J. Gen. Appl. Microbiol., 48, 299–308 (2002) Full Paper Diaphorobacter nitroreducens gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading denitrifying bacterium isolated from activated sludge Shams Tabrez Khan and Akira Hiraishi* Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441–8580, Japan (Received August 12, 2002; Accepted October 23, 2002) Three denitrifying strains of bacteria capable of degrading poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were isolated from activated sludge and characterized. All of the isolates had almost identical phenotypic characteristics. They were motile gram-negative rods with single polar flagella and grew well with simple organic com- pounds, as well as with PHB and PHBV, as carbon and energy sources under both aerobic and anaerobic denitrifying conditions. However, none of the sugars tested supported their growth. The cellular fatty acid profiles showed the presence of C16:1w7cis and C16:0 as the major com- ponents and of 3-OH-C10:0 as the sole component of hydroxy fatty acids. Ubiquinone-8 was de- tected as the major respiratory quinone. A 16S rDNA sequence-based phylogenetic analysis showed that all the isolates belonged to the family Comamonadaceae, a major group of b-Pro- teobacteria, but formed no monophyletic cluster with any previously known species of this fam- (DSM 13225؍) ily. The closest relative to our strains was an unidentified bacterium strain LW1 (99.9% similarity), reported previously as a 1-chloro-4-nitrobenzene degrading bacterium. DNA- DNA hybridization levels among the new isolates were more than 60%, whereas those between our isolates and strain DSM 13225 were less than 50%. The G؉C content of genomic DNA of the new strains was 64 to 65 mol%. Based on these results, we concluded that the PHBV-degrading denitrifying isolates should be classified as a new genus and a new species, for which we pro- CIP؍JCM 11421؍) pose the name Diaphorobacter nitroreducens. The type strain is strain NA10B 107294). We also propose to classify strain DSM 13225 as a genospecies of Diaphorobacter. Key Words——activated sludge; denitrifying bacteria; Diaphorobacter nitroreducens; PHB degradation Introduction water or intentionally added liquid substrates, e.g., methanol and acetate, as electron donors for denitrifi- Biological denitrification is the most widely used mi- cation. On the other hand, several reports have in re- crobial process for nitrogen removal in water and cent years introduced new biotechnology of nitrogen wastewater treatment. Conventional biological nitrogen removal using solid biopolymers, such as poly(3-hy- removal systems use residual organic matter in waste- droxybutyrate) (PHB) and poly(3-hydroxybutyrate-co- hydroxyvalerate) (PHBV), as an alternative substrate * Address reprints requests to: Dr. Akira Hiraishi, Department for microbial growth and denitrification (Biedermann et of Ecological Engineering, Toyohashi University of Technology, al.,1997; Boley et al., 2000; Khan and Hiraishi 2001; Toyohashi 441–8580, Japan. Mergaert et al., 2001; Müller et al., 1992; Schloe et al., E-mail: [email protected] 2000). In this connection, what kinds of PHBV-degrad- 300 KHAN and HIRAISHI Vol. 48 ing denitrifiers exist and actually work in such a new and the Japan Collection of Microorganisms, Wako, denitrification process is a subject of major concern. Japan, respectively. All other strains were from our PHBV-degrading denitrifying bacteria hitherto iso- own collection. lated have been identified as being Acidovorax facilis Growth media and cultivation. A complex medium (Mergaert et al., 2001), Acidovorax sp. (Schloe et al., designated PBY (0.5% Bacto-peptone (Difco Laborato- 2000), and Brevundimonas intermedia (Mergaert et al., ries, Detroit, MI, USA), 0.3% beef extract (Difco) and 2001). In a previous study, we isolated a new PHB- 0.1% yeast extract (Difco)) was used for maintenance, and PHBV-degrading denitrifying bacterium that exhib- preculture, and main culture used for chemotaxonomic Ϫ ited such a high denitrification rate as 19 mg NO3 -N re- and molecular studies. For studying PHBV degrada- moved g dry cellsϪ1 hϪ1 with PHBV as the electron tion, mineral medium RM2 (Hiraishi and Kitamura, donor (Khan and Hiraishi, 2001). A phylogenetic analy- 1984) supplemented with 0.2% PHBV and 0.01% sis based on 16S rDNA sequences showed that this yeast extract (Difco) was used. These media con- bacterium, designated strain NA10BT, belongs to the tained 1.8% agar when used as solidified media. For family Comamonadaceae, a major group of b-Pro- anaerobic denitrifying growth, PBY and PHBV media teobacteria (Willems et al., 1991), but does not form a were supplemented with 0.2% KNO3. Liquid cultures phylogenetic cluster with any species of the genera of were introduced into screw-capped test tubes (20-ml this family. Our attempt to find further similar PHBV-de- capacity) or bottles (100–500 ml capacity) and incu- grading denitrifiers in activated sludge was successful bated aerobically with vigorous shaking on a reciprocal (Khan et al., 2002), and a total of three strains of this shaker. For anaerobic growth, test tubes and bottles new group of the Comamonadaceae have been iso- were completely filled with a medium and incubated lated and characterized. In this article, we report the without shaking. Anaerobic denitrifying growth on agar taxonomic characteristics of the new PHBV-degrading media was performed using the AnaeroPak system denitrifying strains and propose to classify them into a (Mitsubishi Gas Chemicals, Niigata, Japan). All cul- new genus and species with the name Diaphorobacter tures were incubated at 28 to 30°C unless otherwise nitroreducens. specified. Microscopic studies. Morphological properties Materials and Methods were studied using an Olympus BX-50 phase-contrast microscope. For more detailed morphological studies, Bacterial strains. Strains NA10BT, KSP3 and a JEOL-1200EX transmission electron microscope KSP4, all of which were isolated from activated sludge was used. Gram staining was carried out as described taken from sewage treatment plants in Japan (Khan by Magee et al. (1975). and Hiraishi, 2001; Khan et al., 2002), were studied. Physiological and biochemical tests. Unless other- The reference strains used were Acidovorax avenae wise specified, all tests were carried out at 28 to 30°C. subsp. avenae DSM 7227T, Acidovorax facilis DSM Degradation of PHB and PHBV was determined as de- 649T, Acidovorax defulvi DSM 12644T, Acidovorax de- scribed previously (Khan and Hiraishi, 2001). Carbon lafieldii DSM 64T, Acidovorax konjaci DSM 7481T, Aci- nutrition tests were performed in 20-ml screw-capped dovorax temperans DSM 7270T, [Aquaspirillum] test tubes containing 8 ml of mineral medium RM2 (Hi- sinosum DSM 11556T (bracketed nameϭmisclassified raishi and Kitamura, 1984) supplemented with an or- generic name) (Wen et al., 1999), Brachymonas deni- ganic compound as the carbon source. All carbon trificans AS-P1T, Comamonas terrigena IAM 12421T, sources were filter-sterilized and added at a final con- Comamonas testosteroni IAM 12419T and JCM 10170, centration of 0.2% as a neutralized form. For anaero- Delftia acidovorans IAM 12409T, and Rhodoferx fer- bic denitrifying growth, the test tubes were completely T mentans FR2 . In addition, an unidentified bacterium, filled with the medium supplemented with 0.2% KNO3. strain DSM 13225 (ϭstrain LW1) (Katsivela et al., The test tubes were incubated for 10 days with vigor- 1999) were used. The strains with DSM, IAM, and ous shaking for aerobic growth or without shaking for JCM numbers were obtained from the DSMZ anaerobic growth. Growth was monitored by measur- (Deutsche Summlung von Mikroorganismen und Zel- ing the optical density at 660 nm. All other tests were lkulturen GmbH), Braunschweig, Germany, IAM Cul- performed as described by Hiraishi et al. (1995). ture Collection, The University of Tokyo, Tokyo, Japan, Fatty acid and quinone analyses. Whole-cell fatty 2002 Diaphorobacter nitroreducens gen. nov., sp. nov. 301 acids were extracted with methanol-HCl, and gas chro- isolates. matographic analyses of their methyl esters were per- Cultivation on PBY agar media showed that strain formed by NCIMB Japan Co., Shimizu, Japan. In some NA10BT formed slightly rough and raised colonies, cases, 3-OH fatty acids in the samples extracted were whereas strains KSP3 and KSP4 had smooth colonies separated by thin-layer chromatography and analyzed with entire margins. The colonies and cell suspension by gas chromatography as described previously (Hi- were colorless at the early phase of growth but turned raishi et al., 1992). Quinones were extracted, fraction- cream to beige at older stages. ated, and analyzed by spectrochromatography and mass spectrometry as described previously (Hiraishi et Phylogenetic and genetic analyses al., 1996). Almost complete sequences (1,521–1,522 bases) of DNA base composition and DNA-DNA hybridization. the 16S rDNA of strains NA10BT, KSP3, and KSP4 Genomic DNA was extracted and purified by the were determined in our previous studies. The se- method of Marmur (1961). DNA base composition quences of strains KSP3 and KSP4 were identical, (guanine [G]ϩcytosine [C] ratios) was determined by and these were different in only two positions from that the HPLC method (Katayama et al., 1984). DNA-DNA of strain NA10BT. Homology search indicated that the hybridization studies were performed by the quantita- three new isolates had sequence similarities of 95.0–