Method and device for culturing and enriching nitrous oxide reducing microorganisms

Abstract

The invention discloses a method and a device for culturing and enriching nitrous oxide reducing microorganisms. The method comprises the following steps: inoculating natural shallow lake sediments into an anaerobic reactor, and carrying out chemostat culture of nitrous oxide reducing bacteria. The method specifically comprises the following steps: stirring and mixing a nitrate nitrogen-containing culture medium and a sample, culturing for 7 days in dark at 20-30 DEG C, and if the nitrate nitrogen content is obviously reduced, obtaining a preliminarily enriched bacterium mixture; introducing nitrogen into the nitrate-nitrogen-free culture medium for aeration and oxygen removal, and then introducing mixed gas of nitrous oxide and nitrogen to obtain a nitrous oxide-containing culture medium; and continuously introducing a culture medium containing nitrous oxide into the obtained primarily enriched bacterium mixture, continuously discharging the culture medium at the same speed, carrying out chemostat culture in the dark, and controlling the pH value to be 7.49-7.50 in the culture process. According to the method, the nitrous oxide reducing bacteria with relatively high activity can be obtained.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, and in particular to a method and apparatus for culturing and enriching nitrous oxide-reducing microorganisms. Background Technology

[0003] Nitrous oxide (N2O) is the most important greenhouse gas after carbon dioxide (CO2) and methane (CH4). Although its content in the atmosphere is not as high as that of CO2 and CH4, on a century-scale, removing one molecule of N2O is equivalent to removing 300 molecules of CO2. Furthermore, N2O undergoes a photochemical reaction with ozone, thereby destroying the ozone layer. Its oxidation product NOx in the atmosphere exacerbates the formation of acid rain.

[0004] N2O sources include various biological processes such as nitrification, denitrification, nitrifying bacterial denitrification, and the dissimilatory reduction of nitrate to ammonium, as well as abiotic processes such as fossil fuel and biomass combustion. However, the only known biological pathway for reducing N2O in nature is the catalytic reduction of N2O to N2 by nitrous oxide reductase (NOS), and this is the only known pathway. Previously, the reduction of N2O to N2 was only considered to be part of the denitrification reaction. In recent years, it has been gradually realized that this reaction can be completed independently by nitrous oxide reductase microorganisms (N2ORM) possessing NOS. N2ORMs are widely distributed in the natural environment and have been found in rivers, lakes, oceans, and soils.

[0005] NOS by nos Z Gene encoding, usually containing nos Z Nitrous oxide-reducing bacteria are divided into two lineages, typically nosZ For clade Ⅰ nosZ (hereinafter referred to as) nosZⅠ ), atypical nosZ For clade II nosZ (hereinafter referred to as) nosZⅡ Studies have found that typical denitrifying bacteria, such as Bradyrhizobium spp., Pseudomonasspp., and Paracoccus spp., all possess... nosZⅠ And carrying nosZⅡ The bacteria include *Flavobacterium* spp., *Dechloromonas* spp., and *Azospiras* spp., among others. Studies have indicated that... nosZⅡ and nosZⅠ Differences in structure and mechanism of action may lead to variations in N2O reduction under different environmental conditions.

[0006] To further investigate whether niche differentiation exists between the two types and how they differentiate in different environments, and to apply this to N2O emission reduction in natural shallow lakes, it is necessary to design enrichment cultivation conditions and devices that simulate natural lake environments in order to selectively enrich N2ORM. Summary of the Invention

[0007] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for culturing and enriching nitrous oxide-reducing microorganisms.

[0008] Another object of the present invention is to provide an apparatus for implementing the above-described method.

[0009] The objective of this invention is achieved through the following technical solution: A method for culturing and enriching nitrous oxide-reducing microorganisms includes the following steps: (1) Mix the nitrate nitrogen-containing culture medium and the sample, and incubate in the dark at 20-30°C for 7 days. Measure the nitrate nitrogen. If the nitrate nitrogen content decreases by at least 90%, a preliminary enriched bacterial mixture is obtained. Otherwise, replace the lake sediment and repeat the first step. (2) Nitrogen gas is introduced into the nitrate-free nitrogen culture medium to remove oxygen, and then a mixture of nitrous oxide and nitrogen gas is introduced to obtain a culture medium containing nitrous oxide. (3) The culture medium containing nitrous oxide from step (2) is continuously introduced into the preliminarily enriched bacterial mixture obtained in step (1), and the culture medium is continuously discharged at the same rate. The mixture is cultured in the dark, and the pH is controlled at 7.49 to 7.50 during the culture process.

[0010] Furthermore, the sample described in step (1) is sediment from a natural shallow lake.

[0011] Furthermore, the nitrate-containing culture medium in step (1) contains the following components: NO3-N 5 mg / L, CH3COONa 4.1017 g / L, NaCl 0.5 g / L, NH4Cl 0.27 g / L, KH2PO4 0.29 g / L, K2HPO4 0.50 g / L, trace element solution 1 ml / L, and vitamin solution 1 ml / L; Furthermore, the trace element solution contains the following components: EDTA-2Na 50 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5 g / L, CuSO4·5H2O 1.57 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, and CoCl2·6H2O 1.61 g / L; the vitamin solution contains the following components: 4-aminobenzoic acid 0.5 g / L, D-biotin 0.2 g / L, niacin 0.5 g / L, calcium pantothenate 0.5 g / L, pyridoxine hydrochloride 1 g / L, folic acid 0.2 g / L, α-lipoic acid 0.5 g / L, thiamine hydrochloride 0.5 g / L, and cyanocobalamin 0.5 g / L.

[0012] Further, the nitrous oxide and nitrogen mixture mentioned in step (2) is a mixture of 5% N2O and 95% N2 by volume.

[0013] Furthermore, the rate at which the mixture of nitrous oxide and nitrogen is introduced in step (2) is 70 mL / min.

[0014] Furthermore, the nitrate-free nitrogen culture medium described in step (3) contains the following components: CH3COONa 4.1017 g / L, NaCl 0.5 g / L, NH4Cl 0.27 g / L, KH2PO4 0.29 g / L, K2HPO4 0.50 g / L, trace element solution 1 mL / L and vitamin solution 1 mL / L; Furthermore, the trace element solution contains the following components: EDTA-2Na 50 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5 g / L, CuSO4·5H2O 1.57 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, and CoCl2·6H2O 1.61 g / L. The vitamin solution contains the following components: 4-aminobenzoic acid 0.5 g / L, D-biotin 0.2 g / L, niacin 0.5 g / L, calcium pantothenate 0.5 g / L, pyridoxine hydrochloride 1 g / L, folic acid 0.2 g / L, α-lipoic acid 0.5 g / L, thiamine hydrochloride 0.5 g / L, and cyanocobalamin 0.5 g / L.

[0015] Furthermore, the nitrous oxide-containing culture medium introduced in step (2) and the culture medium discharged in step (3) are introduced and discharged using a peristaltic pump at a rate of 4.5 rpm.

[0016] The apparatus for implementing the above method includes a pure nitrogen gas tank (1), a pure nitrous oxide gas tank (2), a nitrogen gas flow controller (3), a nitrous oxide gas flow controller (4), a culture medium supply bottle (5), a first magnetic stirrer (6), a first peristaltic pump (7), a reaction flask (8), a second magnetic stirrer (9), a pH controller (10), a second peristaltic pump (11), a NaOH solution bottle (12), a third peristaltic pump (13), and a waste liquid collection bottle (14). The culture medium supply bottle (5) is connected to the reaction bottle (8) via a first peristaltic pump; the pure nitrous oxide gas tank (2) is connected to the culture medium supply bottle (5) via a nitrous oxide gas flow controller (3); and the pure nitrogen gas tank (1) is connected to the culture medium supply bottle (5) via a nitrogen gas flow controller (3). The NaOH solution bottle (12) is connected to the reaction bottle (8) via the second peristaltic pump (11); the pH controller (10) is connected to the second peristaltic pump (11) and the reaction bottle (8). The reaction flask (8) is connected to the waste liquid collection bottle (14) via the third peristaltic pump (13); The first magnetic stirrer (6) is placed below the culture medium supply bottle (5), and the second magnetic stirrer (9) is placed below the reaction bottle (8).

[0017] Furthermore, the reaction flask is a three-necked flask.

[0018] The steps for implementing the above method using the above-described apparatus are as follows: (1) In the reaction flask (8), the nitrate nitrogen-containing culture medium and the sample are stirred and mixed by the first magnetic stirrer (6), and cultured in the dark at 20-30℃ for 7 days. The nitrate nitrogen is measured to obtain a preliminarily enriched bacterial mixture. (2) Add nitrate-free nitrogen culture medium to the culture medium supply bottle (5), and introduce nitrogen gas for aeration and deoxygenation through the pure nitrogen gas tank (1) and nitrogen gas flow controller (3). Then introduce a mixture of nitrous oxide and nitrogen gas through the pure nitrous oxide gas tank (2), nitrous oxide gas flow controller (3), pure nitrogen gas tank (1) and nitrogen gas flow controller (3) to obtain a culture medium containing nitrous oxide. (3) The culture medium containing nitrous oxide in the culture medium supply bottle (5) is continuously fed into the initially enriched bacterial mixture in the reaction bottle (8) by the first peristaltic pump (8) and the third peristaltic pump (13), and the culture medium is continuously discharged to the waste liquid collection bottle (14) at the same rate; the pH in the reaction bottle (8) is detected by the pH controller (10), and the second peristaltic pump (11) is controlled to transport the NaOH solution in the NaOH solution bottle (12) to the reaction bottle (8) to control the pH to be 7.49 to 7.50.

[0019] The present invention has the following advantages and effects compared with the prior art: This invention designs an enrichment method based on the growth characteristics of microorganisms, which improves the competitive advantage of nitrous oxide-reducing microorganisms and enables the enrichment and cultivation of nitrous oxide-reducing microorganisms. Attached Figure Description

[0020] Figure 1 This is an embodiment of an apparatus used for the enrichment of nitrous oxide-reducing microorganisms. In this apparatus, 1 is a pure nitrogen gas tank; 2 is a pure nitrous oxide gas tank; 3 is a nitrogen gas flow controller; 4 is a nitrous oxide gas flow controller; 5 is a culture medium supply bottle; 6 is a first magnetic stirrer; 7 is a first peristaltic pump; 8 is a reaction flask; 9 is a second magnetic stirrer; 10 is a pH controller; 11 is a second peristaltic pump; 12 is a NaOH solution bottle; 13 is a third peristaltic pump; and 14 is a waste liquid collection bottle.

[0021] Figure 2 This is a graph showing the N2O reduction potential of microorganisms enriched in the sediments of Taihu Lake.

[0022] Figure 3 This is a graph showing the N2O reduction potential of microorganisms enriched in the sediments of Weishan Lake. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0024] Example 1: Enrichment of Nitrous Oxide-Reducing Microorganisms This embodiment uses, as follows: Figure 1 The apparatus shown is used for experiments. The apparatus includes a pure nitrogen gas tank (1), a pure nitrous oxide gas tank (2), a nitrogen gas flow controller (3), a nitrous oxide gas flow controller (4), a culture medium supply bottle (5), a first magnetic stirrer (6), a first peristaltic pump (7), a reaction flask (8), a second magnetic stirrer (9), a pH controller (10), a second peristaltic pump (11), a NaOH solution bottle (12), a third peristaltic pump (13), and a waste liquid collection bottle (14).

[0025] 1) After removing larger impurities such as stones, branches and weeds from the sediments collected from Taihu Lake (31.464932°N, 120.363328°E) and Weishan Lake (34.673662°N, 117.27742°E), the sediments were mixed evenly and 50g of each was weighed out.

[0026] 2) Prepare a nitrate nitrogen-containing culture medium containing the following components: NO3-N 5 mg / L, CH3COONa 4.1017 g / L, NaCl 0.5 g / L, NH4Cl 0.27 g / L, KH2PO4 0.29 g / L, K2HPO4 0.50 g / L, trace element solution 1 ml / L, and vitamin solution 1 ml / L. The solution is deionized water, and the pH is adjusted to 7.5 with sodium hydroxide solution. The medium is then sterilized by high temperature and high pressure steam. The trace element solution contains the following components: EDTA-2Na 50 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5 g / L, CuSO4·5H2O 1.57 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, and CoCl2·6H2O 1.61 g / L. The vitamin solution contains the following components: 4-aminobenzoic acid 0.5 g / L, D-biotin 0.2 g / L, niacin 0.5 g / L, calcium pantothenate 0.5 g / L, pyridoxine hydrochloride 1 g / L, folic acid 0.2 g / L, α-lipoic acid 0.5 g / L, thiamine hydrochloride 0.5 g / L, and cyanocobalamin 0.5 g / L.

[0027] 3) In the pre-culture stage, 600 mL of sterilized nitrate-containing culture medium was placed in the reaction flask (8), and 50 g of Weishan Lake sediment and a magnetic stir bar were added. The second magnetic stir bar (9) was placed below the reaction flask (8). The reaction flask (8) was connected to the waste liquid collection bottle (14) through the third peristaltic pump (13). The pH controller (10) was connected to the second peristaltic pump (11) and the reaction flask (8), and the NaOH solution bottle (12) was connected to the reaction flask (8) through the second peristaltic pump (11). The pH controller could monitor the pH changes of the reaction flask (8). The temperature in the greenhouse was set to 25℃, and the pre-culture was carried out in the dark for 7 days. On the 7th day, the pH was found to rise significantly from 7.5 to 8. The liquid was collected to measure the nitrate nitrogen. The nitrate nitrogen decreased significantly, by 90%, and the enrichment culture stage was entered.

[0028] 4) Prepare a nitrate-free nitrogen culture medium containing the following components: CH3COONa 4.1017 g / L, NaCl 0.5 g / L, NH4Cl 0.27 g / L, KH2PO4 0.29 g / L, K2HPO4 0.50 g / L, trace element solution 1 mL / L, and vitamin solution 1 mL / L; the solution is deionized water, sterilized by high temperature and high pressure steam. The trace element solution contains the following components: EDTA-2Na 50 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5 g / L, CuSO4·5H2O 1.57 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, and CoCl2·6H2O 1.61 g / L. The vitamin solution contains the following components: 0.5 g / L 4-aminobenzoic acid, 0.2 g / L D-biotin, 0.5 g / L niacin, 0.5 g / L calcium pantothenate, 1 g / L pyridoxine hydrochloride, 0.2 g / L folic acid, 0.5 g / L α-lipoic acid, 0.5 g / L thiamine hydrochloride, and 0.5 g / L cyanocobalamin.

[0029] 5) During the enrichment culture stage, place the prepared 10L of nitrate-free nitrogen culture medium in the culture medium supply bottle (5), add a magnetic stir bar and sterilize it, and then place it on the first magnetic stirrer (6). The pure nitrous oxide gas tank (2) is connected to the culture medium supply bottle (5) through the nitrous oxide gas flow controller (3), and the pure nitrogen gas tank (1) is connected to the culture medium supply bottle (5) through the nitrogen gas flow controller (3). Nitrogen gas is aerated at 500mL / min for 30min through the pure nitrogen gas tank (1) and the nitrogen gas flow controller (3) to remove oxygen; then, nitrous oxide mixed gas (5% N2O + 95% N2 by volume percentage) is introduced at a flow rate of 70mL / min through the pure nitrogen gas tank (1), the nitrogen gas flow controller (3), the nitrous oxide gas tank (2), and the nitrous oxide gas flow controller (4). A 0.1 mol / L NaOH solution was prepared and placed in a NaOH solution bottle (12) and aerated with nitrogen to remove oxygen and adjust the pH. The NaOH solution bottle (12) was connected to the reaction flask (8) via a second peristaltic pump (11). A pH controller was set so that when the pH was below 7.49, the second peristaltic pump (11) was adjusted to automatically add aerated NaOH, and when the pH was above 7.50, the second peristaltic pump (11) was adjusted to stop adding NaOH. The culture medium supply bottle (5) was connected to the reaction flask (8) via a first peristaltic pump. The first and third peristaltic pumps were started, and the speed was set to 4.5 rpm to stably input fresh non-nitrate nitrogen culture medium and discharge old culture medium. Chromatography was carried out in the dark.

[0030] 2. N2O reducing capacity test of enriched microbial communities 1) N2O reduction potential The N2O reduction potential was determined by taking the sediment before culture and the mixture of cultured solutions on day 60 and day 120, with three replicates set up for each time point.

[0031] Samples collected at different times were stored at -20℃. After thawing, pre-culture was performed to restore microbial activity: 19.6 mL of the mixture was placed in a 50 mL saline bottle, 0.2 mL of 500 mM sodium acetate solution was added, and nitrogen was used for aeration for 10 min. After capping, 1 mL of nitrous oxide mixture (5% N2O + 95% N2) was introduced for 2 days of pre-culture. The headspace N2O concentration was measured by gas chromatography, and a significant decrease was observed, indicating improved microbial activity. For the sediment samples before culture, a mixture was prepared using a carbon-free, nitrate-free nitrogen medium at a ratio of 1:20 (w:v).

[0032] 2) N2O reduction rate After the culture was completed, 0.2 mL of 500 mM sodium acetate solution was added, and the mixture was aerated with nitrogen for 10 min. Then, 15 mL of a nitrous oxide mixture (5% N₂O + 95% N₂) was introduced, and the mixture was placed in a shaker for culture at 25℃ and 140 rpm. The N₂O concentration was measured at the first hour, and then every 2 hours thereafter. A linear regression was performed with N₂O concentration on the ordinate and culture time on the abscissa; the slope (k) obtained is the N₂O reduction rate.

[0033] 3) N2O reduction potential Biomass determination: The mixture used to determine the reduction rate was filtered through dry filter paper. The crucible and filter paper were dried in a 105℃ oven until constant weight, cooled, and weighed, recording the weight as m1. The crucible and filter paper were then placed in a muffle furnace at 600℃ for 6 hours, cooled, and weighed, recording the weight as m2. The biomass can then be calculated. ).

[0034] N2O reduction potential ( Calculate using the following formula:

[0035]

[0036] The measurement results are as follows Figure 2 and Figure 3 As shown, sediments from different lakes exhibited significantly improved nitrous oxide reduction performance after enrichment culture, indicating that nitrous oxide-reducing microorganisms were effectively enriched and cultured.

[0037] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of culturing an enrichment of nitrous oxide reducing microorganisms, characterized by, The method comprises the following steps: (1) stirring and mixing the nitrate nitrogen-containing culture medium and the sample, culturing for 7 days in darkness at 20-30℃, measuring the nitrate nitrogen, and obtaining a preliminary enriched bacteria mixture if the nitrate nitrogen content decreases by at least 90%, or replacing the lake sediment to perform the first step again if the nitrate nitrogen content does not decrease by at least 90%; (2) introducing nitrogen gas into the nitrate nitrogen-free culture medium to remove oxygen, and then introducing a mixture of nitrous oxide and nitrogen gas to obtain a culture medium containing nitrous oxide; (3) continuously introducing the culture medium containing nitrous oxide obtained in step (2) into the preliminary enriched bacteria mixture obtained in step (1) and continuously discharging the culture medium at the same rate, and performing chemostat culture in darkness, with the pH being controlled to be 7.49-7.50 during the culture process.

2. The method according to claim 1, wherein the sample in step (1) is sediment of a natural shallow lake.

3. The method according to claim 1, wherein the nitrate nitrogen-containing culture medium in step (1) contains the following components: NO3-N 5 mg / L, CH3COONa 4.1017 g / L, NaCl 0.5 g / L, NH4Cl 0.27 g / L, KH2PO4 0.29 g / L, K2HPO4 0.50 g / L, trace element solution 1 mL / L, and vitamin solution 1 mL / L.

4. The method according to claim 3, wherein the trace element solution contains the following components: EDTA-2Na 50 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5 g / L, CuSO4·5H2O 1.57 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L, and CoCl2·6H2O 1.61 g / L; and the vitamin solution contains the following components: 4-aminobenzoic acid 0.5 g / L, D-biotin 0.2 g / L, nicotinic acid 0.5 g / L, calcium pantothenate 0.5 g / L, pyridoxine hydrochloride 1 g / L, folic acid 0.2 g / L, α-lipoic acid 0.5 g / L, thiamine hydrochloride 0.5 g / L, and cyanocobalamin 0.5 g / L.

5. The method according to claim 1, wherein the mixture of nitrous oxide and nitrogen gas in step (2) is a mixture of 5% N2O by volume and 95% N2 by volume; and the introduction rate of the mixture of nitrous oxide and nitrogen gas in step (2) is 70 mL / min.

6. The method according to claim 1, wherein the nitrate nitrogen-free culture medium in step (3) contains the following components: CH3COONa 4.1017 g / L, NaCl 0.5 g / L, NH4Cl 0.27 g / L, KH2PO4 0.29 g / L, K2HPO4 0.50 g / L, trace element solution 1 mL / L, and vitamin solution 1 mL / L.

7. The method according to claim 6, wherein ​ ​ ​ ​ ​ ​ The trace element solution contains the following components: EDTA-2Na 50 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5 g / L, CuSO4·5H2O 1.57 g / L, (NH4)6Mo7O2·4H2O 1.1 g / L and CoCl2·6H2O 1.61 g / L; The vitamin solution contains the following components: 4-aminobenzoic acid 0.5 g / L, D-biotin 0.2 g / L, nicotinic acid 0.5 g / L, calcium pantothenate 0.5 g / L, pyridoxine hydrochloride 1 g / L, folic acid 0.2 g / L, α-lipoic acid 0.5 g / L, thiamine hydrochloride 0.5 g / L and cyanocobalamin 0.5 g / L.

8. The method of claim 1, wherein, The step of passing and discharging the culture medium containing nitrous oxide in step (2) is performed by using a peristaltic pump at a rate of 4.5 rpm.

9. An apparatus for culturing and enriching nitrous oxide-reducing microorganisms according to any one of claims 1 to 8, comprising a pure nitrogen gas tank (1), a pure nitrous oxide gas tank (2), a nitrogen gas flow controller (3), a nitrous oxide gas flow controller (4), a culture medium supply bottle (5), a first magnetic stirrer (6), a first peristaltic pump (7), a reaction bottle (8), a second magnetic stirrer (9), a pH controller (10), a second peristaltic pump (11), a NaOH solution bottle (12), a third peristaltic pump (13), and a waste liquid collection bottle (14); The culture medium supply bottle (5) is connected to the reaction bottle (8) by the first peristaltic pump; the pure nitrous oxide gas tank (2) is connected to the culture medium supply bottle (5) by the nitrous oxide gas flow controller (3), and the pure nitrogen gas tank (1) is connected to the culture medium supply bottle (5) by the nitrogen gas flow controller (3); The NaOH solution bottle (12) is connected to the reaction bottle (8) by the second peristaltic pump (11); the pH controller (10) is connected to the second peristaltic pump (11) and the reaction bottle (8); The reaction bottle (8) is connected to the waste liquid collection bottle (14) by the third peristaltic pump (13); The first magnetic stirrer (6) is placed below the culture medium supply bottle (5), and the second magnetic stirrer (9) is placed below the reaction bottle (8).

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