INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1997, p. 727-734 Vol. 47, No. 3 0020-7713/97/$04.00 + 0 Copyright 0 1997, International Union of Microbiological Societies Amaricoccus gen. nov., a Gram-Negative Coccus Occurring in Regular Packages or Tetrads, Isolated from Activated Sludge Biomass, and Descriptions of Amaricoccus veronensis sp. nov., Amaricoccus tamworthensis sp. nov., Amaricoccus macauensis sp. nov., and Amaricoccus kaplicensis sp. nov. A. M. MASZENAN,’ R. J. SEVIOUR,l* B. K. C. PATEL,2 G. N. REES,3t AND B. M. McDOUGALLl Biotechnology Research Centre, La Trobe University, Bendigo, Ectoria 3550, Faculty of Science and Technology, Grifith University, Nathan, Brisbane, Queensland 4111, and Faculty of Applied Science, University of Canberra, Belconnen, Australian Capital Territory 261 6, Australia Three isolates of gram-negative bacteria, strains Ben 10ZT, Ben 103T, and Ben 104T,were obtained in pure culture by micromanipulation from activated sludge biomass from wastewater treatment plants in Italy, Australia, and Macau, respectively. These isolates all had a distinctive morphology; the cells were cocci that usually were arranged in tetrads. Based on this criterion, they resembled other bacteria from activated sludge previously called “G” bacteria. On the basis of phenotypic characteristics and the results of 16s ribosomal DNA sequence analyses, the three isolates were very similar to each other, but were sufficiently different from their closest phylogenetic relatives (namely, the genera Rhodobacter, Rhodovulum, and Paracoccus in the (Y subdivision of the Proteobacteria) to be placed in a new genus, Amaricoccus gen. nov. Each of the three isolates represents a new species of the genus Amaricoccus; strains Ben 10ZT, Ben 103T, and Ben 104T are named Amaricoccus veronensis, Amaricoccus tamworthensis, and Amaricoccus mucauensis, respectively. An isolate des- ignated Ben 10IT,which was isolated independently by Cech and Hartman in Kaplice, Czech Republic, was also characterized and belongs to the same genus. We propose that the isolate of Cech and Hartman should be placed in another new species, Amaricoccus kaplicensis. Most industrial and domestic wastewaters are treated by the of CaCO,, 0.10 g of Ca(N03)2, 0.05 g of KCl, 0.05 g of K2HP04, 0.05 g of activated sludge process before disposal. However, little is MgSO, * 7H,O, 0.187 g of Na,S * 9H,O, 15 g of bacteriological agar (Difco), and 1.0 ml of a 10X vitamin stock solution. The vitamin stock solution contained (per known about the microbes present in these systems and their liter) 1 mg of calcium pantothenate, 1 mg of niacin, 5 x lop3 mg of biotin, 5 X roles, a situation which reflects the inadequate methods used lop3mg of cyanocobalamin, 5 X mg of folic acid, 1 mg of pyridoxine, 1 mg to study the activated sludge community (1,4,25,40). In 1990, of p-aminobenzoic acid, 1 mg of cocarboxylase, 1 mg of inositol, 1 mg of thia- Cech and Hartman (6) described large numbers of gram-neg- mine, and 1 mg of riboflavin. Three isolates, strains Ben 102=, Ben 103T, and Ben 104T, were cultured from activated sludge biomass from plants in Verona, Italy, ative cocci arranged in tetrads which appeared in a laboratory Tamworth, Australia, and Macau, respectively, after micromanipulation with a scale treatment system fed with glucose. Cech and Hartman Skerman micromanipulator (35). Many attempts to isolate gram-negative “G”- called these organisms “G” bacteria, and although they were like bacteria on a wide range of media routinely used for bacterial isolation from isolated in pure culture, these organisms were not identified activated sludge samples were unsuccessful (32). Inoculated plates of GS me- dium were incubated at 25°C for 2 weeks, and the colony growth of the micro- (7). On the basis of the results of a few phenotypic tests and manipulated organisms was checked daily. Each colony was then streaked out morphology, it was suggested that they might be members of several times onto fresh GS medium to ensure its purity, which was confirmed by the genus Methanosarcina in the domain Archaea (8). Routine microscopy. Strain Ben 10ITwas a gift from J. S. Cech. All cultures were stored microscopic examination of biomass samples from wastewater on GS medium in 20% glycerol at -80°C. Phenotypic characterization. The substrate utilization patterns of all of the treatment plants located around the world revealed the pres- isolates were determined with the BIOLOG system (Special Diagnostic, Mel- ence of large numbers of organisms morphologically similar to bourne, Australia). Cells were grown on GS agar at 25°C for 5 days and then the organisms described by Cech and Hartman (6). Because suspended in physiological saline to the turbidity standard recommended by the these bacterial morphotypes occur frequently in biomass sam- manufacturer. Inoculated GN and GP microplates were incubated at 25°C for 24 h and then examined by using an image analysis system consisting of a ples from activated sludge treatment processes, we attempted Panasonic video camera and video capture software (Mediastation, version 2.5; to obtain several isolates of “G”-like bacteria to determine Ulead Systems, Inc., Torrance, Calif.) with a MIRO 20 video card. Each captured their taxonomic status by a polyphasic taxonomic approach image was edited and analyzed by using the Sigmascan image analysis program (38) and to determine their relationships to the original un- software (Jandel Scientific Software, San Rafael, Calif.), which compensated for any color development in the control wells of the microtiter plates. Cells of each identified isolate of Cech and Hartman. isolate were also inoculated onto API ZYM strips (Bio MCrieux, Lyon, France) that were incubated at 25°C for 4 h, and the results were recorded as recom- MATERIALS AND METHODS mended by the manufacturer. Each isolate was characterized at least in dupli- cate. The Microbact 24E system (Oxoid, Melbourne, Australia) was used to Isolation and maintenance of bacterial strains. All of the strains whose determine selected biochemical properties of the strains, as described below. sources are shown in Table 1 were grown on GS medium of Williams and Unz Catalase and oxidase tests and a test to determine motility by the hanging drop (42), which contained (per liter) 0.15 g of glucose, 0.50 g of (NH,),SO,, 0.10 g method were also performed with all of the isolates by using standard methods (36). Growth responses to temperature and pH were determined by using GS medium. The phenotypic data were analyzed by performing a numerical taxon- omy analysis with simple matching coefficients (Ss,,,) and the unweighted pair group * Corresponding author. Phone: 61-54-447459. Fax: 61-54-447476. with mathematical average (UPGMA) clustering algorithm (37) by using the E-mail: [email protected]. NTSYS-pc software package, version 1.80 (Exeter Software, New York, N.Y.). t Present address: The Murray Darling Freshwater Research Cen- Preparation of specimens for scanning electron microscopy. The isolates used tre, Albury, NSW 2640, Australia. for scanning electron microscopy were fixed overnight in 3% glutaraldehyde in 727 728 MASZENAN ET AL. INT.J. SYST.BACTERIOL. TABLE 1. Sources of the gram-negative isolates used in this study Agrobacterium tumefaciens, which is known to produce cellu- lose fibrils (2), did fluoresce. Strain Source Date of isolation Physiological characteristics. The results obtained in the Ben 10lTu Laboratory scale sequence batch reactor, substrate utilization experiments performed with BIOLOG Kaplice, Czech Republic GN and GP systems showed that the four isolates studied had Ben 102T Full-scale activated sludge plant treating 27 March 1996 very similar carbon utilization patterns, although there were domestic waste, Verona, Italy some differences (Table 2). All of the strains used dextrin, Ben 103T Industrial plant treating malting waste, 15 September 1995 L-arabinose, D-arabitol, cellobiose, D-fructose, L-fucose, D-ga- Tamworth, Australia Ben 104T Full-scale activated sludge plant treating 11 February 1996 lactose, a-D-glucose, rn-inositol, maltose, D-mannitol, D-man- domestic waste, Macau nose, D-psicose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, methyl pyruvate, monomethyl succinate, a- a This strain was kindly provided by J. S. Cech. hydroxybutyric acid, P-hydroxybutyric acid, a-ketobutyric acid, DL-lactic acid, succinic acid, succinamic acid, D-alanine, L-ala- 0.1 M phosphate buffer (pH 7.0). Cells were centrifuged, washed with phosphate nine, L-asparagine, L-glutamic acid, L-serine, maltotriose, pal- buffer, and postfixed in OsO, for 2 h before they were dehydrated in a graded acetone series (0 to 100%). Dehydrated cells were then spread onto glass cov- atinose, D-ribose, salicin, D-tegatose, D-xylose, D-lactic acid methyl ester, L-lactic acid, D-malic acid, L-malic acid, methyl erslips coated with a 2% ethanolic solution of 3-aminopropyltriethoxysilane.The coverslips were critical point dried, coated with gold, and then viewed with a succinate, pyruvic acid, N-acetyl-L-glutamic acid, and adeno- Cambridge Stereoscan model 150 MK2 scanning electron microscope. sine. None of the isolates utilized a-cyclodextrin, N-acetyl-D- Determination of the 16s rDNA sequence. 16s ribosomal DNA (rDNA) am- plification and sequencing were performed for each isolate as described by galactosamine, i-erythritol, a-D-lactose, lactulose, D-raffinose, Blackall (3). PCR-amplified products were purified by using a Magic Wizard D-glucosaminic acid, D-saccharic acid, sebacic acid, L-alanyl-gly- purification kit (Promega, Annadale, Australia), and cycle sequencing reactions cine, glycyl-L-aspartic acid, L-histidine, L-leucine, L-ornithine, L- were performed with an Omnigene Hybaid thermal cycler (Intersciences, Mel- pyroglutamic acid, uridine, inulin, amygdalin, a-methy1-D-ga- bourne, Australia) by using a Ready Reaction dye terminator kit (Applied Biosystems, Melbourne, Australia). The sequencing products were cleaned and lactoside, a-methyl-D-mannoside, sedoheptulosan, stachyose, precipitated as recommended by the manufacturer.