taxonomic characterization of an acidophilic bacterium

1 Taxonomic characterization of an acidophilic bacterium isolated from the acidophilic nitrifying process 2009 MASTER OF ENGINEERING NGUYEN HUYEN THI THANH Student Number: M085807 TOYOHASHI UNIVERSITY OF TECHNOLOGY 2 Taxonomic characterization of an acidophilic bacterium isolated from the acidophilic nitrifying process Abstract. Nitrification and denitrification processes in which ammonia in municipal and industrial wastewater is converted into nitrogen by nitrifying and denitrifying microorganisms, are the centre of nitrogen cycle. Nitrification and denitrification at acidophilic condition is considered as a prospective solution for treatment wastewater containing amonia. Toward this purpose, our laboratory has designed an operator ANSBR (Acidophilic Nitrifying Sequencing Batch Reactor) to treatment wastewater at acidophilic condition with sludge as seed. To understand the roles of microorganisms in this operator, we have studied bacterial community structure and isolated one strain that was first identified as the genus Nitrobacter (reidentified to the genera Dyella). In this study, a new strain isolated from ANSBR (Acidophilic Nitrifying sequencing Batch Reactor) were characterized taxonomically. The strain were characterized using phenotypic analysis and molecular based methods. Physiological test and quinone analysis were carried out as well as 16S rRNA sequence. Using 16S rRNA gene sequence analysis and phylogenetic tree showed this strain was similar to the genus Dyella, Frateuria and Rhodanobacter These Frateuria strains are generally known as acidophilic bacteria and also found in other acidophilic wastewater treatment systems (Kevin et al., 2005). As the result, our strain is Gram-negative, motile, could grow at pH 4.0 – 8.0 and optimum pH was 5.0. This strain grew slowly at 10 or 55 o C, and optimum temperature for for growth was 25 o C. This strain could grow at R2A medium, and very well at NA medium but could not grow at the medium that used for Frateuria, Acetobacter and Glucenobacter. Therefore, based on medium test datas, we found this strain was not the genus Frateuria that can produce a water-soluble brown pigment on glucose-yeast extract-CaCO 3 , and use AE broth – the medium was used to identified the genus Frateuria (Swings et al., 1984). In conclusion, we suggest this strain belongs to the genus Dyella as an acidophilic bacterium. This study with the aim is to provide the first general characteristics of the strain isolated from activated sludge for the further intensive research, particurlarly G/C content and fatty acid analysis, two powerful tools to identified strains at species level. 3 Content Chapter 1. Introduction about acidophilic nitrifying process and microorganism population in this process 1.1. General introduction about nitrifying process 5 1.2. The invention of acidophilic nitrifying process 6 1.3. The genus Dyella, Frateuria and Rhodanobacter 8 1.4. Purpose of our study 9 Chapter 2. Phenotypic characterization of an isolated strain 2.1. Introduction 10 2.2. Materials and methods 12 2.2.1. Test strain 12 2.2.2. Test growth medium 12 2.2.3. Cell morphology and cultural characteristic 12 2.2.4. pH test 12 2.2.5. Temperature test 13 2.2.6. NaCl tolerance test 13 2.2.7. Growth measurement 13 2.2.8. Quinone analysis 14 2.3. Results 16 2.3.1. Test strain and cultivation 16 2.3.2. Test growth medium 17 2.3.3. Cell morphology and cultural characteristic 18 2.3.4. pH test 19 2.3.5. Temperature test 20 2.3.6. NaCl tolerance test 21 2.3.7. Growth measurement 22 2.3.8. Quninone analysis 23 2.4. Discussion 23 Chapter 3. Molecular characteristic of the isolated strains 3.1. Introduction 24 3.2. Materials and methods 27 3.2.1. 16S rRNA analysis 27 4 3.2.2. Phylogenetic tree 32 3.3. Results 32 3.4. Discussion 32 Chapter 4. Concluding remarks 33 Annex 34 Index 36 References 44 5 Chapter 1 : Introduction about acidophilic nitrifying process and microorganism population in this process 1.1.General introduction about nitrifying process Nitrification is the biological oxidation of ammonia with oxygen into nitrite followed by the oxidation of these nitrites into nitrates. Degradation of ammonia to nitrite is usually the rate limiting step of nitrification. Nitrification is an important step in the nitrogen cycle in soil. This process was discovered by the Russian microbiologist Sergei Winogradskyi. The oxidation of ammonium into nitrite is performed by two groups of organisms, ammonia oxidizing bacteria and ammonia oxidizing archaea. Ammonia oxidizing bacteria can be found among the β – proteobacteria and γ – proteobacteria. In soils the most studied ammonia oxidizing bacteria belong to the genera Nitrosomonas and Nitrosococcus. Although in soils ammonia oxidation occurs by both bacteria and archaea, archaeal ammonia oxidizers dominate in both soils and marine environments, suggesting that Crenarchaeota may be greater contributors to ammonia oxidation in these environments. The second step (oxidation of nitrite into nitrate) is mainly done by bacteria of the genus Nitrobacter. NH 3 + CO 2 + 1.5 O 2 + Nitrosomonas → NO 2 - + H 2 O + H + NO 2 - + CO 2 + 0.5 O 2 + Nitrobacter → NO 3 - NH 3 + O 2 → NO 2 − + 3H + + 2e − NO 2 − + H 2 O → NO 3 − + 2H + + 2e − Both steps are producing energy to be coupled to ATP synthesis. Nitrifying organisms are chemoautotrophs, and use carbon dioxide as their carbon source. Nitrification also play an important role in the removal of nitrogen from municipal wastewater. The conventional removal is nitrification, followed by denitrification. The cost of this process resides mainly in aeration (bringing oxygen in the reactor) and the addition of an external carbon source (e.g. methanol) for the denitrification. In most environments, both organisms are found together, yielding nitrate as the final product. It is possible however to design systems in which selectively nitrite is formed (the Sharon process). Together with ammonification, nitrification forms a mineralization process which refers to the complete decomposition of organic material, with the release of available nitrogen compounds. This replenishes the nitrogen cycle. 6 1.2.The invention of acidophilic nitrifying bioreactor Chemolithoautrophic nitrifying bacteria, i.e., ammonia-oxidizing bacteria (AOB), catalyzing the first oxidation step of ammonia to nitrite and nitrite-oxidizing bacteria (NOB) completing the oxidation of the intermediate nitrite to nitrate are known to be sensitive to low pHs. Optimum growth occurs under neutral to moderately alkaline condition (pH 7.5 to 8.0). In liquid pure culture, growth is usually restricted to a low pH of 5.8 (AOB) or 6.5 (NOB) (Watson et al., 1989) and activity ceases typically below pH 5.5 (Hankinson et al., 1988, Jiang et al., 1999). The failure of AOB to cope with acidic conditions is thought to be primarily based on the unavailability of a substrate : with decreasing pHs, ammonia, the substrate of AOB (Suzuki et al., 1974), is increasing protonated. Nitrite, the substrate of NOB, undergoes protonation to nitric acid, which disproportionates to nitrate and gaseous nitric oxide at low pHs (Bock et al., 2001). Furthermore, when present at elevated concentrations under low pHs, free nitric acid negatively affects the growth and activity of nitrifying bacteria (Anthonisen et al., 1976). Despite these limitations, autotrophic nitrifying bacteria have been isolated from, or nitrifying activity has been demonstrated in, acidic environments, such as soils, activated sludge, and biofilms. Numerous nitrifying isolates have been obtained from soild with pHs around 4 (deBoer et al., 2001) even as low as 2.5 (Prosser, J.I 1989). However, the majority of such isolates do not show nitrifying activity in acidic mineral medium (deBoer et al., 2001). In contrast, autotrophic nitrifying activity in soil sample could be maintained at a pH as low as 4 (deBoer et al., 2001). Toward this trend, our laboratory has developed an acidophilic nitrifying sequencing batch reactor called ANSBR to treatment wastewater at acidophilic condition with sludge as seed. Two graduated students in our lab, Kuroki and Matsuba were successful to prove that the addition of yeast extract has affected to keep sludge and the high performance of nitrification in the reactor. To reveal change of the microbial population by the addition of yeast extract, succcessions of the bacterial populations in the acidophilic nitrification sequence batch reactor were evaluated by PCR-DGGE analyses. In addition of yeast extract, predominant bacteria in the reactor were novel microbes affiated with a candidate phylum TM7 that they are functionally unknown. Moreover, a member of our group, Nguyen Minh Giang has isolated a bacterium that first of all, identified to belong to the genus Nitrobacter (similar 98% to Nitrobacter winogradskyi (ATCC 25301) by sequencing 7 a short distance gene (665 bp). The discovery of this genus from sludge at acidophilic condition seems a significant finding. From this view, we take on responsibility to study this strain. Reidentified full gene of this strain by 16S rRNA sequence analysis and phylogenetic tree shows that this strain is closely related to the genus Dyella japonica XD53 T (AB110498). Besides, similar >95% to the genus Frateuria and Rhodanobacter. These genus belong to the family Xanthomonadaeae, class Gammaproteobacteria. Fig. 1-1. The phylogenetic tree of a subset of the γ-Proteobacteria , based on 16S rRNA gene sequence comparision, determined by neighbour-joining. Escherichia coli was used as the outgroup. Bootstrap percentages of 1000 replicates are indicated at nodes 8 1.3. The genera Dyella, Frateuria, Rhodanobacter Our strain is the most closely related to the genus Dyella. In literature about Dyella, there are seven genus : Dyella ginsengisoli (2009), Dyella japonica (2005), Dyella koreensis (2005), Dyella marensis (2009), Dyella soli (2009), Dyella terrae (2009), Dyella yeojuensis (2006). Dyella japonica came from Japan, Dyella ginsengisoli – one of two strains came from China, others came from Korea. Most of these strains were isolated from soil, mesophile and neutrality bacteria, unknown function except Dyella koreensis,Dyella japonica RB28, and Dyella ginsengisoli. Dyella koreensis is identified as β-glucosidase-producing bacterium. More specially, Dyella japonica RB28 is reported to cause human infection. Dyella gingengisoli is used to biodegradation of biphenyl and isolated from activated sludge. Our strain is similar 97% to Dyella japonica XD22, XD10, RB28; 96% to Dyella ginsengisoli LA-4, LA-3 and 97% to Dyella sp CHNCT14, CHNCT13, CHNCT15. The information of all strains of Dyella sp is unpublic. About Dyella ginsengisoli, two strain, as we have said above, are used to biodegradation of biphenyl and isolated from activated sludge. About Dyella japonica, only Dyella japonica RB28 is known as an damage bacterium to human. The genus Frateuria was validly established by Swings et al. in 1980 for bacteria that are polarly flagellated, produce brown pigment, have branched cellular fatty acids, and are Q – 8 equipped Gram-negative, incorporating “Acetobacter aurantius”. Frateuria aurantia is type species and only a species of this genus. The genus Frateuria is recognized as Pseudo-acetic acid bacteria because this strain showed intermediate characteristics to acetic acid bacteria. Up to now, four case of the isolation of Frateuria aurantius, all from flowers and fruits, there are three in Japan, and other in tropical region (Indonesia). Three general characteristics of this genus are acidophilic bacteria, can produce a water-soluble brown pigment on glucose-yeast extract- CaCO 3 , and use AE broth. According to the phylogenetic tree, our strain is closely related to the genus Frateuria sp (DQ419968) and Frateuria WJ69 (AY495959). Both of these strains that are quite different from those above metioned Frateuria, are isolated from wastewater systems that contain heavy metals. The paper of Frateuria sp (DQ419968) is unpublic, but the paper of Frateuria WJ69 was public in 2005, but this isolate is uncharacteristic. In general, the genus Frateuria can be isolated from either flowers and fruits or systems that contain heavy metals. As the genus Dyella, all strains of the genus Rhodanobacter are also not acidophilic bacteria. Only the strain of Dyella koreensis of the genus Dyella is non-motile. On the opposite, all of genus Rhodanobacter are non-motile except Rhodanobacter fulvus. These strains were isolated 9 from soil, only Rhodanobacter thiooxydans sp was isolated from a biofilm on sulfur particles used in an autotrophic denitrification process. In this process, this strain reduced nitrate, but not nitrite. In summary, three above genus belong to the family Xanthomonadaea, Gammaproteobacteria. Therefore, they are relative to some characteristics, the most differences are G/C content, composition of fatty acid. In conclusion, to identified one strain exactly, we need to carry out these experiments. 1.4. Purpose of our study In this study, a strain was isolated from acidophilic nitrifying sequencing reactor was studied taxonomically. Phylogenetic tree indicates this strain is closely related to the genus Frateuria, Dyella and Rhodanobacter Therefore, this study aims to identified and characteristic of this strain to understand their taxonomic position at physiologically and biochemically characteristics as well as DNA-based molecular properties. 10 Chapter 2. Phenotypic characterization of an isolated strain 2.1. Introduction Taxonomy is the science of classification and consists of two major subdisciplines, identification and nomenclature. Although the goal of identification is merely to provide the name of an isolate, most identification systems depend on first determining a number of morphological, biochemical, cultural, antigenic, and other phenotypic characteristics of the isolate before the name can be assigned. Phenotypic analyses have traditionally played an important role in bacterial identification and classification. Characteristics of taxonomic value that are widely used include various aspects of morphology, motility, nutrition and physiology, biochemistry. The morphology of cells normally includes cell shape, cell size, the gram stain reaction. In culture the morphology indicated the status of the cells, both in terms of the cells and in the case of primary isolates the differentiation state may be critical. Cell shape is generally characteristic of a given bacterial species, but can vary depending on growth conditions. Typical examples include coccus (spherical), bacillus (rod-like), spirillum (spiral) and filamentous. Bacteria generally form distinctive cell morphologies when examined by light microscopy and distinct colony morphologies when grown on petri plates. Gram staining is used to differentiate bacterial species into large groups (Gram possitive and Gram negative) based on the physical properties of their cell walls. Bacterial motility styles include motile by flagella, motile by gliding, motile by gas vessels, and nonmotile. Motile by gliding can be observed directly from examination of the tubes following incubation. Growth spreads out from the line of inoculation of the organism is motile. Flagella are readily seen with the electron microscope. In extremely large prokaryote, tufts of flagella can also be observed by phase contrast microscopy. Nutrition test is the test for mechanism of energy conservation (phototroph, chemooraganotroph, chemolithotroph). Phototrophic bacteria – green and purple bacteria use energy from sunlight and carbon from dioxide or organic carbon. Chemoauthotrophic bacteria obtains its nourishment through the oxidation of inorganic chemical compounds as opposed to photosynthesis. Chemolithotrophy is defined as the production of metabolically useful energy by the oxidation of inorganic compounds. Physiological bacteria performs by relationship between bacteria and environment such as oxygen, temperature, pH, and salt tolerances, ability to use various carbon, nitrogen, and [...]... uppermost layer of the water was discarded using Pasteur pipette 12 The bottom layers of chloroform and methanol was filtered into an oval flask using no.2 whattman filter 13 The mixture of chloroform and methanol was evaporated at 37oC using a rotatoryevapotator with a water bath 14 To remove any fraction of water left 2-3 ml of hexane and 1 ml of aceton was again added 15 The contents were transferred... is simple and takes about 2h, and numerous specimens can be analyzed rapidly each day One drawback is that the isolate must be cultured under highly standardized conditions of media and temperature in order to provide a valid basis of comparison woth other fatty acid profiles A second universal system, and the one of choice at present, is one in which all or most of the nucleotide sequence of the 16S... at 4oC 14 Transfer supernatant to micro tube and add chloroform/ isoamyl alcohol 350 µl 15 Suspend completely and centrifuge 12,000 rpm for 5 min at 4oC 16 Transfer supernatant to micro tube and add 3M sodium acetate 35 µl, 100% isopropanol equivalent volume Gently mix and put on freezer at -20oC for over 30 min 17 Centrifuge 12,000 rpm for 10 min at 4oC Remove supernatant and add 70% ethanol 400 µl,... corning tube and little water was also added 16 The tubes were kept for a while to separate water phase from hexane and after the separation hexane layer was transferred to an oval flask with a Pasteur pipette 17 The hexane was evaporated using ratatory evaporator 18 The lipid extract was re-suspended in 1-2 ml of hexane 19 Quinone were eluted through Sep-pack cartridges using a mixture of hexane and 10%... volatile methyl esters, and then separating and quantifying each fatty acid by gas-liquid chromatography A computer compares the resulting fatty acid profile with thousands of others in a huge database and calculates the best match or matches for the isolate The computer can also indicate that the isolate does not closely match any other fatty acid profile, which can lead to discovery of new genera or species... in terms of quantity, quality, and activity The quinone profile method allows good measurement of both fundamental and applied aspects of ecological and environmental microbiology In Proteobacteria, Q-10 is found mostly in the α subclass, Q-8 in the β and γ subclass, Q-9 in the γ subclass 11 2.2 Materials and methods 2.2.1 Test strain This strain was isolated from Nguyen Minh Giang, a member of bioreactor... path of evolution and the (additive) lengths of peripheral and internal branches connecting two terminal nodes indicate the phylogenetic distances between the respective organisms Table 3.1 Characteristics of G + C contents and major fatty acid of type strains of the genus Dyella, Frarteuria and Rhodanobacter Genera Species G + C content (mol%) Frateuria aurantia LMG 1558T Frateuria Major fatty acid... sources.Temperature is one of the most environmental factors affecting growth and survival of microorganisms Classification of bacteria based on temperature includes four groups : psychrophile, mesophile, thermophile, and hyperthermophile Psychrophile can live from -10 to 20oC and optimum temperature is 4oC Mesophile can live from 10 to 45oC and optimum is 39oC Thermophile can live from 40 to 75oC and optimum is... POP6DefaultModule Analysis Module BC – 3100APO6SR – seqOFFftOFF.saz 31 Analysis of sequencing data Sequencing data were compiled by the GENETYX – MAC program (Software development Co., Tokyo, Japan), analyzed for chimera detection with the CHIMERA – CHECK program version 2.7, and compared with those retrieved from Ribosomal Database Project II (Manz et al., 1992) Then the DNA sequencing of strain will be defined and... constant Microorganisms vary in their need for oxygen In fact, microorganisms can be divided into several groups depending on the effect of oxygen Biochemical tests for taxonomy oftenly are fatty acid and quinone Some fatty acid are unique to a group of bacteria For exanple the iso and anteiso saturated fatty acid are widely found in gram positive bacteria Gram negative bacteria very often contain straight . 1 Taxonomic characterization of an acidophilic bacterium isolated from the acidophilic nitrifying process 2009 MASTER OF ENGINEERING NGUYEN HUYEN THI THANH Student. TOYOHASHI UNIVERSITY OF TECHNOLOGY 2 Taxonomic characterization of an acidophilic bacterium isolated from the acidophilic nitrifying process Abstract. Nitrification and denitrification. layer of the water was discarded using Pasteur pipette. 12. The bottom layers of chloroform and methanol was filtered into an oval flask using no.2 whattman filter. 13. The mixture of chloroform