GGTTGGATCACCTCCTTA-3’）. The PCR sewage wastewater. The outstanding characteristics, reaction mixture was composed of Ex Taq Buffer such as its morphological features, growth based on （Takara Bio, Otsu, Japan）, 100 µM of each dNTP, 0.5 the use of carbon sources and phylogenetic relation- µM of each primer, 0.025 units/µl Ex Taq DNA poly- ships in the genera Rhodopseudomonas were investi- merase Hot Start Version（ Takara Bio, Otsu, Japan）, gated. Furthermore, a preliminary study was conducted and one-tenth volume of the bacterial DNA solution. on the use of the isolated strain to treat swine sewage The thermal cycling consisted of an initial denaturation wastewater. of 1 min at 94℃, followed by 25 cycles of 30 s of dena- turation at 94℃, 20 s of annealing at 56℃ and 1.5 min MATERIALS AND METHODS of extension at 72℃, with 7 min of a final extension at 72℃. Microorganism and cultivation method The PCR products were electrophoresed by 1.5 % PSB Strain A was isolated from swine sewage waste- agarose gel in TAE buffer（ 40 mM Tris-acetate, 1 mM water collected from a pig farm in May, 2007（ Isa, ethylenediaminetetraacetic acid, EDTA） under a constant Kagoshima, Japan）. After sampling, PNSB was voltage condition of 100 V with the electrophoresis enriched by inoculating the wastewater into 30 ml of the system GelMate 2000（ Toyobo, Osaka, Japan）. The Basic I medium as described by Hoshino et al.（ 1984）. electrophoresed gels were stained with SYBR Gold An aliquot of the enriched culture was streaked on the （Molecular Probes, Life Technologies, Carlsbad, USA） Basic I double layer agar plates, containing 1.5 % agar and photographed under a blue transilluminator Safe for the bottom layer and 1.2 % agar for the top layer, Imager 2.0（ Invitrogen, Life Technologies, Carlsbad, and cultivated at 30℃ for 14 days under illumination of USA）. The obtained bands were cut from the gel and 12/12 light/dark cycles to obtain the colonies. The the PCR products were purified from the gel pieces obtained colonies were transferred onto the Basic I agar using the MonoFas DNA Purification Kit I（ GL Science, slant medium, cultivated under the same conditions as Tokyo, Japan）. above, and stored at 10℃. Rhodopseudomonas palus- Nucleotide sequences of the PCR-amplified 16S tris NBRC-100419T was purchased from NITE, Japan. rDNA were determined with the BigDye Terminator v3.1 The strains were grown under anaerobic conditions at Cycle Sequencing Kit（ Life Technologies, Carlsbad, 30-35℃, illumination at 12/12 light/dark cycles, in Basic USA）. The primers used were PrSSU.1F, PrSSU.3F（ 5’- I medium. TGCCAGCAGCCGCGGTA-3’）, PrSSU.1R, and PrSSU.2R（ 5’-TGCCAGCAGCCGCGGTA-3’）. The Cell morphology and identification of PSB Strain A obtained sequences were assembled with the program Colonies of the microorganisms were obtained by DNASIS Pro Version 2.7（ Hitachi Solutions, Tokyo, streaking and cultivating the isolates on the agar plate Japan）. media. Scanning electron microscopy was carried out by Closely related sequences were obtained from the using the liquid culture of the microorganisms. Samples GenBank DNA database by using the basic local align- for scanning electron microscopy were prepared as ment search too（l BLAST） program. Phylogenetic anal- follows: briefly, 40 µl of 25 % glutaraldehyde was added ysis with the neighbor-joining method was conducted into 1 ml of the culture medium, and the suspension using the program MEGA Version 5（ Tamura et al., was filtered through a 0.2 µm Nuclepore polycarbonate 2011）. filter（ Whatman Ltd., Kent, UK）. Then, a dehydration process was applied using a series of graded ethanol Accession number solutions（ 1 ml of 50 to 80 % ethanol, four times; then The nucleotide sequences of the partial sequence of 1 ml of 99.5 % ethanol twice）. The filter was gold- the 16S rDNA determined in this study have been coated with a sputter-coating machine（ MSP-Mini submitted to the DDBJ database under the accession Magnetron Sputter, Vacuum Device, Co., Ltd., Ibaragi, number LC066638. Japan） and observed with a scanning electron micro- scope, Miniscope TM-1000（ Hitach High Tech, Co., Use of organic acids by PSB Strains Ltd., Tokyo, Japan）. The growth rate of PSB Strains was measured by a DNA of the isolated PSB Strain A was extracted from spectrophotometer（ HITACHI U-1100, Japan）. The cell CHARACTERIZATION OF PSB 31 growth was determined from the attenuance（ optical Statistical analysis density） of the culture at 660 nm（ OD660）. After Data was subjected to the independent t test method adjusting the OD660 value to 0.5-0.6, 1 ml of PSB to determine significant differences. p<0.05 was consid- culture was inoculated into 20 ml Basic I medium in a ered to be statistically significant. screw-cap tube. Various organic acids were added as the single carbon source in Basic I medium, such as RESULTS AND DISCUSSION acetic acid, propionic acid and n-butyric acid, which was added at a concentration of 20 mM. Furthermore, Isolation and identification of PSB Strain A acetic acid, propionic acid, n-butyric acid and short- Colonies of the photosynthetic bacterium（ PSB） chain fatty acids related to the tricarboxylic acid cycle, Strain A were observed after a 14-day incubation on the such as formic acid, citric acid, α-ketoglutaric acid, Basic I agar plate（ data not shown）. Scanning electron succinic acid, fumaric acid, malic acid, malonic acid microscopic observation showed that the PSB cells and pyruvic acid, were added at a concentration of 200 were rod-shaped with bright spots at both ends of the mM. Except for n-butyric acid, salts of the organic acids cells, and the cell length was 3-4 µm（ Fig.1）. The were applied in this study. 100 µl of 10 % sodium thio- phylogenetic results showed that PSB Strain A was glycolate solution was added as the reductant. most closely related to Rhodopseudomonas faecalis Incubation conditions were as described previously. （Fig.2）, suggesting that the isolate is one of the purple non-sulfur photosynthetic bacteria. Swine sewage wastewater pretreatment Swine sewage wastewater was sampled from the Growth of PSB Strains same site where PSB Strain A was isolated（ Isa, Both PSB Strain A and the NBRC Strain showed Kagoshima, Japan）. The water layer fraction was good growth with acetate, propionate, and n-butyric obtained from the pre-treated wastewater sample by acid at a concentration of 20 mM, and no statistically centrifugation（ 3,000 rpm, 15min）. The undiluted water significant differences were found between PSB Strain layer fraction and its 2-time dilution were applied in the A and the NBRC Strain（ p>0.05）（ Fig.3）. Comparatively, following experiments. PSB Strain A showed better growth with pyruvate, acetate, propionate, succinate and malate at a concen- Incubation experiment using swine sewage tration of 200 mM（ p<0.05）. However, neither PSB wastewater Strain A nor the NBRC Strain showed good growth with Pretreated wastewater and its 2-time dilution was n-butyric acid, formate, citrate and α-ketoglutrate sterilized by autoclaving. After adjusting the OD660 （Fig.4）. Results suggested that PSB Strain A could value to 1.0, 1 ml of PSB culture was inoculated into 20 grow well in a variety of organic acid salts. Better ml of the sterilized undiluted wastewater or its 2-time growth was observed with lower concentrations of dilution. 100µl of 10% sodium thioglycolate solution n-butyric acid, indicating that a high concentration of was added as the reductant. Incubation conditions organic matter might inhibit the growth of photosyn- were as described previously. thetic bacteria. Growth of the genera and species of

Analysis methods The growth rate of PSB Strains in pre-treated waste- water was measured by OD660 as described earlier. After centrifugation（ 15,000 rpm, 10 min）, the superna- tant of the culture medium was used for the following analysis. The concentrations of VFAs（ acetic acid and propionic acid） were analyzed by HPLC（ Column: SHIMADZU, CTO-10A, detector: UV）. Total organic carbon（ TOC） and DOC were tested by the TOC Analyzer（ TOC-V, SHIMADZU, Japan）. The concentra- tion of ammonia nitrogen was analyzed by the indo- phenol method as described by Dora（ 1976）. Nitrite nitrogen and nitrate nitrogen were analyzed by the Auto Analyzer（ BLTEC, SWAAT, Japan） while pH was tested by using pH test paper. FIG. 1．Scanning electron microscopic observation of PSB Strain A. The PSB cells were trapped onto 0.2 µm Nuclepore membranes and sputter-coated. Scale bar, 10 µm. 32 S. OKUNISHI ET AL.

FIG. 2．Unrooted phylogenetic tree of the photosynthetic bacterial isolate based on the 16S rDNA sequences. Bootstrap values are shown at the branching points. Scale bar, genetic distance. Nucleotide sequence information of the photosynthetic bacterial isolate has been registered in DNA Data Bank of Japan（ DDBJ）, accession number: LC066638.

FIG. 3．Growth of strain A（ ） and the type strain FIG. 4．Growth of strain A（ ） and the type strain Rhodopseudomonas palustris（ NBRC 100419T）（ ） in an Rhodopseudomonas palustris（ NBRC 100419T）（ ） in an artificial medium to which a sellected organic acid（ 20mM） artificial medium to which a selected organic acid（ 200 mM） was added as the sole carbon source（ n = 3 ± standard was added as the sole carbon source（ n = 3 ± standard deviation）. deviation）.

Rhodopseudomonas with different carbon sources has reported to be difficult to utilize（ Nakajima et al., 1997）. been reported（ Imhoff et al., 2005）. PSB Strain A could However, in this study, PSB Strain A showed growth comparatively utilize a variety of substrates（ Table 1）. A with n-butyric acid at a concentration of 20 mM. Since long-chain organic acid, n-butyric acid has been acetic acid, propionic acid, and n-butyric acid are the CHARACTERIZATION OF PSB 33

TABLE 1．Growth of purple non-sulfur photosynthetic bacteria belonging to the genus of Rhodopseudomonas with various carbon sources. Carbon Source Organism Butyrate/ References Acetate Citrate Formate Fumarate Malate Ketoglutrate Malonate Propionate Pyruvate Succinate Butyric acid PSB Strain A ＋ ＋ － － ＋ ＋ － ＋ ＋ ＋ ＋ This study R. palustris ＋ ＋ － － ＋ － － ＋ ＋ ＋ ＋ This study NBRC R. palustris ＋ ＋ ＋/－ ＋ ＋ ＋ nd ＋ ＋ ＋ ＋ Imhoff J. F. et al. R. rhenobacensis ＋ ＋ － ＋ ＋ ＋ nd nd － ＋ ＋ Imhoff J. F. et al. R. julia ＋ ＋ － ＋ ＋ ＋ nd － ＋ ＋ ＋ Imhoff J. F. et al. R. cryptolactis ＋ nd nd nd nd ＋ nd nd nd ＋ ＋ Imhoff J. F. et al. aSymbols: ＋, positive in growth; －, negative in growth; ＋/－, variable in different strains; nd, not determined.

TABLE 2．Chemical composition of the pretreated pig farm 3 effluent. Component Concentration 2.5 ) Total organic carbon（ mg/L） 9150.0 2 n m Acetate（ mM） 270.5 6 0

( 6 1.5 Propionate（ mM） 27.0 ＋ NH -N（ mg/L） 1386.0 O D 4 1 pH 8.2 0.5

0 main contents of volatile fatty acids in swine sewage 0 100 200 300 400 wastewater, the results suggest that PSB Strain A Time (h) appears to be a promising agent in the practical treat- ment of swine sewage wastewater. FIG. 5．The change in the growth rate of strain A and type strain Rhodopseudomonas palustris（ NBRC 100419T） in Contents of swine sewage wastewater after undiluted and 2×diluted pig farm effluent（ n = 3 ± standard pre-treatment deviation）, ◆: strain A in undiluted effluent, △: strain NBRC in undiluted effluent, ■: strain A in 2×diluted effluent, ○:

The main contents of pre-treated swine sewage strain NBRC in 2×diluted effluent. wastewater are shown in Table 2. The concentrations of TOC and ammonia nitrogen were 9150.0 mg/L and 1386.0 mg/L, respectively, which are typical features of Strain showed low growth rate in undiluted wastewater. organic wastewater. Analysis of VFAs showed that the Comparatively, PSB Strain A and the NBRC Strain concentrations of acetic acid and propionic acid were entered the logarithmic growth phase after 48 h incuba- 270.5 mM and 27.0 mM, respectively. The results indi- tion in 2 time diluted wastewater. After 330 h incubation, cated that 82 % of TOC was VFAs. Generally, VFAs in the biomass reached a maximum level. PSB Strain A swine sewage wastewater are mainly acetic acid, propi- showed a lower cell concentration compared with the onic acid and n-butyric acid（ Jun, 2000）. In this study, NBRC Strain. the concentration of n-butyric acid was below the detection limit. Similarly, NO3-N and NO2-N were also Reduction of VFA content and TOC below the detection limit. Thus, the nitrogen content Table 3 shows the results of reduction in VFAs. Due was mainly ammonia nitrogen. The pH of swine sewage to the low existing amounts in undiluted swine sewage wastewater was 8.2. wastewater, no reduction in VFAs was observed with either strain while in 2 time diluted swine sewage PSB growth in swine sewage wastewater wastewater, the concentration of acetic acid decreased Fig.5 shows the growth curve of PSB Strains in swine from 158.0 mM to 120.2±2.9 mM and 130.9±1.5 mM sewage wastewater. Both PSB Strain A and the NBRC after 216 h incubation with PSB Strain A and the NBRC 34 S. OKUNISHI ET AL.

TABLE 3．Change in volatile fatty acid〔 VFA（ acetate and propionate）〕 concentrations and reduction rate by strain A and the type strain Rhodopseudomonas palustris（ NBRC 100419T） in 2 time diluted pig farm effluent. VFA concentration（ mM） 0h 216h R. palustris R. palustris VFA A A （NBRC 100419T） （NBRC 100419T） Acetate 158.0 158.0 120.2±2.9 130.9±1.5 Propionate 14.9 14.9 9.3±0.9 10.9±1.6

Together with the high growth rate of PSB Strain A and the NBRC Strain, reduction of VFA and TOC contents was found in 2-time diluted swine sewage wastewater. On the other hand, in undiluted swine sewage wastewater, no significant reduction was observed due to poor growth of the two strains. Similar to the previous results of growth in organic acids, undi- luted swine sewage wastewater containing a high concentration of organic components might not suit the PSB Strains. It has been reported that the high concen- tration of organic matter as well as high pH might inhibit the growth of microorganisms（ Cristina et al., 2008; Suehara et al., 2005）. In this study, pH was 8.2 in both undiluted and 2-time diluted swine sewage wastewater. FIG. 6．The change in the total organic carbon（ TOC） by strain A and type strain Rhodopseudomonas palustris Thus, it was considered that the main reason for the （NBRC 100419T） in undileted and 2×diluted pig farm inhibition of the PSB growth was probably the high effluent（ n = 3 ± standard deviation）, ◆: strain A in undi- concentration of organic matter. luted effluent, △: strain NBRC in undiluted effluent, ■: strain Compared with the standard strain, PSB Strain A A in 2×diluted effluent, ○: strain NBRC in 2×diluted effluent. showed better efficiency in the reduction of acetic acid and propionic acid. The time course of TOC could be divided into two phases, which was coincident with the Strain, respectively. Propionic acid decreased from 14.9 growth curve（ Figs.5 and 6）. It has been reported that mM to 9.3±0.9 mM and 10.9±1.6 mM after 216 h the high cell yield of purple non-sulfur bacteria could be incubation with PSB Strain A and the NBRC Strain, obtained under anaerobic-light conditions with organic respectively. matter. In this photosynthetic process, production of Fig.6 shows the time course of TOC concentration. carbon dioxide was not observed（ Hoshino et al., No significant reduction was observed in undiluted 1984）. Thus, the influence of carbon dioxide on TOC swine sewage wastewater. In 2-time diluted swine was not discussed in this study. While under aerobic- sewage wastewater, TOC concentration showed the dark conditions, oxygen was utilized as the electron same trend with both PSB Strain A and the NBRC acceptor in respiration（ Izu et al., 2001; Ono-Izu et al., Strain: it decreased slowly at the beginning, and then 2004）. Due to the low efficiency of anaerobic metabo- decreased rapidly after 145-h incubation. After 330-h lism, organic matter was assimilated as the electron incubation, the concentration dropped from 4508.0 mg/ donor to produce ATP. Although 10 % sodium thiogly- L to 3104.0±451.5 mg/L, and 3733.5±205.3 mg/L colate solution was added as a reductant in this study, with PSB Strain A and the NBRC Strain, respectively. with the existence of the remaining oxygen, aerobic No statistically significant differences were found metabolism was performed at the beginning. After between PSB Strain A and the NBRC Strain（ p>0.05）. oxygen was consumed, anaerobic metabolism was Rhodopseudomonas palustris has been studied as an considered to start along with the consumption of agent to treat wastewater containing a high concentra- organic matter. tion of organic matter（ Getha et al., 1998; Kim et al., 2004）. In our study, the isolated PSB Strain A showed Reduction of ammonia nitrogen almost the same efficiency. Fig.7 shows the time course of the ammonia nitrogen CHARACTERIZATION OF PSB 35

with the NBRC Strain. The results indicated that PSB 1700 Strain A was efficient in treating wastewater containing high concentration of organic matter. Furthermore, the PSB cells yielded contained a number of valuable

g/L) 1200 components. Thus, it appeared to be a promising agent ( m

N that could be used in swine sewage wastewater treat- - + 4 ment to obtain zero-emission. Further study should H

N concentrate on the optimization of PSB growth effi- 700 ciency to establish a practical treatment system.

200 REFERENCES 0 100 200 300 400 Time (h) Bernet, N., Delgenes, N., Akunna, J.C., and Delgenes, J.P. （2000） Combined anaerobic-aerobic SBR for the treat- ＋ ment of piggery wastewater. Water Res., 34, 611-619. FIG. 7．The change in the NH4 -N concentration by strain A and type strain Rhodopseudomonas palustris（ NBRC Cristina, G., Judith, M., Santiago, V., Pedro, A.G., and Raul, 100419T） in undiluted and 2×diluted pig farm effluent（ n = 3 M.（ 2008） Microalgae-based processes for the biodegra- dation of pretreated piggery wastewaters. Appl. Microbiol. ± standard deviation）, ◆: strain A in undiluted effluent, △: Biotechnol., 80, 891-898. strain NBRC in undiluted effluent, ■: strain A in 2×diluted Dora, S.（ 1976） Determination of ammonia and Kjeldahl effluent, ○: strain NBRC in 2×diluted effluent. nitrogen by indophenol method. Water Res., 10, 31-36. Getha, K., Vikineswary, S., and Chong, V. C.（ 1998） Isolation and growth of the phototrophic bacterium Rhodopseudomonas concentration. The initial concentration was 1609.5 mg/ palustris strain B1 in sago-starch-processing wastewater. L. After 330 h incubation with PSB Strain A and the World J. Microbiol. Biotechnol., 14, 505-511. Guo, J., Yang, C., and Zeng, G.（ 2013） Treatment of swine NBRC Strain, ammonia nitrogen was reduced to 1388.3 wastewater using chemically modified zeolite and biofloc- ±138.4 mg/L, and 1427.7±125.6 mg/L, respectively. culant from activated sludge. Bioresour. Technol., 143, 289- In 2-time diluted swine sewage wastewater, the 297. concentration was reduced from 629.7 mg/L to 424.1 Imhoff, J. F., Hiraishi, A., and Suöling, J.（ 2005） Anoxygenic ±7.4 mg/L and 376.8±82.4 mg/L after 330-h incuba- phototrophicpurple bacteria. In Bergey’s manual of system- atic bacteriology, 2nd ed., Vol.2., The Proteobacteria, part tion with PSB Strain A and the NBRC Strain, respec- A, introductory essays（ D. J. Brenner, N. R. Krieg, J. T. tively. No statistically significant differences were found Staley, and G. M. Garrity eds.）, pp. 119-132, Springer- between PSB Strain A and the NBRC Strain（ p>0.05）. Verlag, NewYork. Generally, high nitrogen concentration has been found Hoshino, Y., and Kitamura, H.（ 1984） Ecology. In Photosynthetic Bacteria（ Kitamura, H., Morita, S., and Yamashita, J. eds.）, in swine sewage wastewater（ Cristina et al., 2008）. pp. 9-21, Gakkai Shuppan Center, Tokyo.（ In Japanese） Analysis of wastewater used in this study also showed Izu, K., Nakajima, F., Yamamoto, K., and Kurisu, F.（ 2001） the same findings（ Table 3）. It has been reported that, Aeration conditions affecting growth of purple nonsulfur under anaerobic conditions with infrared irradiation, the bacteria in an organic wastewater treatment process. enriched PNSB was able to remove 99.6 % NH ＋-N System. Appl. Microbiol., 24, 294-302. 4 Hülsen, T., Batstone, D. J., Keller, J.（ 2014） Phototrophic from primary settled domestic wastewater（ Hülsen et bacteria for nutrient recovery from domestic wastewater. al., 2014）. In this study, regardless of the growth rate, Wat. Res., 50, 18-26. reduction of ammonia nitrogen was found in both undi- Jun, Z.（ 2000） A review of microbiology in swine manure luted and 2-time diluted swine sewage wastewater. odor control. Agr. Ecosyst.Environ., 78, 93-106. Kim, M. K., Choi, K. M., Yin, C. R., Lee, K. Y., Im, W. T., Lim, Comparatively, the removal rates were not so high, J. H., Lee, S. T., 2004. Odorous swine wastewater treat- which were 24.31 % in undiluted swine sewage waste- ment by purple non-sulfur bacteria, Rhodopseudomonas water and 33.82 % in 2-time diluted wastewater. palustris, isolated from eutrophicated ponds. Biotechnol. Lett., 26, 819-822. Kitamura, H., Kurosawa, K., and Kobayashi, K.（ 1980） Animal Conclusion Waste Treatment and Utilization: Proceedings,（ Chung P. A photosynthetic bacteria PSB Strain A was isolated, ed.）, p. 343, Published by Council for Agricultural Planning characterized and used to treat swine sewage waste- and Development. water in this study. Compared with Rhodopseudomonas Kitamura, H., and Maruyama, K.（ 1984） Growth and carbon palustris NBRC 100419T, the isolated PSB Strain A source utilization. In Photosynthetic Bacteria（ Kitamura, H., Morita, S., and Yamashita, J. eds.）, pp. 47-61, Gakkai showed better growth in utilizing a variety of organic Shuppan Center, Tokyo.（ In Japanese） acids. During the treatment with swine sewage waste- Kobayashi, M., and Kurata, S.（ 1978） The mass culture and water, PSB Strain A also showed almost the same effi- cell utilization of photosynthetic bacteria. J. Process Biochem., ciency in the reduction of VFAs and TOC compared 13, 27-30. 36 S. OKUNISHI ET AL.

Kobayashi, M., and Tchan, Y. T.（ 1973） Treatment of indus- Obaja, D., Mace, S., Costa, J., Sans, C., and Mata-Alvarez, J. trial waste solutions and productions and production of （2003） Nitrification, denitrification and biological phos- useful by-products using a photosynthetic bacterial phorus removal in a sequencing batch reactor treating method. Wat. Res., 7, 1219-1224. piggery wastewater. Biores. Technol., 87, 103-111. Lee, M. G., and Kobayashi, M.（ 1992） Deodorization of Ono-Izu, K., Yamamoto, K., and Nakajima, F.（ 2004） swine sewage by addition of a phototrophic bacterium, Optimization of operational conditions for predominance of Rhodopseudomonas capsulata. Soil Sci. Plant Nutr., 38, purple nonsulfur bacteria in organic wastewater treatment 767-770. using photosynthetic bacteria. J. Japan Soc. Wat. Environ., Martinez, J., Burton, C. H., Sneath, R. W., and Farrent, J. W. 27, 261-266. （1995） A study of the potential contribution of sedimenta- Pfennig, N., and Truper, H. G.（ 1974）. The phototrophic tion to aerobic treatment processes for pig slurry. J. Agric bacteria. In Bergey’s Manual of Systematic Bacteriology, Eng Res., 61, 87-96. 8th ed.,（ R. E. Buchanan & N. E. Gibbons. eds.）, pp. Liao, P. H., Gao, Y., and Lo, K. V.（ 1993） Chemical precipita- 24-33, Williams & Wilkins, Baltimore. tion of phosphate and ammonia from swine wastewater. J. Suehara, K., Kawamoto, Y., Fujii, E., Kohda, J., Nakano, Y., Biomass Bioenerg., 4, 365-371. and Yano, T.（ 2005） Biological treatment of wastewater Nakajima, F., Kamiko, N., and Yamamoto, K.（ 1997） Organic discharged from biodiesel fuel production plant with alkali- wastewater treatment without greenhouse gas emission by catalyzed transesterification. J. Biosci. Bioeng., 100, 437- photosynthetic bacteria. Wat. Sci. Tecnol., 35, 285-291. 442. Noparatnaraporn, N., Nishizawa, Y., Hayashi, M., and Nagai, S. Zhan, P., and Liu, W.（ 2013） Use of fluidized bed biofilter and （1983） Single cell protein production from cassava scratch immobilized Rhodopseudomonas palustris for ammonia by Rhodopseudomonas gelatinosa. J. Ferment. Tecnol., 61, removal and fish health maintenance in a recirculation 515-519. aquaculture system. Aquaculture Res., 44, 327-334.