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Jun 28, 2012 - 4Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti ..... Both GJ, Gerards S, Laanbroek J (1992). Kinetics ...

African Journal of Microbiology Research Vol. 6(24), pp. 5126-5133, 28 June, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.1008 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Introducing a novel facultative nitrifying bacterium, "Nitrobacteria hamadaniensis" Mohammad Zare1, Mohammad Hassan Heidari2*, Farkhondeh Pouresmaeili3, Maryam Niyyati4 and Mohammad Moradi5 1

Department of Plant Pathology, Faculty of Agriculture, University of Bu-Ali-Sina, Hamadan, Iran. Cellular and Molecular Biology Research Center, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3 Department of Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 4 Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 5 Department of Microbiology, Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran.


Accepted 5 April, 2012

A new nitrifying bacterium has been identified as "Nitrobacteria hamadaniensis", from a potato farm in Hamadan, Iran. Its morphological and molecular characteristics were examined by electron microscopy, protein purification, SDS-PAGE and 16S rRNA analysis. It was cultured at the different conditions to determine the optimum pH and the generation time. The cells are rod shaped, 0.3-0.4×0.8-1.2 μm in size, and contains polar caps of intracytoplasmic membrane. The strain is lithotrophic and grow slower than heterotrophic strains. The best growth was observed at mixotrophic conditions. It grew at pH range between 6.7 to 8.3 with an optimum pH at 7.6. Based on the growth conditions, the generation time ranged from 7-16 h. The G+C content of this strain was 59 mol%. Also, 16S rRNA gene sequence analysis indicated that the bacterium represents a hitherto unknown line peripherally associated to the Caulobacteriaceae with low G+C relatives. The sequence of nearly complete 16S rRNA gene of the strain is recorded in the GenBank under number AY569007. According to the phylogenetic analysis and phenotypic criteria, it is proposed that the bacterium should be assigned to a new genus Nitrobacteria. Key words: Nitrobacter, nitrobacteria, nitrification, genotype, morphovar, biovar.

INTRODUCTION Nitrifying organisms of the genus Nitrobacter are polymorphic; which are mostly rods to pear shaped and possess polar caps of cytomembranes. The major source of energy and reducing power is from the oxidation of nitrite to nitrate. Some Nitrobacter strains are able to growth in heterotrophic conditions with acetate (Smith and Hoare, 1968), or pyruvate (Bock, 1976) as carbon source. These organisms are also facultative (Bock et al., 1988; Freitag et al., 1987). Nitrite-oxidizing bacteria are ubiquitous in terrestrial and aquatic natural environments under moderate conditions (Bock and Koops, 1992;

*Corresponding author. E-mail: [email protected] Tel: +982123872584. Fax: +9821 22171928.

Laanbroek and Woldendorp, 1995; Both et al., 1992). There are some indications that nitrifying bacteria may also be present in extreme environments such as acid soils (De Boer and Laanbroek, 1989; De Boer et al., 1991; Hakinson and Schmidt, 1988), and acid sulfinic ore (Bock et al., 1992). These bacteria have been found in alkaline environments such as saline soda lakes and soda soil samples in Wadi Natrun, Egypt (Imhoff et al., 1979). The nitrite oxidoreductase consisted of three major proteins with apparent molecular weights of 116.000, 65.000 and 32.000 kDa (Sundermeyer-Klinger et al., 1984). These species of Nitrobacter, that is, Nitrobacter winogradskyi (Watson et al., 1981; Winogradsky, 1892; Engel et al., 1954), Nitrobacter hamburgensis (Bock et al., 1983), Nitrobacter vulgaris (Bock et al., 1990), and Nitrobacter alkalicus (Sorokin et

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al., 1998; Heubuelt 1929) have already been described. This article describes the cultural and the biochemical characteristics of a new strain of bacteria and the result of a phenotypic and phylogenetic analysis based on 16S rRNA gene sequences. MATERIALS AND METHODS Isolation procedures Isolation The strain was isolated from soil in a potato field in Hamadan (Lat. 34˚47′46″ N, Long. 48˚30′57″ E), Iran (March 2004, GenBank accession No. AY569007). Five hundred mg soil was shrilled in a 300 ml flask according to Drews (1968). The cells were grown at 28°C for 2 weeks. Then the cell suspension was inoculated into agar media (basic mineral medium and 18 g agar-agar added to 1 liter of water) (Merck, Germany). Various types of colonies were isolated and grown separately on agar plates with nitrite and mineral salt (Merck, Germany) by multiple subcultures.

Culture conditions The culture media was prepared as described by Drews (1968) and Bock et al. (1990). The basic mineral was supplemented with 400 mg sodium acetate, 1500 mg yeast extract (Difco, USA) and 1500 mg peptone (Merck, Germany) in 1 L of water at pH 7.6 (for aerobic-growth), and nitrate instead of peptone, as an electron acceptor for anaerobic conditions. Master plates (Stock cultures) were prepared under mixotrophic conditions.


diode-array spectrophotometer (Hewlett Packard, USA) was used for the investigation. Proteins were identified from polyacrylamide gels by the method of Francis and Becker (1984).

DNA and 16S rRNA analysis For isolation of DNA, 2 g of wet weight cells were suspended in 5 ml TE buffer (50 mmol Tris, 20 mmol EDTA, pH 8.0) (Merck, Germany). Cell lysates were prepared as described by Kraft and Bock (1984). Total DNA was isolated and purified according to Marmur (1961). The G+C content was calculated from the denaturizing rate according to De Ley (1970). PCR amplification for the nearly complete 16S rRNA gene and sequencing were done as described by Brinkhoff and Muyzer (1997) and Muyzer et al. (1995). Sequences were compared using ARB software (Ludwig et al., 2004). The 16S rRNA gene sequences of the isolates were automatically aligned to sequences stored in the ARB database.

Electron microscopy Cells cultured under mixotrophic conditions were concentrated 100fold, and methods for fixation, embedding, and ultra-thin sections were those described by Bock and Heinrich (1969). Sections were stained with uranyl acetate and lead citrate (Electron Microscopy Sciences, USA). Electron micrographs were taken with a transmission electron microscope (Carl Zeiss EM-900; Zeiss, Germany) at 80 kV accelerating voltage. Negatives were scanned at 1200 dpi resolution, by CanoScan 8800F (Canon, Japan), and pictures were processed using Adobe® Photoshop® software (CS4 Extended, Middle East Version 11.0).

Phylogenic analysis Batch cultivation Batch cultures were grown in 50 ml liquid media. Based mineral medium (Merck, Germany) supplemented with sodium acetate, yeast extract, and peptone for routine and mixotrophic cultivation under aerobic conditions was used (Bock et al., 1990; Drews, 1968). For heterotrophic growth (Bock et al., 1990), the medium was modified as follows: 1000 mg nitrate (Merck, Germany) as an electron acceptor was added to 1 L of medium under anaerobic conditions. All experiments were done at pH 6.7, 7.6, and 8.3 and repeated at least 3 times.

Analytical procedures Protein purification The protein was purified from enzyme extracts and measured exactly as previously described by Bradford (1976), Spector (1978), Davie (1982) and Laemmli (1970). Membranes and nitrite oxidoreductase were isolated and purified according to Sundermeyer-Klinger et al. (1984).

Gel electrophoresis SDS-PAGE (Merck, Germany) was performed as described by Milde and Bock (1984) and Sundermeyer-Klinger et al. (1984). The cytochrome spectra of cell-free extract of the new strain (104) was determined as previously explained by Sorokin et al. (1998). A

In order to establish the precise taxonomic position of unknown bacterium, the entire 16S rRNA sequences of the strain (104) was determined.

RESULTS G+C analysis The G+C content of the new strain 104 DNA was 59%. This value is different from Nitrobacter winogradskyi (61.7 mol%), Nitrobacter hamburgensis (61.2-61.6 mol%), Nitrobacter vulgaris (58.9-59.9 mol%) and Nitrobacter alkalicus (61.5-62.4 mol%) (Bock et al., 1990; Sorokin et al., 1998). The derived 16S rRNA consisted of 1421 nucleotides. The determined sequences were compared with those of other 16S rRNA sequences available in the GenBank. Nitrobacteria hamadaniensis (strain 104) with 95.9% genetic homology with Caulobacter, and 96.3% with Brevundimonas (Table 1). It has 86-87.4% genetic homology to the genus Nitrobacter, which are classified as one of the main classes of Caulobacteriaceae. A phylogenic tree, depicting the relationship of unknown bacterium with Caulobacteriaceae and close relatives,are shown in Figure 1, and the sequence similarities are


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Table 1. Similarity matrix of 16S rRNA sequences.

Afipia clevenlandensis Afipia felis Blastobacter denifricans Bradyrhizobium japonicum Rhodopseudomonas palustris Nitrobacter winogradskyi ATTCC 25381 Nitrobacter winogradskyi ATTCC 14123 Nitrobacter sp. strain R6 Nitrobacter hamburgensis strain x 14 Nitrobacter hamburgensis strain nb 14 Nitrobacter alkalicus strain AN1 Nitrobacter alkalicus strain AN2 Nitrobacteria hamadaniensis strain 104 Brevundimonas diminuta














98.6 96.5 97.9 96.9 97.4 97 96.8 96.6 96.9 97.3 97.4 86.3 87.7

96.5 97.3 96 96.9 96.7 96.4 96.3 96.5 96.9 97.5 85.3 87.1

98.2 97.3 97.1 96.5 96.7 96.8 96.9 97.2 97.1 86 87.8

98.3 98.2 97.9 97.7 97.7 97.9 98.3 98.3 86.3 87.8

97.1 96.9 96.7 97.5 97.2 97.2 97.2 86.6 87.8

98.7 98.8 98 98.4 99.1 99 86.2 87.7

99.3 98 98.3 99.2 99.1 87.1 87.1

98 98.4 99.2 99.1 86.7 87.6

99.5 98.4 98.3 86.1 88

98.6 98.6 86.7 87.6

99.9 87.4 87.8

87.5 87.7


Figure 1. A phylogenetic tree derived from 16S rRNA gene sequences, the tree was created by using the neighbor-joining method and Knuc values, showing the phylogenetic interrelationships between Nitrobacteria hamadaniensis and other close relatives. The bootstrap values are indicated.

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Table 2. Influence of organic compounds on growth of the nitrite-oxidizing strain 104 under anaerobic conditions at different pH values in batch culturea.

Growth condition pH 8.3 pH 7.6 pH 6.7 a

Strain 104 growth Weak Weak Weak

Amounts of NO2 concentration (mmol) 0.15 0.16 0.1

Specific activity of the strain 104 was weak in heterotrophic condition at different pH values.

indicated in Table 1. The comparisons help distinguish the new bacterium located at the periphery of the Caulobacteriaceae.

Protein analysis The protein analysis of cell free extracts of the strain (104) grown at pH 7.6, indicated α-bands (437 and 589 nm in size), presenting nitrite cytochrome spectra comparable to those of other Nitrobacter species. This strongly suggests that the new strain belongs to the Nitrobacteria genus. In addition to α-bands, cytochrome c 550 and cytochrome c oxidase type α3 (maximum 550 nm up to 607 nm) were other prominent features of the strain, respectively. Physiological characteristics Nitrobacteria hamadaniensis grew optimally at 26-28°C and pH 7.6. Colonies on agar plates formed within 3, 4 and 12 d under mixotrophic, heterotrophic, and lithotrophic conditions. Colonies on mineral salt agar plates sized 0.1 mm in diameter, were orange, circular, and swelled. Optimum growth rates were obtained in mixotrophic medium containing nitrite, sodium acetate, yeast extract and peptone. Strain 104 was able to grow under nitrite-oxidizing lithoautotrophic, mixotrophic and heterotrophic conditions at pH 6.7 to 8.3. The stoichiometry analysis, conversion of nitrite to nitrate in batch culture was 96.3-99.1% and nitrate to nitrite, under anaerobic condition was 1-1.6% (Table 2). The organisms grew on mineral medium supplemented with organic compounds such as sodium acetate, yeast extract and peptone as sources of energy and carbon. Batch cultivation at different pH values clearly demonstrated that the nitrite-oxidizing strain (104) isolated from soil belonged to facultative neutrophilic species. It could grow within a pH range of 6.7 to 8.3 (Figure 4). The growth rate at pH below and above 7.6, was extremely slow. During growth at pH above 7, cells started to branch. Their optimum growth was close to their upper pH limit (around 7.6). The main difference of the strain 104 from all four known species of the genus

Nitrobacter (Nitrobacter winogradskyi, Nitrobacter hamburgensis, Nitrobacter vulgaris and Nitrobacter alkalicus) was the rapid growth on culture medium used for cultivation of strain (104) with a starting pH 7.6 and a nitrite concentration of about 1 g. Strain 104 was able to grow in nitrite limited culture media within a broad pH range from 6.7 to 8.3 with an optimum pH 7.6 (Figure 4). The doubling times of autotrophically and mixotrophically grown Nitrobacteria strain 104 was 16 and 7 h at pH 7.6, respectively. This was higher than the rate described for neutrophilic species grown lithoautotrophicaly with nitrite (Bock and Koops, 1992; Keen and Prosser, 1987). Our study shows that organic compounds had an influence on the growth of nitrite-oxidizing strain 104 from soil, at pH 6.7 to 8.3, during 5 d incubation. There were no significant differences between the bacteria activity in heterotrophic with 1000 mgP/L nitrate under anaerobic conditions at different pH values in batch culture (Table 2). The pH profile in the kinetics of oxidation in batch culture was significantly different for the cells grown at different pH values. The profile for the rate of nitrite oxidation (Figure 4) measured with cells grown at pH 6.7 was similar to that measured for Nitrobacter species (Hunik et al., 1993). The curve had its maximum at pH 7.6 and decreased at a pH higher than 8. The nitriteoxidizing activity measured with cells grown at pH 7.6, 6.7, and 8.3 that was maximal at pH 7.6, respectively, and incubation time was 192 h. SDS-PAGE analysis The results of SDS-PAGE of cell-free extracts, based on phenotypic criteria, showed that Nitrobacteria hamadaniensis is composed of 4 bands, 2 strong and prominent bands of ~116 kDa, one 67 kDa, and a 14 kDa band, respectively (Figure 3). DISCUSSION A new species of bacteria was identified, that was different from Nitrobacter winogradskyi (Bock 1976), Nitrobacter hamburgensis (Bock et al., 1983), and Nitro-


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Figure 2. Electron photograph of Nitrobacteria hamadaniensis ultrasturcture. Thin section from grown cell mixotrophically and harvested during exponential phase of growth, showing lamellar membrane system, poly-β-hydroxybutyrate (PHB), and polyphosphate granules (X: 50,000). PHB = Poly-β-hydroxybutyrate. PP = Polyphosphate granules. CM = Cytoplasmic membrane.

Figure 3. SDS-PAGE (12.5% acryl amide) of cell free extract from Nitrobacteria hamadaniensis protein stained with comassie blue R- 250.

bacter vulgaris (Bock et al., 1990). The new bacteria grew at all the different culture conditions, as reported by Steinmueller and Bock (1977), and the growth rates in mixotrophic media could be used for taxonomic purposes. Our observations showed that this new organism, like Nitrobacter vulgaris (Bock et al., 1990) grows by dissimilation and reduces nitrate where nitrate is present as an alternative electron acceptor. The cells of Nitrobacteria hamadaniensis had a similar shape, size, and ultrastructure. This is further evidence to prove the existence of the new Nitrobacteria species. The new isolate is different from most of the other Gram negative

bacteria. They are short rod cells, pear shaped, 0.30.4×0.8-1.2 μm in size, and motile. They tend to form flocks (Figure 2) and/or biofilms on the glass surface of culture flasks. Cell division normally occurres by budding. The cytoplasmic membrane protrudes into the cytoplasm, forming a polar cap of interacytoplasmic membranes. Carboxysomes were found in the strains grown under chemolithotrophic conditions, but this was not observed under mixotrophic conditions. The other typical inclusion bodies were poly-β-hydroxybutyrate and polyphosphate granules. These results correspond to the other four strains, Nitrobacter winogradskyi, (Winogradsky, 1892), Nitrobacter hamburgensis (Bock et al., 1983), Nitrobacter vulgaris (Bock et al., 1990), and Nitrobacter alkalicus (Sorokin et al., 1998). However, in the new strain the upper band was clearly separated. The phenotypic criteria are similar to the recent findings obtained by Samelis et al. (1995). This suggests that each species yields a specific protein profile identical to the respective type strain. Bock et al. (1990) reported that there are significant differences between protein profiles of Nitrobacter winogradskyi, Nitrobacter hamburgensis, Nitrobacter vulgaris and Nitrobacter alkalicus. In conclusion, the new nitrite-oxidizing bacteria strain 104 isolated from soil differs from previously described species by their potential to grow and oxidize nitrite at pH 7.6. The batch continuous cultivation showed their remarkable ability to adapt to abroad range of pH values. Our data shows that the analysis of the whole cell protein was a quick and effective method to distinguish any bacteria among Nitrobacteria hamadaniensis. Species description Description of Nitrobacteria hamadaniensis sp. nov. nitro bacteria. npl (NL. fr. Nitr- + bacteria): the soil bacteria concerned in nitrification. Nitrobacteria




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Figure 4. A-B. Influence of pH and culture condition on dynamic of nitrite oxidation of Nitrobacteria hamadaniensis strain 104 in Batch culture and influence of pH on oxidation activity of washed cells grown in Batch culture at pH 6.7-8.3. The culture started to wash out at D = 0.008 h-1. A. Lithoautotrophic condition. B. Mixotrophic condition. C-D. Influence of pH on the growth of Nitrobacteria hamadaniensis strain 104 in Batch culture with 14.5 mmol nitrite at pH 6.7, 7.6 and 8.3 (Culture wash out at D = 0.008 h-1). C. Nitrite oxidizing activity of cell cultivated at lithoautotrophic with nitrite. D. Nitrite oxidizing activity of cell cultivated at mixotrophic condition.

hamadaniensis = hamadani, ensis. Hamadan Iranian place name; M.L. adj. hamadaniensis of Hamadan. The cells are Gram negative, short rods to pear shaped with a size of 0.3-0.4×0.8-1.2 μm. Each cell contains several carboxysomes and posses a polar cap of cytomembranes to form flattened vesicles. Cells produce extracellular polymers at all growth conditions causing the formation of a biofilm. Facultative lithoautotrophs oxidize nitrite to nitrate under aerobic conditions and reduce nitrate to nitrite under anaerobic conditions. Cells grow chemolithotrophically, heterotrophically, or

mixotrophically. The growth rate in chemo-organic medium is more rapid than in chemolithotrophic medium. The surfaces of colonies on mineral salt agar plates are 0.1 mm in diameter after 12 d at 28°C and pH values between 7.6-7.8. They have two prominent proteins of 116 and 67 kDa, and a 14 kDa protein which appears as a faint band. The G+C content of DNA is 59 mol% and the sequence of nearly complete 16S rRNA gene of strain 104 is stored in the GenBank database and Japan collection of microorganisms under accession numbers, AY569007(http://www.ncbi.nlm.nih.gov/Genbank/index.ht


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ml), and JCM 14789 (http://jcm.riken.go.jp/JCM/ catalogue.shtml), respectively. Nitrobacteria hamadaniensis is deposited in Persian type culture collection under the number, PTCC 1681 (http://www.irost.org/en/ptcc/index.asp?code=1#). ACKNOWLEDGEMENTS We thank M. Piriai and F. Niazi (Department of Anatomy, Faculty of Medicine, Shahid Beheshti University of Medical Sciences) for their excellent technical assistance. REFERENCES Bock E (1976). Growth of nitrobacter in the presence of organic matter II Chemoorganotropic organic Growth of Nitrobacter agilis. Arch. Microbiol., 108(3): 305-312. doi: 10.1007/BF00454857. PMID: 942282. Bock E, Heinrich G (1969). Morphologische und physiologische untersuchungen an zellen Von Nitrobacter winogradskyi Buch. Arch. Microbiol., 69(2): 149-159. doi: 10.1007/BF00409759. Bock E, Koops HP (1992). The genus Nitrobacter and related genera. In The prokaryotes, 2nd ed., vol 1. Edited by Balows A, Truper HG, Dworkin M, Harder W, Shleifer KH. Springer-Verlag, New York, NY., pp. 2302-2309. Bock E, Sundermeyer–Klinger H, Stackebrandt E (1983). New facultative lithoautotrophic nitrite-oxidizing bacteria. Arch. Microbiol., 136(4): 281-284. doi: 10.1007/BF00425217. Bock E, Wilderer PA, Freitag A (1988). Growth of Nitrobacter in the absence of dissolved oxygen. Water Res., 22(2): 245-250. doi: 10.1016/0043-1354(88)90085-1. Bock E, Koops HP, Moller UC, Rudert M (1990). A new facultatively nitrite oxidizing bacterium, Nitrobacter vulgaris sp. nov. Arch. Microbiol., 153(2): 105-110. doi: 10.1007/BF00247805. Bock E, Koops HP, Ahlers B, Harms H (1992). Oxidation of inorganic nitrogen compounds as energy source. In The prokaryotes, 2nd, vol 1. Edited by Balows A, Truper HG, Dworkin M, Harder W, Schleifer KH. Springer-Verlag, New York, pp. 414-430. Both GJ, Gerards S, Laanbroek J (1992). Kinetics of nitirite oxidation in two Nitrobacter species grown in nitrite-limited chemostats. Arch. Microbiol., 157(5):436-441. doi: 10.1007/BF00249101. Bradford MM (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72(1-2): 248-254. doi: 10.1016/00032697(76)90527-3. PMID: 942051. Brinkhoff T, Muyzer G (1997). Increased species diversity and extended habitat range of sulfur-oxidizing Thiomicrospira spp. Appl. Environ. Microbiol., 63(10): 3789-3796. PMID: 9327542. Davie JR (1982). Two-dimensional gel systems for rapid histone analysis for use in minislab polyacrylamide gel electrophoresis. Anal. Biochem., 120(2): 276-281. doi: 10.1016/0003-2697(82)90348-7. PMID: 7046507. De Boer W, Laanbroek HJ (1989). Ureolytic nitrification at low pH by Nitrosospira species. Arch. Microbiol., 152(2): 178-181. doi: 10.1007/BF00456098. De Boer W, Gunnewiek K, Veenhuis PJA, Bock E, Laanbroek HJ (1991). Nitrification at low pH by aggregated chemolithotrophic bacteria. Appl. Environ. Microbiol., 57(12): 3600-3604. PMID: 16348608. De Ley J (1970). Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J. Bacteiol., 101(3): 738-754. PMID: 5438045. Drews G (1968). Mikrobiologisches praktikum fur naturwissenschaftler. Springer Verlag, Berlin-Heidelberg-New York. Engel H, Krech E, Friederichsen I (1954). Beitrage zur kenntnis der

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Winogradsky S (1892). Contributions a la morphologie des organismes de la nitrification. Arch. Sci. Biol., (St. Petersb.) 1:88-137.