CN109321463B - Heat-resistant desulfurization bacterium screening culture solution and screening method thereof - Google Patents
Heat-resistant desulfurization bacterium screening culture solution and screening method thereof Download PDFInfo
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Abstract
The invention discloses a heat-resistant desulfurization bacterium screening culture solution and a screening method thereof, belonging to the technical field of microorganism and environmental engineering. Many heat-resistant desulfurization bacteria are autotrophic bacteria living in hot springs and other environments, and are often difficult to screen due to slow growth speed. According to the invention, by optimizing the screening culture solution of the desulfurization bacteria and adding the non-desulfurization bacteria xanthothermus anaerobacterium flavchermus for assisting in screening the desulfurization bacteria, the culture solution obtained by the invention can screen out the desulfurization bacteria with low content which can not be screened out by a common desulfurization bacteria culture solution, the problem that the desulfurization bacteria in a sample are leaked and screened is effectively avoided, the screening efficiency of the heat-resistant desulfurization bacteria is greatly improved, particularly, the screening of the heat-resistant desulfurization bacteria in a hot spring with the hot spring water temperature not more than 75 ℃ is very efficient, and a simple, easy and efficient screening method is provided for screening the heat-resistant desulfurization bacteria.
Description
Technical Field
The invention relates to a heat-resistant desulfurization bacterium screening culture solution and a screening method thereof, belonging to the field of microorganism and environmental engineering.
Background
The electric power structure of China is mainly coal, and coal combustion becomes one of the main sources of sulfur pollution in China. The sulfur contained in coal can be divided into inorganic sulfur and organic sulfur according to occurrence forms, wherein the inorganic sulfur (accounting for 60-70% of the total sulfur) mainly exists in a pyrite form, and in addition, a small amount of sulfate and trace elemental sulfur are also included. The desulfurization method includes physical desulfurization, chemical desulfurization, microbial desulfurization and the like.
The microbial desulfurization is to convert sulfur in coal into other forms which are easy to remove before combustion by utilizing the metabolic process of desulfurization microbes, thereby achieving the aim of desulfurization. Compared with the traditional physical desulfurization and chemical desulfurization, the microbial desulfurization has the advantages of low production cost, mild reaction conditions, high removal rate, small environmental pollution, strong specificity and the like. Therefore, microbial desulfurization is receiving increasing attention and attention.
The obtainment of desulfurization microorganisms is a prerequisite for the realization of microbial desulfurization. Temperature is an important factor in chemical reactions, and generally, the reaction speed is faster at higher temperatures. The hot spring contains a large amount of heat-resistant bacteria, many of which are autotrophic bacteria with desulfurization capability, and the bacteria have the characteristic of autotrophic growth while desulfurization, so that the content of organic matters in coal can be finally increased, and the calorific value of the coal can be increased. Autotrophic bacteria are typically cultured in enriched culture media that do not contain organic carbon sources. Autotrophic bacteria generally have a slow growth rate and are difficult to screen. Most of the bacteria in the hot spring are attached to the surface of solid such as sand and the like, and the content of the bacteria in the spring water is lower. The collection of hot spring sample sometimes has certain difficulty, it is difficult sometimes to reach the hot spring source in order to obtain the sample that bacterial content such as grit is high, can only gather the spring that flows out or draw by oneself, bacterial content is generally very little in these water samples, in addition, often can reach the laboratory that can screen the cultivation through a period of time after the hot spring sample is gathered, the vitality of this in-process microorganism often can decline, the possibility of leaking the sieve when above reason causes the desulfurization fungus of screening in the hot spring sample is very big.
Therefore, a method for screening heat-resistant desulfurization bacteria with high efficiency is urgently needed.
Disclosure of Invention
In order to solve the problem that the desulfurization bacteria in a sample are easy to leak due to low growth speed and low content of the desulfurization bacteria in the sample when screening heat-resistant desulfurization bacteria, the invention designs the culture solution with optimized components, and the culture solution is matched with anaerobic bacillus flavivithermus for use, so that the screening rate of the desulfurization bacteria in the sample is greatly improved when screening the desulfurization bacteria.
The invention firstly provides a screening culture solution of heat-resistant desulfurization bacteria, which comprises the following components in parts by weight: KH (Perkin Elmer)2PO4 0.4-0.5g/L，K2HPO4 0.5-0.6g/L，MgCl2 0.03-0.05g/L，CaCl20.05-0.07g/L, 4-6g/L NaCl, 0.05-0.06g/L nitrogen source, 0.3-0.4g/L sodium thiosulfate pentahydrate, 0.1-0.2g/L sodium bicarbonate and 0.1-0.2mL/L trace element solution; wherein the culture solution contains Bacillus thermoanaerobicus at concentration of 0.8-1.2 x 10 per ml5And (4) cells.
Further, the nitrogen source comprises three compounds of sodium nitrate, ammonium chloride and ammonium nitrate, wherein the concentration of the sodium nitrate, the concentration of the ammonium chloride or the concentration of the ammonium nitrate is more than or equal to 0.01 g/L.
Further, preferably, the nitrogen source comprises 0.02g/L of sodium nitrate, 0.02g/L of ammonium chloride and 0.02g/L of ammonium nitrate.
Further, the trace element solution is EDTA.Na2 0.5-0.7g/L；(NH4)6Mo7O24·4H2O 0.01-0.2g/L、CoCl·6H2O 0.015-0.02g/L、MnCl·4H2O 0.03-0.04g/L、CuSO4·5H2O 0.01-0.02g/L、ZnSO4·7H2O 0.13-0.14g/L，FeSO4 0.3-0.4g/L。
Further, the Bacillus thermoanaerobicus Anaxybacter flavithermus is not limited to a specific strain or subspecies.
The invention also provides a screening method of the heat-resistant desulfurization bacteria, which utilizes the screening culture solution of the heat-resistant desulfurization bacteria to screen.
Further, the screening method specifically comprises the following steps:
(1) preparing a screening culture solution of heat-resistant desulfurization bacteria;
(2) adding hot spring water with volume fraction of 1-3% into the culture solution, culturing at + -5 deg.C corresponding to the temperature of hot spring, with the highest culture temperature not exceeding 65 deg.C, and culturing at 65 deg.C when the temperature of hot spring to which the sample belongs is higher than 70 deg.C;
(3) and when the pH of the culture solution is reduced, the culture solution is preliminarily judged to be successfully screened, and when sulfur smell and floating sulfur simple substances further appear, the culture solution is judged to be successfully screened.
Furthermore, when the pH of the culture solution rises to above pH 9 after 5-7 days of culture, it indicates that there is no heat-resistant desulfurization bacterium in the hot spring water sample.
Furthermore, the hot spring water is hot spring water with the temperature not higher than 75 ℃.
The method for identifying whether the desulfurization bacteria grow is as follows: according to the experience of this subject group, if the culture solution does not grow, a small amount of precipitate is generated in the culture solution after 5 to 6 days of culture, which is formed by the chemical reaction of the metal ions in the culture solution with carbonate and the like, and at the same time, the pH of the culture solution rises, and according to the experience, if the pH of the culture solution reaches 9 or more, the growth of the desulfurization bacteria is not possible, and it can be determined that the desulfurization bacteria do not exist in the hot spring sample. Therefore, the present invention uses an increase in culture broth pH of up to 9 as a marker of selection failure.
If the thiobacillus grows, the pH of the culture solution is firstly reduced within 9 days, then the culture solution has obvious sulfur taste, floating faint yellow elemental sulfur appears on the surface of the culture solution after the culture solution is cultured for 1-2 days, and a small amount of the culture solution is taken for microscope observation to see a large amount of live bacteria. As the culture medium used in the invention does not contain organic matters, the carbon source of the culture medium is NaHCO3The bacteria thus selected are all autotrophic bacteria or groups of bacteria, the bacteria or groups of bacteria growingThe energy source is obtained during the bacterial desulfurization of sodium thiosulfate. S in sodium thiosulfate can be removed by the desulfurizing bacteria2-Converted to elemental sulfur, so that floating elemental sulfur is visible on the surface of the broth if the culture of the desulfurizing bacteria is successful. S metabolism by desulfurization bacteria is firstly to convert S2-The sulfur can be further oxidized to obtain sulfate radical and the generation of sulfuric acid is accompanied, so that the pH is reduced, when the desulfurization bacteria exist, the reason that the pH is reduced at the early stage of screening can appear, and then the elemental sulfur is accumulated to a certain extent along with the increase of the number of the desulfurization bacteria to become macroscopic floating elemental sulfur. Therefore, when the pH of the culture solution is reduced, the culture solution can be preliminarily judged to be successful in screening the desulfurization bacteria, and when sulfur smell and floating sulfur simple substances are further generated, the culture solution is judged to be successful in screening the desulfurization bacteria.
The culture can be easily expanded to culture in a common desulfurization medium because of its high activity and large bacterial count, and can be used for other desulfurization experiments.
The invention has the following beneficial effects:
(1) according to the invention, the composition of the screening culture solution is optimized, and the auxiliary bacterium Anoxybacillus flavithermus is added, so that low-content desulfurization bacteria which cannot be screened by a common desulfurization bacteria culture solution can be screened, and the problem that the desulfurization bacteria in a sample are leaked to be screened is effectively avoided.
(2) The method is simple and easy to implement, has good screening effect, can screen the desulfurization bacteria in hot spring water with the temperature not more than 75 ℃, and only a few samples can obtain the desulfurization bacteria when other culture solutions (including the common desulfurization bacteria culture solution and the optimized desulfurization bacteria screening culture solution) are adopted for culture.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
Common desulfurization bacterium culture solution: KH (Perkin Elmer)2PO4 0.46g/L，K2HPO4 0.55g/L，MgCl2 0.05g/L，CaCl20.07g/L, NaCl 5g/L, nitrogen source (ammonium sulfate 0.06g/L), sodium thiosulfate pentahydrate 0.33g/L, sodium bicarbonate 0.16g/L, and trace element solution 0.1 mL/L.
The optimized screening culture solution of the desulfurization bacteria comprises the following steps: KH (Perkin Elmer)2PO4 0.46g/L，K2HPO4 0.55g/L，MgCl2 0.05g/L，CaCl20.07g/L, NaCl 5g/L, nitrogen source 0.6g/L (sodium nitrate 0.02g/L + ammonium chloride 0.02g/L + ammonium nitrate 0.02g/L), sodium thiosulfate pentahydrate 0.33g/L, sodium bicarbonate 0.16g/L, and trace element solution 0.1 mL/L.
Screening culture solution for heat-resistant desulfurization bacteria: KH (Perkin Elmer)2PO4 0.46g/L，K2HPO4 0.55g/L，MgCl2 0.05g/L，CaCl20.07g/L, NaCl 5g/L, nitrogen source 0.6g/L (sodium nitrate 0.02g/L + ammonium chloride 0.02g/L + ammonium nitrate 0.02g/L), sodium thiosulfate pentahydrate 0.33g/L, sodium bicarbonate 0.16g/L, and trace element solution 0.1mL/L, wherein the culture solution comprises yellow-heat anaerobic bacillus with concentration of 10 per milliliter of the culture solution5And (4) cells.
Wherein, the microelement solution in the three culture solutions is as follows: EDTA.Na2 0.51g/L；(NH4)6Mo7O24·4H2O 0.015g/L、CoCl·6H2O 0.017g/L、MnCl·4H2O 0.043g/L、CuSO4·5H2O 0.015g/L、ZnSO4·7H2O 0.133g/L，FeSO4 0.33g/L。
Common bacterial culture medium: 0.5% of yeast powder, 1% of peptone, 0.5% of glucose and 2% of sodium chloride. HCl and NaOH are used to adjust the pH to be basically consistent with the required pH of the cultured microorganism. The pH value of the culture medium used for culturing the anaerobic bacillus flavothermis is 6.5-7.0.
The chemical reagents used in the invention are all common analytical pure reagents and are purchased from Shanghai chemical reagent company of the national drug group.
And (3) culturing the desulfurization bacteria in the hot spring: firstly, preparing the culture solution according to the given composition, subpackaging the culture solution in a 250mL triangular flask after the preparation, sealing the triangular flask by using an aluminum foil, filtering the culture solution for sterilization, sterilizing the triangular flask and the aluminum foil at high temperature, and adding a small amount of hot spring water samples into the culture solution. Mixing spring water with the culture solution, and culturing at + -5 deg.C of the original temperature of hot spring. The addition amount of spring water is 1-3% of the volume of the culture solution, the volume of the spring water is too small, the spring water is easy to leak the screen, and the volume of the spring water is too large, so that the culture solution can generate precipitate to cause the pH value of the culture solution to rise rapidly, the screening fails, and some components in the spring water can react with the components in the culture solution to form the precipitate.
EXAMPLE 1 Effect of screening for desulfurization bacteria from Hot spring samples Using Heat-resistant desulfurization bacteria screening Medium
200mL of heat-resistant desulfurization bacterium screening culture solution is added into a sterilized 250mL triangular flask, 2mL of hot spring water sample is added, and the culture is carried out at the temperature corresponding to the hot spring water to screen desulfurization bacteria.
The five hot spring water samples are all from geological parks of the hot sea country in Tengcong county, Yunnan province, and are as follows:
01: the hot water of the pond at the downstream of the pregnant well (the temperature is 70 ℃ when sampling);
02: a hot spring water sample (50 ℃) in a sulfur pond (above a large rolling pot);
03: a pond water sample (45 ℃) at the downstream of the mouth of the toad;
04: cucumber Banlangtang water sample (45 ℃);
05: water sample of warm water in outdoor ditch of cucumber Banlang Tang (45 ℃ in sampling)
The culture conditions are shown in Table 1.
TABLE 1 results of screening of desulfurization bacteria using the Heat-resistant type desulfurization bacteria screening culture solution
As can be seen from Table 1, after hot spring samples are added into the screening culture solution of heat-resistant desulfurization bacteria and cultured for 3-5 days, the pH of the culture medium is reduced in 5 samples, and when the culture is continued, the culture solution has obvious sulfur taste, and floating sulfur simple substances appear on the surface of the culture solution. Further microscopic examination can show that a large amount of bacteria exist in the culture solution, the form of the bacteria is obviously different from that of the previously added yellow-heat anaerobic bacillus, and the screened heat-resistant desulfurization bacteria can be seen.
As a control, the culture results of the heat-resistant type desulfurization bacterium screening culture solution without adding the hot spring water sample at 45 ℃, 50 ℃ and 65 ℃ showed that the culture solution showed a pH increase after 6 days of culture, the pH of the culture solution reached 9 at day 9, and no sign of growth of desulfurization bacteria was observed when the culture solution was taken out and observed with a microscope. The original Bacillus thermoanaerobicus strain in these culture solutions was not observed, and it was considered that the previously added bacteria had lysed during 9 days of culture. When the culture is started, the culture medium contains the yellow-heat anaerobic bacillus, and the yellow-heat anaerobic bacillus cannot grow in the desulfurization medium per se and can only promote the growth of other desulfurization bacteria in the hot spring according to the culture result, which is consistent with the condition that the yellow-heat anaerobic bacillus is a heterotrophic bacillus and the desulfurization medium does not contain organic matters.
EXAMPLE 2 Effect of screening for desulfurization bacteria from Hot spring samples with other culture solutions
1) Screening effect of screening culture solution by using optimized desulfurization bacteria
The optimized screening culture solution of the desulfurization bacteria is essentially a culture solution without adding auxiliary screening bacteria in the preparation process of the high-efficiency screening culture solution of the heat-resistant desulfurization bacteria.
200mL of the optimized desulfurization bacteria screening culture solution is added into a sterilized 250mL triangular flask, 2mL of hot spring sample is added, the culture is carried out at the temperature corresponding to hot spring water, and the screening of the desulfurization bacteria is carried out, wherein the hot spring water sample is shown in example 1. The culture results are shown in Table 2.
TABLE 2 results of screening of desulfurization bacteria with the optimized desulfurization bacteria screening culture solution
As can be seen from Table 2, when the samples are inoculated in the optimized screening culture solution for culture, the pH of the samples 01-05 shows the sign of pH increase after the samples are cultured for 5-6 days, the pH of the culture solution reaches 9 after the samples are cultured for 8-9 days, and microscopic examination does not show any sign of microbial growth, which indicates that the desulfurization bacteria cannot be screened. Therefore, if the auxiliary screening bacteria of the anaerobic bacillus flavedo is not added in the optimized screening culture solution of the desulfurization bacteria, the effect of screening the desulfurization bacteria is not ideal.
2) Screening Effect of other culture solutions containing no auxiliary bacteria
In the early stage of the research work of the invention, the screening of the desulfurization bacteria is carried out by adopting the common desulfurization bacteria culture solution, but the desulfurization bacteria can not be screened. The composition of the medium was subsequently changed. Among these culture liquids prepared by different methods, a medium having a different nitrogen source is typical, and examples thereof include the following media:
to 800mL of deionized water were added: KH (Perkin Elmer)2PO4 0.3-0.7g，K2HPO4 0.4-0.6g，MgCl2 0.02-0.08g，CaCl20.03 to 0.09g, 3 to 8g of NaCl, 0.05 to 0.06g of nitrogen source (0.05 to 0.08g of sodium nitrate or 0.05 to 0.08g of ammonium chloride or 0.05 to 0.08g of ammonium nitrate or 0.05 to 0.08g of ammonium sulfate, or a combination of nitrogen sources is used, wherein the total amount of the nitrogen source is still 0.05 to 0.08g), 0.2 to 0.7g of sodium thiosulfate pentahydrate, 0.1 to 0.3g of sodium bicarbonate, 0.1 to 0.2mL of trace element solution, and water is added to 1L. Filtering and sterilizing after the preparation.
Solution of trace elements: EDTA.Na was added to 10L of deionized water2After 3-8g of the mixture is fully dissolved, the following components are added in sequence: (NH)4)6Mo7O24·4H2O 0.1-0.2g、CoCl·6H2O 0.1-0.3g、MnCl·4H2O 0.3-0.6g、CuSO4·5H2O 0.1-0.3g、ZnSO4·7H2And (3) fully dissolving O1-2 g and FeSO 42-5 g, and filtering and sterilizing.
In the research process, culture solutions with various components are used, and the culture solution without the auxiliary bacterium, namely the thermoanaerobacterium flavum cannot be screened from the five samples to obtain the desulfurization bacteria.
3) Screening effect of different culture solutions and auxiliary bacteria
In order to screen for microorganisms that may be helpful in screening for desulfurization bacteria, theA large number of microorganisms are tested, and 1000 strains of various microorganisms are mainly obtained from an industrial microorganism resource data platform of colleges and universities in China, Jiangnan university. These microorganisms were cultured and added to the various types of desulfurization media described in example 2 in such an amount that the final bacteria content per ml of the culture was about 105And (4) cells. The culture solution containing the other bacteria is inoculated with a hot spring-derived sample, and cultured at a temperature corresponding to the temperature of the hot spring-derived sample (not higher than 65 ℃).
The results show that only the components of the screening culture solution for heat-resistant desulfurization bacteria can screen the heat-resistant desulfurization bacteria. 3 strains of yellow fever anaerobic bacillus are collected in a data platform (http:// www.cicim-cu. jiangnan. edu. cn) of industrial microbial resources of colleges and universities in south Jiangnan, and the serial numbers of the yellow fever anaerobic bacillus are respectively as follows: CICIM B6808, CICIM B6809 and CICIM B6951. The three strains are used as auxiliary screening strains and added into an optimized screening culture solution of the desulfurization bacteria to be cultured together, so that heat-resistant desulfurization bacteria can be obtained through screening, and the heat-resistant desulfurization bacteria cannot be obtained through screening by combining the other bacteria and different culture solutions.
The culture solution capable of screening heat-resistant desulfurization bacteria comprises the following components: KH (Perkin Elmer)2PO4 0.4-0.5g/L，K2HPO40.5-0.6g/L，MgCl2 0.03-0.05g/L，CaCl20.05-0.07g/L, 4-6g/L NaCl, 0.05-0.06g/L nitrogen source, 0.3-0.4g/L sodium thiosulfate pentahydrate, 0.1-0.2g/L sodium bicarbonate and 0.1-0.2mL/L trace element solution; wherein the addition concentration of the Bacillus thermoanaerobicus is 0.8-1.2 x 10 per ml of culture solution5A cell, wherein the composition of the nitrogen source includes, but is not limited to, table 4 below:
TABLE 4 composition of nitrogen source in culture broth for screening heat-resistant desulfurization bacteria obtained by the present invention
The heat-resistant desulfurization bacteria screening culture solution containing the nitrogen source is added into 5 hot spring water samples in the embodiment 1 for culture, the pH of the culture medium is reduced after 5 samples are cultured for 4-8 days, obvious sulfur taste is generated in the culture solution after the culture is continued, floating sulfur simple substance is generated on the surface of the culture solution, a large amount of bacteria are found in the culture solution through microscopic examination, the form of the bacteria is obviously different from that of the previously added yellow-heat anaerobic bacillus, and the screened heat-resistant desulfurization bacteria can be seen. It can be seen that the culture solutions obtained in example 2.3 can be used for screening heat-resistant desulfurization bacteria in hot springs, but the screening effect is inferior to that of the culture solution for screening heat-resistant desulfurization bacteria optimized in example 1 in combination with Thermoanaerobacterium flavum.
Example 3 screening results of desulfurization Medium supplemented with different Thermoanaerobacters flavus
In example 2, the three strains which were obtained from the data platform of industrial microbial resources in university, china, south china and were able to significantly promote the growth of desulfurization bacteria belong to the group of yellow fever anaerobic bacillus in classification, but the three strains are significantly different in colony morphology and also significantly different in 16s rDNA sequences. It is presumed that the growth of the desulfurization bacteria may be promoted by the Bacillus thermoanaerobicus. The inventor purchases another two strains of Thermoanaerobacters flavicans (numbered as XQ03734 and XQ03738) from Xianqin Biotechnology Limited company to perform further tests, and the results show that the culture medium obtained by adding the two strains into the optimized screening culture solution of the desulfurization bacteria has the phenomenon of pH reduction after 4 days of culture after adding hot spring samples, the culture solution has obvious sulfur taste after being cultured for one day, a great amount of bacteria can be seen by microscopic examination of the culture solution, and the bacteria have different forms from the Thermoanaerobacters flavicans, so that the heat-resistant desulfurization bacteria are screened. In conclusion, on the basis of the optimized screening culture solution for the desulfurization bacteria, the anaerobic bacillus flavothermis generally has the effect of improving the screening efficiency of the desulfurization bacteria in hot springs, and has no obvious relation with the specific strains used.
Example 4 Effect of Using culture solution for efficient screening of Heat-resistant desulfurization bacteria
In order to further examine the effect of the selection culture solution for heat-resistant desulfurization bacteria of the present invention, more hot spring samples were collected from different locations and tested, and the results are shown in table 5.
TABLE 5 results of screening of other samples for desulfurization bacteria using the heat-resistant desulfurization bacteria screening culture solution
TABLE 6 results of screening of desulfurization bacteria from other samples with the optimized heat-resistant type desulfurization bacteria screening culture solution
Description of the samples:
a, preparing first-class water from a hot spring bathing pool in Shangquan county of Shannan province. The temperature of the hot spring is about 55 ℃, and the culture temperature for screening the desulfurization bacteria is 50 ℃.
B big rolling pot hot spring of wax palm hot spring group of Longling county of Yunnan province (the hot spring does not boil when sampling). The temperature of the hot spring is about 80 ℃, and the culture temperature for screening the desulfurization bacteria is 65 ℃.
C hot spring head water behind hot spring town small pine source hot spring restaurant in east sea county of Jiangsu province. The source is in a deep well, the water temperature is not measurable, the estimated temperature is 70 ℃, and the culture temperature for screening the desulfurization bacteria is 65 ℃.
D, a water outlet of bathing pool spring water of army of Chozacun county, Tengchong province is at 45 ℃, and the culture temperature for screening the desulfurization bacteria is 45 ℃.
E, water sample is taken from the spring water outlet (above the bathing pool) of the spring town of Nanjing, the water temperature is estimated to be 50 ℃, and the culture temperature is 45 ℃.
As can be seen by comparing tables 5 and 6, when the heat-resistant desulfurization bacteria screening culture solution is used for screening desulfurization bacteria, 4 of the 5 samples can be screened. When the desulfurization culture medium without the yellow-heat anaerobic bacillus is used for screening, only the desulfurization bacteria are screened from the stock spring pool of the mall, which shows that the content of the desulfurization bacteria in the hot spring water sample is higher, so that the desulfurization bacteria can be screened without adding auxiliary bacteria, but the screening time is obviously prolonged. No heat-resistant desulfurization bacteria could be screened for sample B in any of the media. The hot spring temperature of sample B was 80 ℃ and the bacteria in this hot spring, which is particularly high, were not suitable for screening using the method of this patent.
In conclusion, the method for screening the heat-resistant desulfurization bacteria by adopting the high-efficiency screening culture solution for the heat-resistant desulfurization bacteria has obviously higher efficiency.
Example 5 Effect of subculturing Heat-resistant desulfurization bacteria
In example 4, A, C, D, E in table 5, the bacterial solution obtained by screening 4 samples by the method of the present invention was further inoculated with 1% inoculum size of a screening culture solution of heat-resistant desulfurization bacteria, an optimized screening culture solution of heat-resistant desulfurization bacteria, and a culture solution of common desulfurization bacteria, respectively, and subcultured. The results show that the pH of the culture solution of the 4 cultures is reduced after the culture is carried out for 3 days after the culture is carried out in the three culture solutions, obvious sulfur taste appears at the 4 th day, obvious floating sulfur simple substance exists on the surface of the culture solution, and a large amount of living bacteria can be seen through microscopic examination. These bacterial solutions can be continuously and stably passaged. It can be seen that the desulfurization bacteria obtained from hot spring by the present invention were not dependent on the method of the present invention in the re-cultivation, probably because of the large number of viable bacteria in the culture and the high activity.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A screening culture solution for heat-resistant desulfurization bacteria is provided, wherein the culture solution is：KH2PO4 0.4-0.5g/L，K2HPO4 0.5-0.6g/L，MgCl2 0.03-0.05g/L，CaCl20.05-0.07g/L, 4-6g/L NaCl, 0.05-0.06g/L nitrogen source, 0.3-0.4g/L sodium thiosulfate pentahydrate, 0.1-0.2g/L sodium bicarbonate and 0.1-0.2mL/L trace element solution; wherein the culture solution also comprises Bacillus thermoanaerobicus with concentration of 0.8-1.2 x 10 per ml5And (4) cells.
2. The screening culture solution of heat-resistant desulfurization bacteria of claim 1, wherein the nitrogen source comprises three compounds of sodium nitrate, ammonium chloride and ammonium nitrate, and the concentration of each of the sodium nitrate, the ammonium chloride and the ammonium nitrate is greater than or equal to 0.01 g/L.
3. The culture solution for screening heat-resistant desulfurization bacteria according to claim 1 or 2, wherein the nitrogen source is composed of 0.02g/L sodium nitrate, 0.02g/L ammonium chloride, and 0.02g/L ammonium nitrate.
4. The selection medium for heat-resistant desulfurization bacteria according to any one of claims 1 to 3, wherein the solution of trace elements is EDTA.Na2 0.5-0.7g/L；(NH4)6Mo7O24·4H2O 0.01-0.2g/L、CoCl2·6H2O 0.015-0.02g/L、MnCl2·4H2O 0.03-0.04g/L、CuSO4·5H2O 0.01-0.02g/L、ZnSO4·7H2O 0.13-0.14g/L，FeSO40.3-0.4g/L。
5. A method for screening heat-resistant desulfurization bacteria by using the screening culture solution for heat-resistant desulfurization bacteria according to any one of claims 1 to 4, which comprises the following steps:
(1) preparing a screening culture solution of heat-resistant desulfurization bacteria;
(2) adding hot spring water with the volume fraction of 1-3% into the culture solution, wherein the culture temperature is +/-5 ℃ corresponding to the hot spring, and the hot spring water is hot spring water with the temperature not exceeding 75 ℃; the culture temperature is not more than 65 ℃;
(3) and when the pH of the culture solution is reduced, the culture solution is preliminarily judged to be successful in screening the desulfurization bacteria, and further, sulfur smell and floating sulfur simple substances are judged to be successful in screening the desulfurization bacteria.
6. The method of claim 5, wherein when the pH of the culture medium rises to pH 9 or higher after 5 to 7 days of culture, it is determined that there is no heat-resistant desulfurizing bacterium in the hot spring water sample.
7. The method for screening heat-resistant desulfurization bacteria according to claim 6, wherein the culture temperature is set to 65 ℃ when the hot spring temperature is higher than 70 ℃.
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