KR101255096B1 - Method for Enrichment Culture of Ammonia-Oxidizing Archaea Through Co-culturing with Sulfur-Oxidizing Bacteria and Novel Ammonia-Oxidizing Archaea Isolated from Marine By Using the Same Method - Google Patents

Abstract

The present invention relates to a method for enrichment culture of ammonia-oxidizing archaea comprising co-culturing with sulfur-oxidizing bacteria, and to isolate it from the ocean using the method. Novel ammonia oxidizing archaea SJ strain (KCTC 11878BP). By using the culture method of the present invention, it is possible to isolate and identify various ammonia-oxidizing archaea, which greatly contributes to the circulation of ammonia in the ecosystem, to easily obtain these archaea resources, and to analyze the characteristics and functions of ammonia-oxidizing archaea. It can also be useful for research. In addition, the novel ammonia-oxidizing archaea of the present invention can be effectively used for the purification of the marine environment contaminated with ammonia.

Description

Method for Enrichment Culture of Ammonia Oxidized Archaea by Coculture with Sulfated Bacteria and Novel Marine Derivative Ammonia-Oxidized Archaea Isolated by the Method of Sulfur-Oxidizing Bacteria and Novel Ammonia-Oxidizing Archaea Isolated from Marine By Using the Same Method}

The present invention relates to a method for enriching culture of ammonia-oxidized archaea using co-culture with sulfated bacteria and a novel marine-derived ammonia-oxidized archaea isolated by the method.

Archae has been known as an extreme microorganism because most of the cultured archaea has been cultivated from extreme environments such as acidic environments, high heat environments, and high salinity environments. In addition, anaerobic methane production is limited to archaea, and this metabolic capacity has not been reported in other domains so far. The prejudice against archaea as an extreme microorganism has radically changed as molecular ecological techniques using polymerase chain reaction (PCR) have been applied to the field of environmental microorganisms. That is, novel archaea 16S rRNA gene sequences were found from seawater in studies using domain-specific PCR primers (DeLong 1992, PNAS. 89: 5685-5689; Fuhrman et al 1992, Nature 356: 148-149). Following this monumental discovery, a surprisingly wide variety of archaea 16S rRNA gene sequences were found even in a variety of tragic environments (Schleper et al 2005, Nat. Rev. Microbiol. 3: 479-488). This means that archaea, like bacteria, are widely distributed in any environment, rather than in certain extreme areas. Molecular ecological studies are reporting on the diversity and abundance of archaea, but there are still many mysteries about the physiological and ecological roles of these catastrophic archaea. Ammonia oxidation, which has long been regarded as a property confined to the bacterial domain, has been estimated to be characteristic of crenarchaeal groups I.1a and I.1b archaea, based on recent meta-gnome analysis, and this estimate is based on Sargasus seawater. Discovery of Archaea amoA-like Gene in Environmental Shotgun Sequencing Studies of Seawater and Ca. Supported by genetic analysis of Cenarchaeum symbiosum (Bock and Wagner 2001; Treusch et al 63 2005; Venter et al 2004; Hallam et al 66 2006). Molecular ecological studies show that ammonia-oxidizing archaea dominates ammonia-oxidizing bacteria in the North Sea and in seawater, soil and marine sediments (Wuchter et al 2006; Chen et. al 2008; Leininger et al 2006; Park et al 2008b). Significant evidence on the ammonia oxidation of autotrophic archaea is characterized by the characterization of " Ca. Nitrosopumilus maritimus SCM1," a mesophilic Crenarchaeon (group I.1a), first cultured from an aquarium, and the concentration of related archaea from the North Sea waters. The culture result was then obtained by enrichment of thermophilic ammonia oxidizing archaea (AOA) (Konneke et al 2005; Wuchter et al 2006; de la Torre et al 2008; Hatzenpichler et al 2008). Microorganisms in marine sediments are very important in the biogeochemical cycle because they are present in very large quantities (Whitman et al 1998). Nitrification is essential for the nitrogen cycle in marine sediment, and it is metabolically combined with denitrification and anaerobic ammonia oxidation to remove nitrogen in the form of molecular nitrogen and to produce nitrous oxide, also known as greenhouse gas. (Dalsgaard et 80 al 2005, Tal et al 2005). Compared to studies of nitrification of archaea in seawater, the information so far known about archaea nitrification in marine sediments is very limited. Cultivation of ammonia-oxidizing archaea is essential but very difficult to study the metabolic activity of archaea in environments such as soil and marine sediment.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a method for culturing ammonia oxidizing archaea (AOA), which is known as an egg culture microorganism. As a result, co-culturing with sulfur-oxidizing bacteria (SOB) can successfully enrich the ammonia-oxidizing bacteria (AOA) and analyze its microbiological properties. The present invention has been accomplished by experimentally proving that it can be done.

It is therefore an object of the present invention to provide a method for enriching culture of ammonia-oxidizing archaea.

In addition, another object of the present invention is to provide a novel marine-derived ammonia-oxidizing archaea isolated by the above-mentioned thickening culture method.

Another object of the present invention is to provide a composition for use in purifying a marine environment contaminated with ammonia, which comprises the ammonia-oxidizing archaea as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a method for enriching culture of ammonia-oxidizing archaea comprising co-culturing with sulfur-oxidizing bacteria. do.

The present invention is based on the experimental demonstration that co-culture of ammonia-oxidizing archaea, known as non-cultured microorganisms, with sulfur-oxidizing bacteria can successfully enrich the ammonia-oxidizing archaea.

As used herein, the term “archaea” is another group of organisms that is distinguished from bacteria in prokaryotes. That is, as a class of prokaryotes, some of which are similar to bacteria and some of which are similar to eukaryotes, are largely classified into basophils, methane-producing bacteria, and hyperthermic bacteria.

As used herein, the term "ammonia-oxidizing archaea" means archaea that has the ability to perform nitrification to oxidize ammonia to its oxidized form nitrite.

The term "nitride action" herein is ammonium nitrogen in the soil, seawater or ocean sediments (NH ₄ -N) a nitrite nitrogen by the action of microorganisms (NO ₂ -N) through the nitrate nitrogen (NO ₃ - N) means to be converted. The nitrification effect by the microorganism is known to be possible only if the microorganism has an enzyme called ammonia monooxigenase.

In the present invention, the term "enrichment culture" is a culture that predominates by selectively advantageously forming the growth of the microorganism to be separated by adjusting the selection medium or selection conditions, when a small number of microorganisms to be separated are present Means the way.

According to the method of the present invention, by co-culturing with sulfur-oxidizing bacteria, it is possible to culture so that ammonia-oxidizing archaea predominates in the culture solution.

As used herein, the term "sulfur-oxidizing bacteria" refers to bacteria that grow by using a reduced sulfur compound as an electron donor. According to a preferred embodiment of the present invention, the sulfur-oxidizing bacterium is a bacterium using the reduced form of the sulfur compound as a substrate. The reduced form of the sulfur compound includes sulfide, thiosulfate, polythionate, elemental sulfur or sulfite, more preferably thiosulfate , Sulfite or tetrathionate. According to a preferred embodiment of the present invention, the concentration of the sulfur compound in the reduced form in the co-culture medium is in the range of 0.1-5.0 mM, most preferably in the range of 0.1-2.0 mM.

As used herein, the term "co-culture" means culturing under the same conditions in the same medium.

According to a preferred embodiment of the present invention, the co-culture medium comprises natural sea water. According to another preferred embodiment of the invention, the co-culture medium comprises ammonia. According to another preferred embodiment of the present invention, the concentration of ammonia in the co-culture medium is 0.1-8.0 mM, more preferably 0.1-4.0 mM. According to a preferred embodiment of the present invention, the co-cultivation in the present invention comprises the step of passage in fresh medium after completion of ammonia oxidation by ammonia oxidizing archaea. After the ammonia oxidation is completed in the medium by the ammonia oxidizing archaea in the medium, the subculture of the ammonia oxidizing archaea is further promoted by passage in a fresh medium. More preferably, the passaging is performed continuously until the ammonia oxidizing archaea reaches a desired ratio.

According to another aspect of the invention, the invention provides an ammonia-oxidizing archaea SJ strain (Accession No .: KCTC 11878BP) isolated from the ocean.

According to yet another aspect of the present invention, the present invention provides a composition for use in the purification of an ammonia-contaminated marine environment comprising the ammonia-oxidized archaea SJ strain (Accession Number: KCTC 11878BP) as an active ingredient. .

The novel ammonia-oxidized archaea SJ strain (Accession No .: KCTC 11878BP) of the present invention is a strain cultured and isolated from the ocean by co-culture with the sulfur-oxidizing bacteria of the present invention described above. Ammonia-oxidized archaea SJ strain of the present invention was deposited on February 25, 2011 by the Korean Collection for Type Cultures of the Korea Research Institute of Bioscience and Biotechnology with accession number KCTC 11878BP.

The novel ammonia-oxidized archaea SJ strain (KCTC 11878BP) of the present invention has much better ammonia oxidation activity compared to the ammonia oxidative archaea isolated from the existing ocean. The new strain of the present invention is capable of completely oxidizing ammonia even at a relatively high concentration of ammonia of 3 mM, and exhibits ammonia oxidation activity, even at a high concentration of ammonia of 8 mM. According to one preferred embodiment of the present invention, the ammonia-oxidized archaea SJ strain (KCTC 11878BP) of the present invention has a nucleic acid base sequence of the rRNA gene represented by SEQ ID NO: 1 sequence.

SJ strain of the present invention (KCTC 11878BP) is promoted ammonia-oxidation activity and growth by co-culture of sulfur-oxidized bacteria.

The novel ammonia-oxidizing archaea of the present invention can be effectively used for the purification of the marine environment contaminated with ammonia. The marine environment is meant to include any environment, including sea water, and may include, for example, a seawater aquarium, a cage farm on land and sea, and a high salt wastewater treatment plant.

In summary, the features and advantages of the present invention described above are as follows.

(Iii) According to the method of the present invention, not only can ammonia-oxidizing archaea known as a non-culturing microorganism be enriched and cultured using microbial-microbial interaction, but also it can maintain the growth of this bacterium.

(Ii) The method of the present invention is capable of stably enriching and culturing archaea predominant in an actual separation environment, and establishes a method for culturing and maintaining ammonia-oxidizing archaea that contributes to the circulation of ammonia in the ecosystem. Can be used for securing and function research.

(Iii) The ammonia-oxidized archaea SJ strain (KCTC 11878BP) of the present invention can be effectively used for the purification of marine environment contaminated with ammonia.

The present invention relates to a method for enrichment culture of ammonia-oxidizing archaea comprising co-culturing with sulfur-oxidizing bacteria, and to isolate it from the ocean using the method. Novel ammonia-oxidized archaea SJ strain (KCTC 11878BP). By using the culture method of the present invention, it is possible to isolate and identify various ammonia-oxidizing archaea, which greatly contributes to the circulation of ammonia in the ecosystem, to easily obtain these archaea resources, and to analyze the characteristics and functions of ammonia-oxidizing archaea. It can also be useful for research. In addition, the novel ammonia-oxidizing archaea of the present invention can be effectively used for the purification of the marine environment contaminated with ammonia.

Figure 1 shows the oxidation of ammonia by ammonia-oxidized archaea (AOA) after oxidation of thiosulfate by sulfated bacteria (SOB) in SJ enriched culture. Panel (A) shows the change in concentration of ammonia, nitrite, nitrate, thiosulfate and dissolved oxygen, respectively. Panel (B) shows the copy number of bacterial and archaea 16S rRNA genes. Error bars represent standard deviation of the mean of three experiments.
Figure 2 shows a photomicrograph of archaea cells in SJ culture. Panel (A) shows FISH analysis by staining archaea cells with Cy5-labeled probe (red, Arch915) and bacterial cells with fluorescein-labeled probe (green, EUB 338). Panel (B) shows total cells stained with DAPI (blue). The same position was selected for panel (A) and panel (B) in the FISH image. Scale bars correspond to 5 μm. Panel (C) is a transmission electron micrograph of the cells stained negatively. The inserted photo is an enlarged form. The scale bar corresponds to 0.5 μm. Panel (D) is the photograph which observed the thinly fragmented cell with the transmission electron microscope. Scale bars correspond to 0.2 μm.
Figure 3 is a phylogenetic analysis of the archaea 16S rRNA gene sequence (about 900 bp) obtained in SJ and AR culture. Branch patterns supported by more than 60% bootstrap values (1,000 repetitions) by neighbor-joining were indicated by their respective bootstrap values. Numbers in parentheses indicate the frequency of duplicate clones. The scale bars represent 5% estimated sequence divergence.
FIG. 4 shows that the archaea-specific 16S rRNA gene copy number in SJ and AR enrichment cultures is dependent on ammonia concentration. Ammonia was also oxidized at ammonia concentration of 4 mM, but slightly incomplete oxidation was observed. Error bars represent standard deviation of the three experimental means.
Figure 5 shows the phylogenetic tree of the archaea amoA gene sequence (about 600bp) obtained in SJ enriched culture and AR enriched culture. Branch patterns supported by more than 60% bootstrap values (1,000 repetitions) by near-junction means are indicated with their respective bootstrap values. The numbers in parentheses indicate the frequency of duplicate clones. Based on the origin of the reference sequence, cluster groups are indicated on the right side of the figure. Scale bars represent 2% putative sequence divergence.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

1. Collection of Samples

Samples of marine sediment (approximately 100 g of sediment and 1 L of seawater) were taken from the East Sea (E 128 ° 35 ', N 38 ° 20'; 650 m deep) and Svalbard (North Pole, E 016 ° 28 ', N 78 ° 21'; Depth Harvested using a sterile glass bottle at 78 m). Each sample was named SJ and AR sample. The collected precipitate sample was mixed well into a slurry, and then about 10 ml of the slurry was transferred to a sterile conical tube and stored at 4 ° C. in the laboratory.

2. Culture and Chemical Analysis of Ammonia Oxide Archaea (AOA)

Cultivation of bacteria containing ammonia oxidizing archaea (AOA) was carried out using ammonium sulfate (1 mM), sodium thiosulfate (0.1 mM to 1 mM), sodium bicarbonate (2.5 mM), Potassium phosphate (0.1 mM), trace element mixture (1X) and vitamin solution (1X) were added and in natural seawater medium, including sterile East Sea beach water, under aerobic conditions (Widdel and Bak. 1992; Widdel and Bak. 1992). The pH of the medium was adjusted to 8.2 using NaOH or HCl. Marine sediment slurry (0.5 g) to be inoculated in 10 ml of medium was taken from the East Sea of Korea and the Arctic Sea of Svalbard and placed in a Hungate tube sealed with butyl-rubber stoppers. The cultivation was conducted under a static condition where the inverting was performed every day without continuous shaking. After ammonia was oxidized (typically after about two weeks), 5% of the total culture volume was transferred to a new marine medium at 25 ° C., dark. After batch culture, the pH of the medium did not change significantly (changed from pH 8.0 to pH 8.2). Ammonia and thiosulfate were measured via color change (Solorzano 1969, Voroteliak et al 1993). Nitrite and nitrate were measured using ion chromatography (ICS-1500, Dionex, Sunnyvale, CA) equipped with OnGuard II Ag Cartridge (Dionex) (Manning and Bewsher 1997). Dissolved oxygen (DO) and pH were measured using 3-Star DO (Thermo, Beverly, MA) and S20 383 SevenEasy pH Meter (Mettler Toledo, Switzerland), respectively.

3. Measurement of kinetics of ammonia oxidation and oxygen absorption

Kinetic measurements of oxygen uptake and ammonia oxidation were performed on AR cultures with modifications suitable to known methods (Bodelier et al 1996, Martens-Habbena et al 2009, Park et al 2010). Oxygen uptake was measured in an Oxygraph system (Hansatech Instruments Ltd., England) equipped with an S1 Clark type polarographic oxygen electrode disk, a 2 ml DW1 borosilicate glass reaction / sample vessel, a magnetic stirrer bar, and equipped with Oxygraph Plus software. . Oxygen microsensors were polarized continuously for at least 3 hours before use. All measurements were made in a water bath recycled at 28 ° C. Activity measurements were made using cells in the late phase of the exponential phase, monitoring ammonia oxidation and nitrite production in AR culture. After taking 10 ml of the culture from the culture, it was immediately transferred to a pre-heated glass tube in a 28 ° C. water bath. Sub-samples were then filled into a 2 ml DW1 electrode glass vessel equipped with an oxygen electrode disk and carefully sealed with a plunger assembly. Oxygen uptake was continuously monitored after initial equilibration at least 40 minutes. The required ammonium was added from the concentrated solution using a Hamilton syringe. Apparent half-saturation constants (Km) and maximum velocity (Vmax) for oxygen and ammonia were measured from a plot of oxygen absorption rate.

4. Fluorescence in situ hybridization (FISH) and transmission electron microscope (TEM) analysis

FISH analysis was performed on paraformaldehyde-fixed samples according to the method described by the prior art (Amann et al. 1995). Cells were recovered from 10 ml culture and resuspended in PBS (130 mM NaCl, 10 mM sodium phosphate, pH 7.5), followed by fixation by adding cold paraformaldehyde solution (4% in PBS) in 3 volumes. Store at 16 ° C. for 16 hours. Cells were harvested and washed three times with PBS solution to remove residual fixative solution and then concentrated 10-fold in PBS solution. Resuspension of immobilized cells was mixed with an equal volume of cold ethanol and the mixture was stored at -20 ° C.

For FISH analysis of bacteria and archaea, paraformaldehyde immobilized samples were subjected to Cy3-labeled archaea specific probes (Arc915) (Alm et al., 1996) and FAM-labeled bacterial specific probes (EUB338) (Amann et al. , 396 1990). All cells were visualized using DAPI. Samples were observed with a Nikon 80i (Nikon, Tokyo) equipped with a bedside lens.

To analyze ammonia oxidative archaea (AOA) from the enriched culture by transmission electron microscopy (TEM), 10 ml cultured cells were mixed with a mixture of 2.5% paraformaldehyde and 1.5% glutaraldehyde buffered with 0.1 M phosphate (pH 7.2). Fix for 2 hours at 4 ° C., post-fix for 1 hour in 1% osmium tetroxide in the same buffer, and then dehydrate in 70% to 100% continuous ethanol Transfer to propylene oxide and embed in Epon-812 (TAAB, England) (Luft 1961). Ultrathin sections made of ULTRACUTE (Leica, Austria) ultra microtome were double stained (uranyl acetate and lead citrate) and examined using a CM 20 electron microscope (Philips, Netherlands) (Reynolds 1963).

5. Genome DNA and Total RNA Extraction

Genome DNA and total RNA were extracted to measure the copy number of specific genes and their expression in genome DNA. Cells were collected by centrifugation from 50 ml of culture at 2 mid days of ammonia oxidization (approximately 0.5 mM ammonia remaining) and 2 days after ammonia depletion and stored at −70 ° C. until further analysis. DNA was isolated from frozen cells using the Genomic DNA Prep Kit (Solgent, Korea) according to the manufacturer's instructions. RNA was isolated from frozen cells using the RNeasy mini kit (Qiagen, Germany) and cDNA was synthesized using the SuperScript First Strand Synthesis System (Invitrogen, San Diego, CA) according to the manufacturer's instructions. The concentrations of DNA, RNA and cDNA were measured using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, 418 DE).

6. Preparation of clone libraries and phylogenetic analysis of 16S rRNA, archaea amoA and carbon fixation genes

The key carbon immobilization genes (acc, mce and 4-budh) of the bacterial and archaea 16S rRNA, putative archaea amoA, and the 3-hydroxypropionate / 4-hydroxybutyrate pathway are shown in the table below. Amplification using the primers described in 1. To design primers for PCR amplification, the putative key carbon immobilized gene sequences of the 3-hydroxypropionate / 4-hydroxybutyrate pathway were selected from Berg et al. (2007). PCR conditions for all genes were 5 minutes at 94 ° C; 30 cycles of 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 45 seconds at 72 ° C; 7 minutes at 72 ° C. Clone libraries of PCR product and purified using the PCR Purification System Kit (Solgent, Korea) according to the instructions of the manufacturer using the T & A Cloning Vector Kit (Real Biotech Corporation, Taiwan) Lai transfected in ligation and, E. coli DH5α cells Prepared by conversion. Positive putative clones were transferred to 96-well plates containing LB (Luria broth) with ampicillin (100 μg / ml). Clones were incubated overnight at 37 ° C. and stored in LB / glycerol at −70 ° C. prior to PCR screening and sequencing. The library clone uses two M13 universal primers M13F (5'-GTTTCCCAGTCACGAC-3 ': SEQ ID NO: 34) and M13R (5'-TCACACAGGAAACAGCTATGAC-3': SEQ ID NO: 35) to PCR for the presence of an insert Direct screening by PCR conditions were 5 minutes at 94 ° C; 30 cycles of 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 30 seconds at 72 ° C; 5 minutes at 72 ° C. Positive clones were randomly selected from each library and PCR products were purified using a PCR Purification Kit (Solgent, Korea). PCR products were directly sequenced using ABI 441 PRISM® BigDye ™ Terminator Cycle Sequencing Ready Reaction kits (Applied 442 Biosystems, Foster City, Calif.) And ABI PRISM 3730xl DNA Analyzer (Applied 443 Biosystems, Foster City, Calif.).

For phylogenetic analysis, gene sequences of relevant taxons were obtained from the GenBank database and multiple alignments were performed using the Clustal X program (Thompson et al 1997). Phylogenetic trees were created using the MEGA 3 software program (Kumar et al 2004).

7. Quantification of Gene Copy Numbers Using Real-time PCR

All quantitative real-time PCR experiments were performed using a MiniOpticon real-time PCR detection system (Bio-Rad Laboratories, Hercules, Calif.) And built-in Opticon Monitor Software version 3.1 451 (Bio-Rad Laboratories, Hercules, Calif.). 15 minutes at 95 ° C. to amplify all genes; 40 cycles of 20 seconds at 95 ° C., 20 seconds at 55 ° C., and 20 seconds at 72 ° C. were used, and the measurement was detected between each cycle. A standard curve was prepared for each response using a standard of reference genes ranging from 0 to 10 ⁹ gene copies for each response. The standard curve shows the correlation between the known copy number of the gene and the cycle threshold (Ct) value as known. Specificity of the real-time PCR reaction was confirmed by analysis of melting curves, measurement of the size of the reaction product using gel electrophoresis, and sequencing of the reaction product. PCR amplification primer information for each target gene is shown in Table 1 above.

8. Bicarbonate incorporation analysis

To measure the bicarbonate incorporation of ammonia oxidative archaea (AOA) during ammonia oxidation, ¹³ C-labeled bicarbonate (99% ¹³ C; Cambridge Isotope 466 Laboratories, Andover, MA; 5 mM) Hungate tube sealed with butyl-rubber stopper after thiosulfate (0.1 mM or 1.0 mM thiosulfate) is completely depleted, but before archaea growth begins, to rule out absorption of ¹³ C-labeled bicarbonate by (SOB) To 10 ml medium. The ratio of ¹³ C-labeled bicarbonate concentration to unlabeled bicarbonate concentration was determined by measuring the bicarbonate concentration before and after spiking ¹³ C-labeled bicarbonate. Control cultures were those cultured under the same conditions without addition of ¹³ C-labeled bicarbonate. Total cells were recovered by centrifugation before complete ammonia oxidation and lyophilized for archaea membrane lipid analysis. Lyophilized cells were sonicated with methanol and three dichloromethanes. The extracts were combined and water was removed using a small pipette Na ₂ SO ₄ column. To determine the amount of ¹³ C incorporation into archaeological membranes, biphytane was released from GDGTs present in lipid extracts by treatment with HI / LiA1H4, gas chromatography (GC) and isotope ratio-monitoring GC / Measurement was made using a mass spectrometer.

Experiment result

1. Establishment of enrichment culture method of ammonia-oxidizing archaea using a combination of sulfur-oxidizing bacteria

In previous studies we reported that ammonia-oxidizing archaea was present in marine sediments in Korea. Attempts to enrich the ammonia-oxidizing archaea using a medium containing only ammonia as the only energy source with or without antibiotics have not been successful. Following this attempt, we find that in most cases, the independent trophic bacteria are sulfur-oxidizing bacteria (SOB), hydrogen-oxidizers, methanotrophs, light Attempts have been made to promote the growth of ammonia-oxidizing archaea through coculture with phototrophs and lignin-decomposers. As a result, promotion of oxidation of ammonia by ammonia-oxidizing archaea was found only in co-culture medium with sulfur-oxidizing bacteria containing thiosulfate as an auxiliary energy source in addition to ammonia. For about 20 months, after ammonia oxidation was completed, successive passages every two weeks resulted in the development of ammonia-oxidized archaea (AOA) into dominant bacteria in the culture medium. Eukaryotic rRNA genes were not amplified from any culture. That is, the present inventors, by using the co-cultivation method with the sulfur-oxidizing bacteria described above, from the sediments of the coastal sediment and Svalbard (North Pole) in the East Sea of Korea, each of the archaea belonging to the crenarchaeal group I.1a ammonia oxidative archaea (AOA) Successfully enriched. In the present specification, the culture from the sediment in the East Sea of Korea is referred to as SJ culture, and the culture from the Svalbard (North Pole Region) sediment is referred to as AR culture.

In the thickening method of the present invention using co-culture with sulfur-oxidizing bacteria, the oxidation of ammonia and the increase in the number of co-occurring archaea 16S rRNA genes, which occur simultaneously, are accompanied by a sharp decrease in oxygen concentration (typically by 10%). Together, it started immediately after 0.1 mM thiosulfate was consumed completely by sulfur-oxidizing bacteria (see panel A in FIG. 1). After 1 mM ammonia was consumed in the culture, the ammonia-oxidizing archaea became a dominant bacterium. These findings indicate that 16S rRNA of ammonia-oxidative archaea accounted for 81% of the total prokaryotic 16S rRNA gene copy (see panel B and Table 2 of the Figure 1), according to the results of FISH analysis. This is clearly supported by the results of ammonia-oxidizing archaea accounting for at least 80% of DAPI-stained cells (see FIGS. 2A and 2B).

According to the electron microscopic observation, most of the ammonia-oxidizing archaea of the present invention showed a long, rod-shaped cell morphology (0.15-0.2 μm in width and 0.5-1.0 μm in length; panel of FIG. 2). C and panel D).

According to the phylogenetic tree analysis, the ammonia-oxidizing archaea cultured in the present invention was found to be closely related to the archaea (Park et al 2008b) identified in the marine sediment and " Ca. Nitrosopumilus maritimus" (Fig. 3). Reference). The average identity of the archaea 16S rRNA gene sequence (size of about 900 bp) in clones of SJ and AR cultures was 99.3 ± 0.4%. The closest species to the ammonia-oxidative archaea of the SJ and AR cultures of the present invention was " Ca. Nitrosopumilus maritimus" with 16S rRNA gene sequence identity of 99.1 ± 0.4% (see Figure 3).

The experimental data of the Examples herein demonstrate that the identified archaea belonging to crenarchaeal group I.1a is the only ammonia-oxidizing archaea in the enrichment culture of the present invention.

First, the archaea 16S rRNA gene copy number increased synchronously with the amount of ammonia consumed (see FIG. 4). Crenarchaea is the only microorganism that grows during ammonia oxidation. As a result of denaturing gradient gel electrophoresis (DGGE) analysis and analysis of clone libraries from 16S rRNA genes, 16S rRNA gene sequences associated with crenarchaeal group I.1b or pSL12 clade, possibly other marine ammonia oxidative archaea, were not detected (see FIG. 3). ).

Second, the expression of the archaea amoA gene was observed during the oxidation process of ammonia and decreased 2-3 times after ammonia was depleted (Table 2). For AR enriched cultures, the archaea amoA gene expression decreased from 4.1 X 10 ³ to 4.5 X 10 ¹ copies per ng RNA as ammonia decreased.

Third, beta- and gamma-proteaobacterial amoA genes were not amplified by PCR in genome DNA and cDNA extracted from enriched cultures.

Fourth, the addition of bacterial antibiotics (streptomycin or kanamycin) during the oxidation process of ammonia results in the growth of ammonia oxidizing bacteria and the accumulation of nitrite, which leads to the conversion of ammonia to nitrite by the growth of ammonia oxidizing bacteria. It is to prove that. In addition, similar to the results of thermophilic ammonia oxidative archaea " Ca. Nitrososphaera gargensis" in the presence of 50 μM allylthiourea, nitrite inhibitors (100 μM chlorite and 50 μM nitrapyrin) When added, the ammonia oxidation and archaea growth of archaea were completely inhibited.

Since aggregates were formed at the time of culturing and measuring the growth rate of ammonia oxidizing archaea based on FISH was unsuccessful, the copy number of the 16S rRNA gene was used to indirectly estimate the growth rate. The maximum growth rate (0.57 / day for SJ culture and 0.65 / day for AR culture) obtained under the culture conditions of this experiment (25 ° C., pH 8.2, 1 mM ammonia) was “ Ca N. maritimus” (0.78 / day). Slightly lower than that of (Konneke et al. 2005). However, this growth rate is higher than that of the native bacterioplanktonic community (0.05-0.3 / day). Ammonia was observed to be incomplete but consumed (oxidized) at an ammonia concentration of 4 mM (see FIG. 4).

The growth of sulfated bacteria oxidizing thiosulfate in the culture of this experiment was essential for the activity of ammonia oxidative archaea. When the culture was transferred to a medium without thiosulfate, only about 20% of ammonia was oxidized in two weeks, and the ammonia oxidation activity was not resumed. Sulfite, tetrathionate and elemental sulfur, reducing sulfur compounds other than thiosulfate, were tested as substrates of sulfur oxidizing bacteria. In contrast, sulfur did not have a growth promoting effect. Thiosulfate concentrations of 0.1-2.0 mM were used to maintain optimal activity of the ammonia oxidizing archaea. However, at a concentration of 5 mM thiosulfate, the ammonia oxidation activity of the ammonia oxidizing archaea was no longer observed.

2. Dynamics of Ammonia Oxidation and Oxygen Absorption

Nitrification rate was measured during the batch growth experiment of ammonia oxidative archaea (see FIG. 1). Specific oxidation rate of ammonia is 2.8 fmol / cell / day for SJ culture 3.0 fmol / cell / day for AR culture. It was. Oxidation of ammonia was severely inhibited by shaking during incubation, but the degree of thiosulfate oxidation was not affected. These results suggest that the activity of sulfated bacteria induces levels of dissolved oxygen below a certain level. The level of dissolved oxygen actually measured decreased to low levels (below 3 mg / liter) before initiation of ammonia oxidation (see panel A in FIG. 1). This suggests that the ammonia oxidizing archaea in the rich culture of the present invention can be well adapted in a low level oxygen environment, which is typical of surface deposits in the ocean.

To measure affinity (Km value) for oxygen and ammonia in AR culture, oxygen uptake coupled with ammonia oxidation was measured respiratoryly. The kinetics of ammonia oxidation and oxygen consumption by AR culture showed the kinetics of Michaelis-Menten-type. The results of this experiment show that the affinity of ammonia oxidative archaea to oxygen and ammonia in AR culture was much greater than that of ammonia oxidizing bacteria (AOB), and that the The maximum rate was much lower than that of ammonia oxidizing bacteria (AOB). The ammonia affinity of ammonia oxidizing bacteria is wide, ranging from about 30 to 2,000 μM.

3. Carbon Immobilization in Ammonia Oxide Archaea (AOA) Concentration Cultures

As a result of quantifying 16s rRNA gene copies, increasing the concentration of bicarbonate in the culture of this experiment did not cause any serious effects on bacterial or archaea growth. To investigate the carbon fixation pathway of ammonia oxidative archaea, which was enriched in culture, it was labeled with [ ¹³ C] -bicarbonate in SJ and AR enrichment cultures. This resulted in significant amounts of ¹³ C-labels incorporated into the characteristic crenarchaeal membrane lipids, providing reliable evidence for autotrophic growth of ammonia oxidizing archaea (see Table 4). The degree of ¹³ C labeling of archaea lipids was not dependent on the amount of thiosulfate added (see Table 4). The 3-hydroxypropionate / 4-hydroxybutyrate cycle has been estimated to be an independent nutrient carbon immobilization pathway in various Crenarchaea, including mesophilic ammonia oxidative archaea, such as "Ca. C. symbiosum". We describe two important key genes of the 3-hydroxypropionate / 4-hydroxybutyrate cycle in enriched cultures: 4-hydroxybutyryl-CoA dehydratase; 4 -BUDH) and genes encoding acetyl-CoA carboxylase (ACC), respectively, could be amplified, which were 81-92% of the genes of " Ca. N. maritimus" and Had 86-92% nucleotide sequence identity. The expression levels of these two genes were significantly lower than the expression levels of amoA (see Table 2). Unexpectedly, the gene encoding methylmalonyl-CoA epimerase (MCE), another key gene in the 3-hydroxypropionate / 4-hydroxybutyrate cycle, could not be amplified (Table 1 Reference).

4. Oxidation of Nitrite Salts

During the oxidation of ammonia, nitrite accumulation could not be observed at any point during the batch culture, but it was observed that nitrate was accumulated almost stoichiometrically in the medium (Panel A of FIG. 1). However, it was confirmed that nitrite was accumulated when bacterial antibiotics (streptomycin or kanamycin) were added to the concentrated culture during ammonia oxidation. This suggests that nitrite-oxidizing bacteria (NOB) immediately oxidizes the nitrites produced by ammonia-oxidizing archaea, but bacterial 16S rRNA gene libraries in SJ cultures using normal bacterial specific primers in SJ cultures. As a result, only one clone related to Nitrospina was identified. PCR amplification experiments of 16S rRNA genes from a population of marine nitrite-oxidizing bacteria (ie, Nitrospina , Nitrospira and Nitrococcus ) detected sequences related to Nitrospina , which accounted for 0.5% of the total copy of the bacterial 16S rRNA gene (SJ enrichment) and 2.8 % (AR thickening culture) is comprised (refer Table 2).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Korea Biotechnology Research Institute KCTC11878BP 20110225

<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Method for Enrichment Culture of Ammonia-Oxidizing Archaea          Through Co-culturing with Sulfur-Oxidizing Bacteria and Novel          Ammonia-Oxidizing Archaea Isolated from Marine By Using the Same          Method <160> 35 <170> Kopatentin 1.71 <210> 1 <211> 835 <212> DNA <213> Ammonia Oxidizing Archaea <400> 1 acctgactgc tatcggattg atactaagcc atgcgagtca ttgtagtaat acaaggcaga 60 cggctcagta acgcgtagtc aacctaacct atggacggga ataacctcgg gaaactgaga 120 ataatgcccg atagaacact atatctggaa tggtttgtgt tccaaatgat ttatcgccgt 180 aggatgggac tgcggcctat cagtttgttg gtgaggtaat ggcccaccaa gactattaca 240 ggtacgggct ctgagaggag tagcccggag atgggtactg agacacggac ccaggcccta 300 tggggcgcag caggcgagaa aactttgcaa tgtgcgaaag cacgacaagg ttaatccgag 360 tggtttctgc taaaggaacc ttttgtcagt cctagaaaca ctgacgaata aggggtgggc 420 aagttctggt gtcagccgcc gcggtaaaac cagcacctca agtggtcagg atgattattg 480 ggcctaaagc acccgtagcc ggctctgtaa gttttcggtt aaatctgtac gctcaacgta 540 caggctgccg ggaatactgc agagctaggg agtgggagag gtagacggta ctcggtagga 600 aggggtaaaa tcctttgatc tattgatgac cacctgtggc garggcggtc taccagaaca 660 cgtccgacgg tgagggatga aagctggggg agcaaaccgg attagatacc cgggtagtcc 720 cagctgtaaa ctatgcaaac tcagtgatgc attggcttgt ggccaatgca gtgctgcagg 780 gaagccgtta agtttgccgc ctgggaagta cgtacgcaag tatgaaactt aaagg 835 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 cagcmgccgc ggtaa 15 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 gctttcrtcc ctcaccgt 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 ttccggttga tccygccrg 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 tccggcgttg amtccaatt 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 tcgcagcttr scacgycctt c 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 7 gctctaatta tgacagtata c 21 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 8 aycatgttga ayaatggtaa tgac 24 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 9 staatggtct ggcttagacg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 10 acatacagat ggatggccgc 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 11 gcatcaaata caagcacatt acc 23 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 12 catatttgtc atctgaatat ttgta 25 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 13 gcaagtatwg gwccttctgc 20 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 14 gataatwtct ttcatwtcat awggttg 27 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 15 aytcwggwgg tgcamgaat 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 16 gaaccwggca tgttaccwgg 20 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 17 cgatatgcrt tttgyttttg yctwat 26 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 18 caaaatgaya thytmaaaga 20 <210> 19 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 19 ccagcagccg cggtaat 17 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 20 ctaccagggt atctaatc 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 21 agagtttgat cmtggctcag 20 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 22 tacggytacc ttgttacgac tt 22 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 23 atggtccgcg aactcatc 18 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 24 agctcgcacc gatggatg 18 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 25 cctgcgctcc atgctccg 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 26 ggggtttcta ctggtggt 18 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 27 cccctckgsa aagccttctt c 21 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 28 ggngactggg acttctgg 18 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 29 gaasgcngag aagaasgc 18 <210> 30 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 30 ggtgagtggg ytaacmg 17 <210> 31 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 31 gctagccact ttctgg 16 <210> 32 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 32 gaaactgcga atggctc 17 <210> 33 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 33 cygcaggttc acctac 16 <210> 34 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 34 gtttcccagt cacgac 16 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 35 tcacacagga aacagctatg ac 22

Claims (5)

A method for enriching ammonia-oxidizing archaea comprising co-culturing with sulfur-oxidizing bacteria, wherein the medium of the co-culture is 0.1-8.0 mM. And ammonia.
The method of claim 1, wherein the sulfur-oxidizing bacteria using a reduced sulfur compound as a substrate, the sulfur compound is a sulfide (sulfide), thiosulfate (polythionate), polythionate, Elemental sulfur or sulfite.
The method of claim 1, wherein the medium of co-culture comprises seawater, and further comprising the step of subcultured in fresh medium after completion of ammonia oxidation by ammonia-oxidizing archaea.
Ammonia-oxidized archaea SJ strain having ammonia oxidation activity isolated from the ocean (Accession No .: KCTC 11878BP).
Composition for use in the purification of the marine environment contaminated with ammonia comprising the ammonia-oxidized archaea SJ strain (Accession No .: KCTC 11878BP) of claim 4 as an active ingredient.

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