An Aerobic Formate-Utilizing Bacterium, Cupriavidus Sp., Isolated from Activated Sludge of Wastewater Treatment
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Ann Microbiol (2014) 64:869–873 DOI 10.1007/s13213-013-0663-4 SHORT COMMUNICATION An aerobic formate-utilizing bacterium, Cupriavidus sp., isolated from activated sludge of wastewater treatment Retno Wahyu Nurhayati & Yoshihiro Ojima & Saori Kitatsuji & Prayoga Suryadarma & Masahito Taya Received: 22 December 2012 /Accepted: 2 May 2013 /Published online: 1 June 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract An aerobic formate-assimilating bacterium, de- by oxygen, through the following reaction: HCOO- +H+ + o -1 noted as strain FAB, was newly isolated from activated 1/2 O2 → CO2 +H2O, ΔGc =−281 kJ mol , or by another + - + sludge of wastewater treatment. Phylogenetic analysis based oxidant such as NAD ; HCOO + NAD → CO2 + NADH, o -1 on 16S rDNA sequence assigned the isolate to the genus ΔGc =−22 kJ mol (Metzler 1977; Tishkov and Popov Cupriavidus. Scanning electron micrography revealed that 2004). Moreover, oxidation of formate to carbon dioxide is this bacterium has a coccal morphology, and from some catalyzed by formate dehydrogenase which is abundantly physiological assays, the bacterium was characterized to available in anaerobic bacteria (Ferry 1990). A recent report be Gram-negative, nitrate-reduction-positive and catalase- from Kim et al. (2010) gave the detailed evidence that a positive. In addition to formate, strain FAB was able to single strain of bacterium could grow using formate as a sole utilize fructose, acetate or pyruvate as a preferred carbon carbon source in an anoxic environment. Their work has source. Compared with a close relative, Cupriavidus shed light on the ability of several hyperthermophilic ar- necator, our isolate exhibited a greater growth rate on for- chaea, such as Thermococcus sp., to convert formate to mate under an aerobic condition. hydrogen. To our knowledge, there are a few reports dealing with Keywords Formate assimilation . Aerobic bacterium . pure cultures with formate as a sole carbon and energy Cupriavidus sp. source and, particularly, under an aerobic condition (Quayle and Keech 1959; Kelly et al. 1979; Friedrich et al. Formate, HCOO-, is the simplest organic compound that 1979; Niekus et al. 1980). Alcaligenes eutrophus, recently contains single carbon as a backbone (C1 compound). named as Cupriavidus necator (Vandamme and Coenye Formate oxidation can be considered energetically enough 2004), is a well-known chemolithotroph which is capable to support microbial growth. This process can be facilitated of growing on formate (Friedrich et al. 1979). In addition, Cupriavidus oxalaticus or Ralstonia oxalaticus, taxonomi- cally re-classified from Pseudomonas group by Sahin et al. : : : : (2000), which was originally screened for oxalate utiliza- R. W. Nurhayati Y. Ojima S. Kitatsuji P. Suryadarma tion, has been proven as a formate-assimilating bacterium. M. Taya (*) Division of Chemical Engineering, Department of Materials In the present study, we report the isolation of a bacteri- Engineering Science, Graduate School of Engineering Science, um from activated sludge utilizing formate as a sole carbon Osaka University, 1-3 Machikaneyama-cho, and energy source under an aerobic condition. The isolate Toyonaka, Osaka 560-8531, Japan (named as strain FAB) was characterized and identified in e-mail: [email protected] terms of phylogenetic, morphological and physiological Present Address: analyses. The current study will contribute to exploring the P. Suryadarma diversity of aerobic microorganisms with formate-utilizing Department of Agroindustrial Technology, ability under a limited nutritional condition. Faculty of Agricultural Technology, Bogor Agricultural University, Jl. Raya Darmaga Kampus IPB Darmaga, Activated sludge, collected from an aeration tank in a Bogor 16680 West Java, Indonesia wastewater treatment plant (located in Toyonaka, Osaka, 870 Ann Microbiol (2014) 64:869–873 Japan) was used as inocula. Enriched cultivation was cultivation with notable turbidity was selected in these pro- performed in an incubator with shaking (120 rpm) at cesses for further examination. 30 °C, with the inoculants added to 5 mL of sterile enrich- An isolated strain was identified based on 16S rDNA ment medium in test tubes. Enrichment medium, denoted as gene sequence. Total DNA of cells was extracted according MF medium, contained 1 g yeast extract, 2.96 g sodium to Marmur (1961). The 16S rDNA gene was amplified using formate, 2 g (NH4)2SO4,13.6gKH2PO4,0.25g the forward primer BSF8/20 (5’-AGAGTTTG MgSO4·7H2O, 0.5 mg FeSO4·7H2O, 1 mg thiamine, and ATCCTGGCTCAG-3’) and the reverse primer 1492R (5’- 0.5 mg trace minerals (NiCl2,CoSO4,MnSO4,ZnCl2, GGTTACCTTGTTACGACTT-3’)inathermalcycler CuCl2) per liter of deionized water, with minor modification (Perkin-Elmer, USA) under the following conditions: initial of the M63 medium (Miller 1972). Initial pH of medium denaturation at 94 °C for 2 min, then 30 repeated cycles of was adjusted to 6.0. The inocula were grown by means of 30 s at 94 °C, 90 s at 55 °C, and 120 s at 68 °C. A PCR batchwise subculturing, and yeast extract concentration was product was extracted and purified using a Gel Extraction decreased stepwise from 1 g L−1 to ultimately zero. kit (Qiagen, USA). An amplified DNA fragment was se- From the enriched cultivation lasting for over 20 days, an quenced by a commercial service using a genetic analyzer aliquot of culture broth was taken and inoculated onto yeast (ABI PRISM®; Life Technologies, USA). The BLAST pro- extract-free MF agar plates. After incubating for 3 days at gram was used to search sequence homology, and a phylo- 30 °C, morphologically distinct colonies were picked up and genetic tree was created using ClustalX2 and ClustalW2 tested for their ability to assimilate formate. Survival iso- software. lates were cultured in the MF liquid medium without yeast The cell morphology was observed with a scanning elec- extract, in the same way as employed in the enriched culti- tron microscope (SEM) (S-5000L; Hitachi, Japan). Cells vation. The colony isolation and liquid cultivation were were fixed in 2.5 % glutaraldehyde for 2 h and dehydrated repeated several times for purifying each isolate. The with serial gradients of aqueous ethanol solutions. After Fig. 1 Phylogenetic relationship and morphological property of strain FAB. a Phylogenetic tree derived from similarities of 16S rDNA sequence. The tree was created based on the neighbor-joining method and Knuc values. Bootstrap values from 1,000 replicates are shown on the branch points. The selected species have been referred to (Nishihara et al. 1998) and the sequence data were cited from the GenBank database. Bacillus subtilis (X60646) was used as an out-group species. b SEM images of strain FAB Ann Microbiol (2014) 64:869–873 871 platinum sputtering (5 nm in thickness), the specimen was et al. 2012). C. necator NBRC 102504 (obtained from Japan subjected to SEM observation. Society for Culture Collections, Chiba, Japan) was used as a Gram-staining was carried out using a Hucker-Conn’s reference strain. modified method (Hucker and Conn 1923). Catalase assay Through the enriched cultivation and repeated colony was conducted according to a method described by Aebi isolation on formate medium, a bacterial strain, denoted as (1984), using crude enzyme extracted from cells, based on FAB, was screened based on its active proliferation. BLAST the decrease of H2O2 which was monitored by a spectro- analysis revealed a high homology (99 %) of our isolate photometer. Nitrate reduction test was conducted, according with Cupriavidus necator. A phylogenetic tree was to a protocol as described elsewhere with a minor modifi- constructed based on 16S rDNA sequence. Our isolate was cation (Knapp and Clark 1984). found to be assigned to β-proteobacteria (Fig. 1a). The high For testing the ability to utilize selected carbon sources, homology to C. necator suggested that strain FAB belongs isolated cells were pre-cultured in LB medium overnight at to the genus Cupriavidus. 30 °C. The culture was centrifuged (12,000 rpm, 4 °C) for Table 1 summarizes the morphological characteristics of 10 min and then washed using MF medium without yeast strain FAB as well as those of C. necator as a reference. Our extract and carbon source. Cells were suspended in the strain was identified as a Gram-negative bacterium which medium so as to give an optical density at 660 nm, OD660 was positive in motility, catalase activity, and nitrate reduc- ≅1.0, and then inoculated at 2 % (v/v) into 5 mL of same tion. Scanning electron micrographs (Fig. 1b) exhibited the medium, containing 2 g L−1 of each carbon source. The morphological property of bacterium. Strain FAB was a culture tubes were incubated with shaking (120 rpm) at coccus-shaped bacterium of 1.0 μm in diameter. As reported 30 °C for 24 h. by Makkar and Cassida (1987), C. necator has a coccoid rod The time profile of the cultures on formate were exam- shape with 0.7–0.9 μm × 0.9–1.3 μm in size. Hence, the ined under almost the same conditions as mentioned above, using 200-mL baffled conical flasks containing 40 mL of medium on a rotary shaker (120 rpm). Oxygen mass transfer coefficient, KLa, was determined according to Cooper et al. (1944), based on the oxidation of sodium sulfite in a liquid phase. Cell growth was monitored by measuring OD660 of culture broth and dry cell weight (g L−1) was calculated by multiplying the OD660 value with a pre-determined factor of 0.45 (strain FAB) or 0.54 (Cupriavidus necator). Formate concentration was analyzed as described elsewhere (Ojima Table 1 Morphological characteristics of strain FAB and its close relative, C. necator FAB C. necator Motility + + Gram stain - - Cell shape Coccus Coccoid rod Catalase test + + Nitrate reduction + + Carbon source utilization Glucose +/− - Galactose +/− - Fructose + + Sucrose +/− +/− Pyruvate + + Acetate + + Glycerol +/− - Fig. 2 Time profiles of cell growth and formate concentration in – − Characters scored as + positive, negative, and +/ equivocal aerobic cultures of strain FAB and C.