CN104152375B - One strain deamination deodorization bacterial strain QDN01 and the application in biological deodorizing thereof - Google Patents

Abstract

The invention discloses a deammonization and deodorization bacterial strain QDN01 and its application in biological deodorization. The present invention screens a deammonization and deodorization bacterial strain from grass-root black soil, and studies its biological characteristics and denitrification of chicken manure. Through the analysis of physiological and biochemical characteristics and 16SrDNA sequence, the deaminating and deodorizing bacterial strain was identified as Enterobacter hallii, and it was preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M2013670. Studies have shown that the QDN01 strain isolated by the present invention has stable biological characteristics and fast reproductive ability, especially can grow rapidly in chicken manure added with starch, and the growth amount reaches 250 billion cfu/ml, and can denitrify well at the same time Deodorization has obvious advantages compared with the prior art. Therefore, the proposal of the present invention provides a very feasible and practical technical solution for the harmless biological treatment of poultry manure, and has a good development prospect.

Description

A Deamination and Deodorization Bacteria QDN01 and Its Application in Biological Deodorization

technical field

The invention relates to a deammonization and deodorization bacterium, and also relates to the application of the strain in the harmless biological treatment of poultry manure and industrial waste water, especially in the deamination and deodorization of poultry manure. The invention belongs to the technical field of biological deodorization.

Background technique

With the rapid development of global industrial and agricultural products and the continuous improvement of human living standards, odors also come from the generated waste. Odor pollution is a kind of perceived pollution. Its harmful gases directly affect people's life and health. Therefore, odor pollution has been recognized as one of the six major public hazards after noise pollution. There are many ways to remove odor, and microbial odor removal has become one of the methods with high efficiency, environmental protection and low consumption, and has exerted great potential in environmental pollution.

The main cause of the stench is the toxic and harmful gases such as ammonia, hydrogen sulfide, methyl sulfide, and methane produced by the fermentation of livestock and poultry manure and the decomposition of sulfur-containing proteins, which increase the pollution in the air and reduce the air quality. To solve this problem, a large number of scholars have carried out a series of researches.

According to the characteristics of malodorous gas purification methods, the methods of purifying odor pollutants can be divided into: (1) physical methods, such as masking neutralization method, diffusion dilution method, condensation method, water absorption method, adsorption method; (2) chemical method , such as chemical washing, combustion, oxidation, etc.; (3) biological methods, such as biological filtration, biological absorption, soil composting, etc.

Biological deodorization is a method developed in recent years to deal with malodorous gases. It uses the biochemical action of microorganisms to convert malodorous substances into odorless or less odorous substances to achieve the purpose of deodorization. The principle is that malodorous substances are first dissolved in water, then absorbed by microorganisms into the microorganisms, and decomposed as their nutrients, thereby producing odorless substances.

The biological deodorization process roughly has the following three stages:

In the first stage, the process of transferring the malodorous gas from the gaseous state to the liquid state follows Henry's law.

In the second stage, the aqueous solution of the malodorous gas is adsorbed by the microbial flora and absorbed into the organism, so that the malodorous components are removed from the water, and the speed is close to the speed of general chemical reactions.

In the third stage, the malodorous substances ingested by microbial cells become the energy source of microorganisms, and are decomposed, utilized and turned into cellular substances through microbial metabolism to be removed. Nitrogen-containing malodorous components such as amines and ammonia are partly used to form microbial cell proteins, and partly converted into nitrous acid or nitric acid. Nitrogen-free substances such as butyric acid, phenol, and formaldehyde are decomposed into CO ₂ and H ₂ O, etc. Sulfur-containing malodorous substances are oxidized to S, SO ₃ ^2- , SO ₄ ^2- by the action of fungi, molds, and sulfur-oxidizing bacteria, and become the source of microbial energy. Biological deodorization methods can be divided into biological filtration method (fixed state), biological absorption method (suspended state), soil composting method and biological deodorizer, etc.

The principle of the filtration method is to make the collected exhaust gas pass through the solid carrier (filler) covered with microorganisms such as bacteria, fungi, and actinomycetes under suitable conditions. The odorous substances are first absorbed by the filler, and then oxidized and decomposed into carbon dioxide and carbon dioxide by the microorganisms on the filler. water and other substances to complete the deodorization process of exhaust gas. The method has the advantages of simple equipment and low operating costs; however, the reaction conditions are not easy to control and occupy a large area. It is suitable for treating large volume and low concentration of odor.

The absorption method is also called the biological washing method. It uses the washing method to transfer the malodorous components to the water, and then uses the activated sludge to treat the contaminated water with microorganisms to effectively absorb and decompose the odor components. The reaction conditions of this method are easy to control and the operating cost is low, but a large amount of oxygen is required to maintain high-efficiency operation.

The raw materials of the soil composting method are mainly sludge, feces, garbage and other substances, which are mixed and then subjected to aerobic fermentation heat treatment to suppress the generation of odor. The deodorization efficiency of this method is higher, but the cycle is longer.

Biological deodorizers generally refer to domestication and screening of highly efficient deodorizing microorganisms from activated sludge or soil in sewage treatment plants for odor control. The biological agent developed according to the principle of microbial deodorization is to immobilize the screened high-efficiency microorganisms on a certain carrier, and the purpose of removal can be achieved when the odorous substances pass through

The main odor components that endanger the normal growth of livestock and poultry and affect human health in the breeding industry are NH ₃ and H ₂ S. Therefore, these two gases are often used as the main detection indicators of farm environmental pollution, and are also important to measure the effect of microbial deodorants. index. Common microorganisms that can utilize ammonia mainly include nitrifying bacteria. Nitrifying bacteria are inorganic chemoautotrophic microorganisms that use CO ₂ as a carbon source to synthesize organic matter and play a very important role in the nitrogen cycle in nature. The nitrification they perform converts ammonia nitrogen into nitrite, which in turn converts nitrite into nitrate. The process of oxidizing NH ₃ to NO ^3- is the result of the continuous action of two types of bacteria, ammonia oxidizing bacteria and nitrite oxidizing bacteria. Ammonia-oxidizing bacteria convert ammonia nitrogen to nitrite, and nitrite-oxidizing bacteria oxidize nitrite to nitrate. Both types of bacteria belong to the family Nitrobacteriaceae. Nitrifying bacteria play a very important role in the nitrogen cycle in nature. Nitrifying bacteria can convert ammonia nitrogen into nitrite through nitrification, and then further convert nitrite into nitrate, which is an important step in the complete mineralization of nitrogenous substances and plays an important role in the nitrogen cycle of the ecosystem.

After the 1970s, various countries have successively carried out extensive research in the field of biological deodorization, among which Germany and Japan have achieved the most remarkable results. Since the mid-1980s, high-efficiency deodorizing bacteria targeting specific odor pollutants have been discovered one after another, and when applied to the background flora of an actual deodorization system, a dominant bacterial community with higher deodorization efficiency can be formed. At present, Japan is more researched on the control of sulfur-containing odors. The "Thiobacillus sulfur bacteria TK-M" discovered by the Institute of Microbial Technology of the Japan Industrial Technology Institute has a very strong ability to decompose sulfur-based odor substances. Ono Katsushi used the Bacillus subtilis isolated from the soil, which has a strong ability to decompose oily wastewater. This bacterium has a good inhibitory effect on the odor of oil, and has now been made into a deodorant product. Kurita Industry and Tokyo Institute of Technology have developed microbial deodorants such as Nitrosomonas, which use peat as a carrier. Filling this into a reaction tank can remove hydrogen sulfide, ammonia and other odorous components. The Japanese Institute of Microbial Technology dried the activated sludge of the sewage plant at 30-40°C and crushed it to make a deodorant, which was filled into a column tube. When hydrogen sulfide, mercaptan and other odors pass through, it can have a good removal effect. .

Microorganisms have the characteristics of wide sources, rapid reproduction, easy cultivation, strong adaptability to the environment, and easy variation. Under certain conditions, they can be domesticated and have good application prospects in many aspects. Paneray et al. Soil microbial treatment of H ₂ S waste gas" ^[9-10] , followed by the application of 20-type cell immobilization technology (such as embedding method) to biological treatment devices, it is expected that odor control technology, especially biological treatment technology This is also a direction for environmental protection workers to work hard in the future.

The invention mainly produces a bacterial strain for degrading ammonia nitrogen and nitrate nitrogen through selective cultivation and domestication, and studies its biological characteristics and denitrification and deodorization effects, thereby providing a new microbial deodorizing bacterial agent.

Contents of the invention

One of the objects of the present invention is to domesticate a deammonizing and deodorizing bacterial strain capable of effectively degrading ammonia nitrogen and nitrate nitrogen through selective cultivation;

Two of object of the present invention is to provide the application of described bacterial strain in biological deodorant;

The third object of the present invention is to provide a deodorant bacterial agent, which contains the above-mentioned deamination and deodorization bacterial strain;

Four of purpose of the present invention is to provide a kind of method that chicken manure is carried out deamination deodorization.

The purpose of the present invention is achieved by the following technical means:

The present invention screens a deammonization and deodorization bacterial strain from grass-root black soil, and studies its biological characteristics and denitrification of chicken manure. Through physiological and biochemical characteristics and 16SrDNA sequence analysis, the deaminating and deodorizing bacterial strain was identified as Enterobacter hormaechei (Enterobacter hormaechei), named Enterobacter hormaecheiQDN01, and preserved in the China Center for Type Culture Collection, the address is Wuhan University, and the preservation number is CCTCC NO: M2013670, the deposit date is December 17, 2013.

The present inventor has investigated the growth curve of QDN01 bacterial strain and the growth situation of bacterial strain under different temperature (15 ℃～40 ℃), pH value (pH=5～9), different C/N (15 :1～73:1) the growth of the strain, and the degradation of ammonia nitrogen and nitro nitrogen by the strain of nitrification and heterotrophic nitrification 72h. Studies have shown that the most suitable growth temperature for QDN01 strain is 30°C, the optimum growth pH for deaminated strains is about 8, and the best growth rate for strains is 25:1 when the medium C/N is different. When the QDN01 strain is cultured for 30-32 hours, the The quantity is basically stable, the removal rate of ammonia nitrogen reaches 64.75%, and the removal rate of nitro nitrogen reaches 70.62%. In addition, the bacterial strain of the present invention is used for the deodorization of chicken manure, and the results show that the isolated QDN01 strain of the present invention can grow rapidly in chicken manure added with starch, and the growth amount can reach 250 billion cfu/ml, and at the same time, it can be well Compared with the existing technology, the denitrification and deodorization of chicken manure has obvious advantages.

Therefore, the present invention also proposes the application of the deamination and deodorization bacterial strain in biological deodorization.

In the present invention, preferably, the biological deodorization includes removing peculiar smell in livestock manure and malodorous industrial wastewater, and more preferably, the livestock manure is chicken manure.

For the convenience of use, the present invention obtains a biological deodorizing bacterial agent by absorbing the bacterial liquid containing the above-mentioned deaminating and deodorizing bacterial strain QDN01 on bentonite, and adding brown sugar at the same time.

In the present invention, preferably, the biological deodorizing bacteria agent is prepared by absorbing the bacteria liquid containing the above-mentioned deaminating and deodorizing bacterial strain QDN01 on bentonite to make 1.0×10 ¹² (bacteria)/g Bacteria preparation, obtained after adding 1% commercially available brown sugar by mass ratio simultaneously.

Practice has proved that the deodorizing bacteria agent is evenly sprinkled on the chicken manure excretion place below the breeding cage (the chicken manure uses starch to adjust the C/N ratio to 20-30/1), and the usage amount is 200g bacteria agent/kg chicken manure Starch was sprayed once every 3 days. The on-site sensory evaluation was conducted by the breeding personnel and volunteers of the demonstration sites. The air quality in the breeding houses of the 4 demonstration sites improved significantly, the choking ammonia odor disappeared, and the rancid smell was reduced. The volunteers It means that the time to stay in the breeding room has been extended. Based on the on-site evaluation results, the bacterial agent can effectively reduce the ammonia content in the breeding house and improve the air quality. Simultaneously, 20 days of monitoring have been carried out for the egg output of the demonstration points. After the deodorizing bacteria agent of the present invention was applied at 4 monitoring points, the egg output increased by 4-6% on average, and the increase in output was relatively large. It is beneficial to the improvement of the chicken body condition, thereby increasing the egg production.

Therefore, the present invention proposes the application of the biological deodorizing bacteria agent in biological deodorization.

Wherein, the biological deodorization includes removing peculiar smell in livestock manure and malodorous industrial wastewater, preferably, the livestock manure is chicken manure.

Further, the present invention also proposes a method for deaminating and deodorizing chicken manure, which is characterized in that it comprises the following steps:

(1) Adsorb the bacterium solution containing the deamination and deodorization bacterial strain QDN01 of the present invention on bentonite, and add brown sugar simultaneously to obtain the biological deodorization bacterium;

(2) Use starch to adjust the C/N ratio of chicken manure to 20-30:1;

(3) Sprinkle the deodorant bacterium agent of step (1) evenly in the chicken manure after step (2), and sprinkle once every 3 days;

(4) Evaluation of the deodorizing effect.

In the present invention, preferably, the bacterial solution containing the deaminated and deodorized bacterial strain QDN01 is adsorbed on bentonite to make a bacterial preparation of 1.0×10 ¹² /g, and at the same time, 1% commercially available brown sugar is added by mass ratio , to obtain deodorant bacteria.

In the present invention, preferably, adopting starch to adjust the chicken manure C/N ratio is 25:1, and the deodorizing bacteria agent of step (1) is 200g bacteria agent/m ³ (chicken manure starch) evenly sprinkled on the In the chicken manure after step (2).

The above studies show that the QDN01 strain isolated by the present invention has stable biological characteristics, fast reproductive ability, good deamination and deodorization ability, and can grow rapidly especially in chicken manure added with starch, and the growth amount reaches 250 billion cfu/ ml, and can denitrify and deodorize well at the same time, which has obvious advantages compared with the prior art. The proposal of the invention provides a very feasible and practical technical solution for the harmless biological treatment of poultry manure, and has a good development prospect.

Description of drawings

Fig. 1 is the bacterium colony form of QDN01 bacterial strain growing 24h and diluting 10-8 on N1 selection medium;

Fig. 2 is the phylogenetic tree (vector diagram) of deamination deodorizing bacteria;

Fig. 3 is the growth curve of QDN01 bacterial strain;

Fig. 4 is the influence of temperature on the growth of QDN01 bacterial strain;

Fig. 5 is the influence of pH on the growth of QDN01 bacterial strain;

Fig. 6 is the influence of C/N on the growth of QDN01 bacterial strain;

Fig. 7 is the change situation of growth and deammonia nitrogen of QDN01 bacterial strain under OD600;

Figure 8 is the change of growth and denitrification nitrogen of QDN01 strain under OD600;

Figure 9 is the number of bacteria observed under the hemocytometer for the QDN01 strain;

Fig. 10 is the pH change in the chicken manure starch under three kinds of different carbon-to-nitrogen ratios;

Fig. 11 is the change of pH in chicken manure starch under three different carbon-nitrogen ratios by adding the same amount of QDN01 strain;

Fig. 12 is the comparison of deodorization effect in the chicken manure starch that does not add QDN01 bacterial strain;

Fig. 13 is the comparison of deodorizing effect in the chicken manure starch that adds QDN01 bacterial strain;

Figure 14 is the change of ammonium nitrogen content over time in chicken manure starch without adding the QDN01 bacterial strain;

Fig. 15 is that the ammonium nitrogen content changes with time in the chicken manure starch that adds QDN01 bacterial strain;

Fig. 16 is that the nitrate nitrogen content changes with time in the chicken manure starch that does not add QDN01 bacterial strain;

Fig. 17 is that the nitrate nitrogen content changes with time in the chicken manure starch that adds QDN01 bacterial strain;

Fig. 18 is the growth curve in the chicken manure starch of QDN01 bacterial strain;

Figure 19 is the change of the number of bacteria in chicken manure starch of different QDN01 bacterial strains;

Figure 20 shows the changes in the D600 absorbance of the bacteria suspension in chicken manure starch with different QDN01 strains.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Isolation, acclimatization and identification of deamination and deodorization bacterial strain QDN01

1. Materials and methods

1.1 Medium

Ammonia enriched medium (N1 selective medium): 36.00g sucrose, 2.00g KH ₂ PO ₄ , 0.50g MgSO ₄ , 2.00g NaCl, 10.00ml ammonia water, 0.10g FeSO ₄ , 5.0ml 1% ZnSO ₄ , agar 20g, 1000ml distilled water, pH=7.0, sterilized at 121°C for 20min, C/N 7:1.

Liquid NH ₃ selective medium (N2 medium): N1 medium does not contain agar, C/N is 7:1.

Denitrification medium (DM medium): C ₆ H ₅ Na ₃ O ₇ 2H ₂ O 3.00g, KNO ₃ 0.7210g, KH ₂ PO ₄ 1.0g, MgSO ₄ 7H ₂ O 1.0g, distilled water to 1000ml, adjusted to pH=7.0, sterilized at 121°C for 20min.

Heterotrophic nitrification medium: C ₆ H ₅ Na ₃ O ₇ 2H ₂ O 5.0g, (NH ₄ ) ₂ SO ₄ 0.45g, K ₂ HPO ₄ 0.25g, FeSO ₄ 7H ₂ O 0.0025g, NaCl 0.125g , MgSO ₄ 0.06g, MnSO ₄ ·H ₂ O 0.001g, distilled water to 1000ml, adjusted to pH=7.0, sterilized at 121°C for 20min.

1.2 Isolation and acclimatization of deamination and deodorization strains

Grass root black soil with higher oxygen content in wetlands was taken (Jennifer G.Allen, Marc W.Beutel, Douglas R.Call, et al. Effects of oxygenation on ammonia oxidation potential and microbial diversity in sediment from surface-flow wetland mesocosms[J] .Bioresource Technology, 2010,101(4):1389-1392.) 100g and 500ml distilled water were put into 1000ml Erlenmeyer flask at 150r/mim, shaken for 1h, and stood still; 50ml supernatant was put into 150ml N2 culture medium, 130r /min, shake at a constant temperature of 30°C for 3 days; then take 10ml of the turbid solution and directly add it to 190ml of N2 culture solution, repeat every 3 days, and repeat 8 times. Use a pipette gun to take 1ml of the enriched bacterial solution and dilute it to 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , respectively, take 0.1ml and spread it on the N1 selection medium, culture at 30°C for 48 hours, and select the form Repeated domestication and cultivation of single colonies with different appearances.

1.3 Observation of strain morphology

Observe the shape, edge, color, transparency, etc. of the colony on the N1 selection medium, and use the safranin staining method to take pictures under an optical microscope to observe the shape and size of the bacteria.

1.4 Physiological and biochemical tests

Using Gram staining, methyl red test, VP test, indole test, H2S production test, lysine decarboxylate, arginine dihydrolase, oxidase test, glucose _acid production, acetate utilization, Citrate, phenylalanine deaminase, gelatin liquefaction test, urease hydrolysis test, oxidation-fermentation test, ornithine decarboxylase, spore staining, glucose gas production, nitrate reduction, motility test, malonate test Physiological and biochemical identification of deamination and deodorization bacteria. For specific experimental methods, refer to the eighth edition of "Berger's Handbook of Bacteria".

1.5 Sequence analysis of strain 16SrDNA

Take the bacterial liquid in the logarithmic phase, collect the bacterial cells after centrifugation, extract the total genomic DNA, design and synthesize primers according to the sequence of bacterial 16SrDNA, forward primer: 5'-AGAGTTTGATCMTGGCTCAG-3', reverse primer: 5'-TACGGYTACCTTGTTACGACTT- 3'. PCR reaction system: 10×Ex Taq buffer 2.0 μl, 2.5mM dNTP Mix 1.6 μl, 5p Primer1 0.8 μl, 5p Primer2 0.8 μl, Template 0.5 μl, 5u Ex Taq 0.2 μl, ddH ₂ O 14.1 μl, Total volume 20 μl. PCR reaction conditions: Denaturation at 95°C for 5 minutes and then cycle: 95°C for 30s → 55°C for 30s → 72°C for 1.5min → 72°C for 10min, a total of 24 cycles; finally annealing at 10°C ∞. After purification, the amplified product was sequenced by Shanghai Meiji Biological Co., Ltd., and the obtained sequence was compared with the 16S rDNA sequence already in the nucleic acid database in Genbank to establish a phylogenetic tree for similarity comparison analysis.

1.6 Effects of Environmental Conditions on the Growth of Deammonization and Deodorization Strains

1.6.1 Growth curve of deaminated and deodorized bacterial strains: Take 2ml of freshly cultivated bacterial seed liquid and transfer it to 98ml of N2 liquid medium, shake at a constant temperature of 30°C (130r/min), at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48h Measure the growth of the strain at a wavelength of 600nm OD value, the growth of the strain was studied with the change of OD value.

1.6.2 The influence of temperature on the growth of bacterial strains: Take 2ml of freshly cultured bacterial seed liquid and transfer it to 98ml of N2 liquid medium, and shake it at 15, 20, 25, 30, 35, and 40°C respectively (130r /min) after culturing for 24 h, the optical density value (OD ₆₀₀ nm) of the bacteria was measured at a wavelength of 600 nm to study the effect of temperature on the growth of the strain.

1.6.3 The effect of pH on the growth of the strain: Take 2ml of the freshly cultured bacterial seed solution and transfer it to 98ml of N2 liquid medium, and shake it at pH 7, 7.5, 8, 8.5, and 9 respectively (130r/min) After culturing for 24 hours, the optical density (OD ₆₀₀ nm) of the bacteria was measured at a wavelength of 600 nm to study the effect of pH on the growth of the strain.

1.6.4 The effect of C/N on the growth of strains: by changing the addition of sucrose and ammonia water, adjust the C/N of N2 medium to 15, 25, 35, 46, 54, 64, and 73 respectively, and take freshly cultivated bacteria 2ml of the seed solution was transferred to 98ml of N2 liquid medium with C/N of 15, 25, 35, 46, 54, 64, and 73, respectively, and cultured with shaking (130r/min) for 48h, and the bacteria were measured at a wavelength of 600nm every 4h. The optical density value (OD ₆₀₀ nm) of the body was used to study the effect of C/N on the growth of the strain.

1.7 Denitrification Performance Determination of Bacterial Strains

1.7.1 Performance measurement of deaminated nitrogen: Take 2ml of freshly cultivated bacterial seed liquid and connect it to 98ml of heterotrophic nitrification medium, culture at 130r/min, 30°C for 96h, take 2ml every 6h, and measure the bacteria at a wavelength of 600nm Then centrifuge the sample at 4000r/min for 10min, take the supernatant and use Nessler's reagent spectrophotometry ( _HJ535-2009 , Determination of Ammonia Nitrogen in Water Quality Nessler's reagent spectrophotometry [S]) , detect the content of ammonia nitrogen in the solution at a wavelength of 420nm.

1.7.2 Performance measurement of denitrifying nitrogen: Take 2ml of freshly cultured bacterial seed liquid and connect it to 98ml DM medium, culture at 130r/min, 30°C for 96h, take 2ml every 6h and measure the light of the bacterial cell at a wavelength of 600nm Density value (OD ₆₀₀ nm), then the sample was centrifuged at 4000r/min for 10min, and the supernatant was taken by ultraviolet spectrophotometry (Shaanhong, Zhang Qingzhong, Zhang Xiaojuan, etc., storage, analysis methods and other factors have a great influence on the determination of nitrate nitrogen in soil. Effect[J]. Journal of Analytical Testing, 2013,32(12):1466-1471.) The content of nitro nitrogen in the solution was detected at a wavelength of 210nm.

2. Results

2.1 Isolation and domestication analysis of QDN01 strain

The present invention uses the grass-root black soil to screen bacterial strains, and the purpose is to avoid screening out pathogenic bacteria with the same function as feces. Through the enrichment and cultivation of selective medium, and by boric acid absorption titration (Wu Pengming. Ambient Air Quality Assurance Manual [M]. China Environmental Science Press, 1989: 184-211) screen out the best deamination effect single strain and do further experiments.

2.2 Observation on Morphology and Physiological and Biochemical Tests of Deamination and Deodorization Bacteria

It can be seen from Figure 1 that the single strain is 2 to 3 mm in diameter, milky white, oval in shape, smooth in surface, raised, translucent, without exudate, negative in Gram stain, and rod-shaped. Physiological and biochemical identification results are as follows:

Table 1 Physiological and biochemical test identification results of deamination and deodorization bacteria

Note: If the test result is positive, it will be indicated by the number "+"; if the result is negative, it will be indicated by the number "-".

2.3 Sequence Analysis of 16SrDNA of QDN01 Strain

The QDN01 strain was amplified by 16SrDNA, and its sequence size was about 1282bp. Use universal primers:

27F: 5'-AGAGTTTGATCCTGGCTCA-3', 1492R:

5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction system is: 10×Ex Taq buffer 2.0μl,

2.5mM dNTP Mix1.6μl, 5p Primer10.8μl, 5p Primer20.8μl, Template0.5μl,

5u Ex Taq 0.2μl, ddH2O 14.1μl, Total volume 20μl.

The sequence is shown in Figure 2 through the homology comparison analysis of the phylogenetic tree. It has a high homology of 99% with the 16SrDNA sequence in the genus Enterobacterhormaechei. It is Enterobacter hormaechei QDN01, which is preserved in China Center for Type Culture Collection with the preservation number CCTCC NO: M2013670.

2.4 Analysis of the influence of environmental conditions on the growth of QDN01 strain

2.4.1 The growth curve of deaminization and deodorization strains:

It can be seen from Figure 3 that 0-8h is the lag phase of the strain, and the growth of the logarithmic phase is 8-32h; the OD ₆₀₀ value does not change much when cultured for 32-48h, indicating that the strain has entered the stable growth phase at this time. The results showed that the lag phase and logarithmic phase of the QDN01 strain were shorter, and the stable phase was longer. The concentration of the strain could be high and relatively stable for a long time, which was beneficial to its degradation of the target substance.

2.4.2 Effect of temperature on the growth of QDN01 strain:

It can be seen from Figure 4 that the most suitable ambient temperature for the QDN01 strain is between 25°C and 35°C, and when the temperature is 30°C, the growth rate of the strain is the highest; after exceeding 30°C, the growth rate of the strain gradually decreases, therefore, The optimal temperature for this strain is 30°C. This also shows that temperature is one of the important indicators to measure the growth, reproduction and metabolism of strains. If the temperature is too high, it will lead to the inactivation of protein and nucleic acid; if the temperature is too low, the bacterial viability will be correspondingly inhibited, and the metabolic function of the cells will be relatively weakened. Therefore, the temperature requirement of the strain has a suitable growth range.

2.4.3 The effect of pH on the growth of QDN01 strain:

It can be seen from Figure 5 that the optimum pH of the QDN01 strain is about 8, and it grows better in the range of pH 7.5-8.5, while the environment of strong acid or strong alkali may relatively affect the growth of the strain. This also shows that pH is one of the important indicators of the adaptability of strains to the environment, and the optimum pH for the growth of each strain is often limited to a small range.

2.4.4 Effect of C/N on the growth of QDN01 strain

C/N is one of the indicators that reflect the nutrient requirements of the strain in the environment. Too high or too low will inhibit the growth of the strain to a certain extent. It can be seen from Figure 6 that when the C/N ratio of the strain is 25:1, the growth rate of the strain is relatively high and the reproductive ability is fast. With the increase of C/N, the OD value of the strain gradually decreased, which indicated that C/N had a certain influence on the denitrification and heterotrophic nitrification of the QDN01 strain, which was the same as the effect of C/N on the N produced by aerobic denitrifying bacteria. The effect of ₂ O is consistent (Takaya N, Catalan-Sakairi MAB, Sakaguchi Y, et al. Aerobic denitrifying bacteria that produce low levels of nitrogen oxide [J]. Applied and Microbiology Biotechnology, 2003, 69(6): 3152-3157. ), therefore, the optimal C/N content is selected as 25:1.

Denitrification Performance Analysis of 2.5QDN01 Strain

2.5.1 Performance determination of deaminated nitrogen

In this paper, Nessler's reagent is used to detect the content of ammonia nitrogen in the solution by spectrophotometry, and the initial concentration is 8 mg/l by dilution. It can be seen from Figure 7 that the strain reached a stable phase at about 30 hours, and the OD ₆₀₀ value after dilution was 0.042, which indicated that the QDN01 strain reproduced rapidly. The concentration of ammonia nitrogen in the medium decreased from 8mg/l to 2.82mg/l, and the removal rate reached 64.75% after culturing in the different nitrifying medium for 96 hours. This data shows that the QDN01 strain has better performance in removing ammonia nitrogen.

2.5.2 Denitrification performance measurement:

The content of nitrate nitrogen in the solution is detected by ultraviolet spectrophotometry, and the initial concentration is 10mg/l by dilution. It can be seen from Figure 8 that the strain reached a stable phase at about 30 hours, and the maximum value of OD ₆₀₀ after dilution was 0.041. After 96 hours of cultivation, the concentration of nitrate nitrogen in the medium decreased from 9.87mg/l to 2.90mg/l, and the removal rate reached 70.62%. This data shows that the QDN01 strain has a good ability to denitrify nitrogen.

The above research shows that the bacterial strain QDN01 screened by the present invention has a fast and vigorous growth rate, reaches a stable period in about 30 hours, and has a strong ability to remove ammonia and out-of-market nitrogen. QDN01 strain can grow in the medium with ammonia nitrogen and nitrate nitrogen concentration of 100mg·L ^-1 . In liquid medium, the strain has good removal ability of ammonia nitrogen and nitrate nitrogen, reaching 64.75% and 70.62% respectively. The results show that the removal ability of QDN01 strain to nitrate nitrogen is stronger than the removal ability to deammonia nitrogen, but the difference is not big, indicating that the strains have a relatively average denitrification ability and there will be no imbalance; the growth curve is drawn to explore the culture conditions of the strain To provide evidence, the QDN01 strain has a short delay period, fast logarithmic phase, strong reproductive ability, quickly reaches a stable period, and a long stable period, indicating that the inoculated strains are in a physiologically active period and have strong vitality. The culture conditions reached the best condition when the temperature was 30℃, the pH was 7.5-8.5, and the C/N ratio was 25:1. The strain is widely used, can remove peculiar smell in livestock manure, bad smell in industrial waste water, etc., has fast growth, wide propagation conditions, and is easy to be mass-produced into a corresponding bacterial agent and put on the market.

Example 2 Application of deamination and deodorization bacterial strain QDN01 in deamination and deodorization of chicken manure

1. Materials and equipment

1.1 Materials

Fresh chicken manure, QDN01 strain

1.2 Drugs and equipment

Drugs: glucose, concentrated sulfuric acid, hydrochloric acid, sodium hydroxide, 95% ethanol, mercury iodide, potassium iodide, sucrose, normal saline, sodium dihydrogen phosphate, disodium hydrogen phosphate, soluble starch, ammonia water, magnesium sulfate, iron sulfate, sulfuric acid Zinc;

Equipment: SHZ-D(III) circulating water vacuum pump, electronic balance, PB-10pH, THA-82A desktop constant temperature oscillator, electric heating constant temperature water bath, glass instrument airflow dryer, electric heating constant temperature blast drying oven, TU-1810 UV-Vis Spectrophotometer, Sterilizer

1.3 Instruments

Test tubes, colorimetric tubes, cuvettes, Erlenmeyer flasks, centrifuge tubes, plastic droppers, beakers, pipettes, glass rods, volumetric flasks, etc.

2. Solution preparation

2.1 Preparation of Nessler's reagent

Weigh 16.0g of sodium hydroxide, dissolve in 50ml of water, and cool to room temperature.

Weigh 7.0g of potassium iodide and 10.0g of mercury iodide, dissolve in water, then slowly add the solution to the above 50ml of sodium hydroxide solution under stirring, and dilute to 100ml with water. Stored in polyethylene bottles, tightly closed with rubber stoppers or polyethylene caps, stored in a dark place, valid for 1 year.

2.2 Preparation of Potassium Sodium Tartrate Solution

Weigh 50.0g of potassium sodium tartrate and dissolve it in 100ml of water, heat and boil to remove ammonia, fully cool and dilute to 100ml.

2.3 Preparation of ammonia nitrogen standard solution

Ammonia nitrogen standard stock solution: Weigh 3.8190g of ammonium chloride (ammonium chloride, superior grade, dry at 100-105°C for 2h), dissolve in water, transfer to a 1000ml volumetric flask, dilute to the mark, and store at 2-5°C Keep for 1 month.

2.4 Preparation of chicken manure starch with different carbon-to-nitrogen ratios

The C/N of chicken manure used in this experiment was 15:1, the molar concentration of C in the starch used was 37mol/kg, and the mass fraction of C was 44.44%. Based on this, the C/N in the system was adjusted.

Treatment Ⅰ is chicken manure + starch with a C/N ratio of 20:1, treatment II with chicken manure + starch with a C/N ratio of 25:1, and treatment III with chicken manure + starch with a C/N ratio of 30:1 , Three sets of fermentation tanks compost at the same time. Water is added to make the water content of the mixture 65%, and the shake bottle is fermented at 28 degrees Celsius.

3. Experimental methods and steps

3.1 Determination of ammonium nitrogen by Nessler's reagent method

(1) Water sample pretreatment: Colorless and clear water samples can be directly measured; water samples with high chroma, turbidity and more interfering substances need to undergo pretreatment steps such as distillation or coagulation precipitation.

(2) Drawing of standard curve: pipette 0, 0.50, 1.00, 3.00, 5.00, 7.00 and 10.0mL of ammonium standard solution into a 50mL colorimetric tube, add water to the marked line, add 1.0mL potassium sodium tartrate solution, and mix well. Add 1.5mL of Nessler's reagent and mix well. After standing for 10 minutes, measure the absorbance at a wavelength of 420nm with a cuvette with an optical path of 10mm and water as a reference.

After subtracting the absorbance of the zero-concentration blank tube from the measured absorbance, the corrected absorbance was obtained, and a standard curve of ammonia nitrogen content (mg) against the corrected absorbance was drawn.

(3) Determination of water samples: Take an appropriate amount of water samples (so that the ammonia nitrogen content does not exceed 0.1mg), add it to a 50mL colorimetric tube, dilute to the marked line, add 1.0mL potassium sodium tartrate solution (distilled pretreated Water samples, water samples and standard tubes do not add this reagent), mix well, add 1.5mL of Nessler's reagent, mix well, let stand for 10min.

(4) Blank test: replace the water sample with ammonia-free water, and make a blank test for the whole procedure.

(5) calculation

After subtracting the absorbance of the blank experiment from the absorbance measured by the water sample, the ammonia nitrogen content (mg) was found from the standard curve.

Ammonia nitrogen (N,mg/L)＝m×1000/V

In the formula: m——the ammonia nitrogen content (mg) of the sample tube obtained from the calibration curve;

V——the volume of water sample (mL).

And use a spectrophotometer to measure the D600 of the suspension of deaminating and deodorizing bacteria

3.2 Determination of nitrate nitrogen by ultraviolet method

(1) UV and visible spectrophotometers and quartz cuvettes.

(2) Nitrate nitrogen standard stock solution 100 μg/ml: Weigh 0.3609 g of potassium nitrate obtained by baking at 105° C. for 2 hours, dissolve in water, transfer to a 500 ml volumetric flask, and dilute to volume with water.

(3) Potassium nitrate standard solution preparation

Use a pipette to draw 25ml of stock solution into the volumetric flask, and distill the volume to 250ml with distilled water (the content of nitrate nitrogen in the solution is 10mg/l)

(4) Drawing of calibration curve:

Add 0.00, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00 ml of nitrate nitrogen standard solution to 10 25ml colorimetric tubes, put them into 50ml volumetric flasks respectively, and dilute to the mark with water , then add 2 ml of 10% sulfuric acid solution and shake well. Concentrations are 0.00, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00mg/l

(5) When measuring, measure its absorbance at 220 nanometers, and add the liquid to be tested into the cuvette at a time not to exceed 3/4 volume. Blank is distilled water

(6) Take 5ml of bacterial liquid and centrifuge at 4000r/min for 10min, take the supernatant after centrifugation, and put it into a quartz cuvette to detect the absorbance therein (three parallel samples)

3.3 Sensory determination method for odor of mixture of chicken manure and starch

Index determination. The 6-level classification method is used to divide the gas odor, which are Ms0: no odor; Msl: barely feel the odor; Ms2: weak odor; Ms3: obvious odor; Ms4: strong odor; Ms5: Unbearable stench. Logged at every sample.

Odor is represented by *. *More means stronger odor.

3.4 Determination of the number of bacteria in the medium by hemocytometer method

In this experiment, the counting area of the counting board was 80 grids.

Determination steps:

(1) Depending on the concentration of the bacterial suspension to be tested, add sterile water to dilute appropriately, and the number of bacteria per cell can be counted as the degree.

(2) Take a clean hemocytometer plate and put a cover glass on the counting area.

(3) Shake the bacterial suspension evenly, take a little with a dropper, and drop a small drop (not too much) from the grooves on both sides of the middle platform of the counting plate along the lower edge of the cover glass, so that the bacterial suspension can be used The surface tension of the liquid fills the counting area, so as not to generate air bubbles, and absorb the excess bacterial suspension flowing out of the groove with absorbent paper. You can also drop the bacterial suspension directly on the counting area (do not let the platforms on both sides of the counting area be stained with the bacterial suspension, so as not to increase the depth of the counting area after adding a cover glass), and then add a cover glass ( Do not create air bubbles).

(4) Let stand for a while, let the cells settle on the counting plate and no longer drift with the liquid. Place the hemocytometer on the stage of the microscope and clamp it firmly. First find the counting area under the low-power lens, and then switch to the high-power lens to observe and count. Since the refractive index of living cells is similar to that of water, the intensity of light should be weakened when observing.

(5) When counting, if the counting area is a counting area composed of 25 middle squares, to the four middle squares, it is necessary to count the number of bacteria in the central middle square (that is, 80 small squares). In order to ensure the accuracy of counting and avoid repeated counting and missing records, there should be uniform regulations on the statistics of cells settled on the grid line during counting. If the bacteria are located on the double lines of the large grid, when counting, count the upper line but not the lower line, and count the left line instead of the right line to reduce errors. That is, the cells located on the upper line and the left line of this grid are included in this grid, and the cells on the lower line and right line of this grid are included in the corresponding grid according to regulations.

(6) For germinated bacteria, when the bud reaches half the size of the mother cell, it can be counted as two bacteria. Repeat the counting of each sample 2-3 times (the value should not differ too much each time, otherwise the operation should be repeated), and calculate the number of cells contained in each mL (g) of bacterial suspension according to the formula.

(7) After the measurement is completed, remove the cover glass and rinse the hemocytometer with water. Do not scrub or wipe with hard objects, so as not to damage the grid scale. After washing, dry it by yourself or blow dry with a hair dryer, and put it in the box for storage.

Counting formula:

Red blood cell count/L=N*25/5*10*10^6*200

N is: the total number of RBCs in the five squares

N/5 is: the average number of RBCs in the 5 middle squares (pink area) (and then extended to the number of RBCs in each middle square in the central large square)

N*25/5 is: the total number of RBCs in the central large square (ie: the total number of RBCs of 0.1mm (ul))

N*25/5*10 is: total number of 1mm(ul)RBC

N*25/5*10*10^6 is: the total number of RBCs in 1L

*200 is the dilution factor of blood

4. Results

4.1 Changes of strain number of QDN01 strain in chicken manure starch

As can be seen from Figure 9, the number of bacteria in the whole culture process changes with the number of days in the three treatment groups, and the trend is the same. When the carbon-nitrogen ratio is 25:1, the bacteria The longest in the same time period, due to the counting problem, all the above data are plotted under the multiple of 106, ^on the 9th day, the strain growth in the bacterial liquid with a carbon-nitrogen ratio of 25:1 can reach 2500 100 million cfu/ml.

4.2 PH changes of chicken manure starch added with QDN01 strain

As can be seen from Figures 10 and 11, Figure 10 is the control group (CK), that is, without adding the QDN01 strain, wherein the pH value has been declining from the first day to the ninth day, compared with Figure 11 in the same period, the same Under the treatment of adding QDN01 strain, the decline was even greater, and finally maintained at 7.4-7.6.

4.3 The deodorizing effect of adding QDN01 strain on chicken manure starch

It can be seen from Figures 12 and 13 that the treatment of chicken manure starch without adding QDN01 strain in the treatment of CK in each carbon-to-nitrogen ratio, the decline in odor is very slow compared with the addition of QDN01 strain. The smell is not gone. However, the odor of the carbon-nitrogen ratio of 25:1 in the QDN01 strain has disappeared on the seventh day, while the odor in the other two treatments has been very weak, and the odor of the 20:1 treatment has also disappeared on the ninth day , indicating that the deodorization effect of adding QDN01 strain is obvious.

4.4 Changes of ammonium nitrogen of QDN01 strain in chicken manure starch

Poultry manure contains many nitrogen substances, which will be rapidly degraded into NH ₄ ⁺ -N during the fermentation process. Part of the NH ₄ ⁺ -N is further converted into NO ₃ ^- -N and organic nitrogen by microorganisms, and most of the NH ₄ ⁺ -N that is too late to be converted is volatilized as gas in an environment with pH>7, which is not only the main cause of N loss during cultivation It is also the main cause of poultry manure odor.

It can be seen from Figures 14 and 15 that the NH ₄ ⁺ -N content of each treatment continued to decrease, and the organic nitrogen NH ₄ ⁺ -N was converted from the QDN01 strain to NO ₃ ^— N, which rapidly increased the NO ₃ ^— N content. The order of NH ₄ ⁺ -N content in Laige period was treatment 25:1 < treatment 30:1 < treatment 20:1 < CK, and the conversion rate reached 31.2%, indicating that the treatment group with a carbon-nitrogen ratio of 25:1 had a better deodorizing effect .

Changes of nitrate nitrogen in chicken manure starch of 4.5QDN01 strain

It can be seen from Figures 16 and 17 that the NH ₄ ⁺ -N content of each treatment continued to decrease, and the organic nitrogen NH ₄ ⁺ -N was converted from the QDN01 strain to NO ₃ ^— N, which rapidly increased the NO ₃ ^— N content. NO ₃ ^— N content in different periods. At the end of the culture, the NO ₃ ^— N content was the highest in the carbon-nitrogen ratio 25:1 treatment group, and the other two treatments were roughly the same. So the carbon-nitrogen ratio 25:1 treatment group has a very high conversion rate, which can reach 39.5%.

4.6 Changes of growth curve of QDN01 strain in chicken manure starch

It can be seen from Figure 18 that the three treatments grew relatively slowly in the chicken manure and starch medium within 1-3 days, the growth rate increased significantly within 3-7 days, and reached stability after 7 days. Moreover, among the three treatments, the carbon-nitrogen ratio 25:1 treatment group is the most obvious, and the range of increase is the highest. It can be proved that the carbon-nitrogen ratio of 25:1 is the best carbon for the chicken manure and starch where the QDN01 strain is located. Nitrogen ratio.

4.7 Select the appropriate inoculation amount QDN01 strain growth changes in chicken manure starch

The changes in the number of bacteria and the OD value of different QDN01 strains in the chicken manure starch medium can be seen from Figures 19 and 20. The numbers are different, so the multiples of change of the QDN01 bacterial strain can be compared. In the treatment of 5% of the inoculated amount, the number of bacteria is about 7 times that of the initial number after 24 hours. In the remaining 5 treatments, they are all at 4 ~ 5 times, 9% access amount is only about 3 times, as can be seen from Figure 20, when the treatment of 5% access amount measures its absorbance with a spectrophotometer at 600 nanometers, the magnitude of increase is the largest, as shown in Figure 19- 20 It can be seen that the inoculation amount of 5% is the most suitable inoculation amount of QDN01 strain in chicken manure starch.

The application effect of embodiment 3QDN01 bacterial strain in breeding chicken coop

1. Method

(1) Based on chicken manure, starch is used to adjust C/N to 25:1;

(2) Take 2ml of the seed solution of the deaminated and deodorized bacterial strain QDN01 and transfer it to 98ml of N2 liquid medium with a C/N ratio of 25:1:, shake and cultivate at 130r/min for 24h, centrifuge at 300r/min, and use sterile Wash with water, and prepare 1.0×10 ¹² cells/ml cell suspension;

(3) Adsorb the cell suspension obtained in step (2) on bentonite to make 1.0×10 ¹² (bacteria)/g bacterial preparation, and add 1% commercially available brown sugar by mass ratio simultaneously to obtain deodorant bacteria agent;

(4) Sprinkle the deodorant bacterial agent of step (3) evenly on the chicken manure excretion place under the breeding cage after step (1), the usage amount is 200g bacterial agent/m ³ chicken manure starch, and spread in 3 days 1 time, the deodorizing effect was evaluated by artificial odor evaluation and egg production.

2. Experimental location

The Hongqi Farm in Qiqihar City, Damingtun Breeding and Incubation Center, Nianzishan Fine Breed Farm, and Heilongjiang Tianyu Green Agricultural Engineering Co., Ltd. are the experimental sites, all of which are laying hen breeding rooms.

3. Experimental results

3.1 Deodorizing effect

Through the on-site sensory evaluation by the breeders and volunteers of the demonstration sites, the air quality in the breeding houses of the four demonstration sites has improved significantly, the choking ammonia odor has disappeared, and the rancid smell has been reduced. The volunteers said that they can stay in the breeding room for a longer time up. Based on the on-site evaluation results, the bacterial agent can effectively reduce the ammonia content in the breeding house and improve the air quality.

3.2 Effect on egg production

A 20-day monitoring was carried out on the egg production of the demonstration sites. After applying this deodorant fungicide at the 4 monitoring sites, the egg production increased by an average of 4-6%. The improvement of the situation, thereby increasing the egg production.

Claims (8)

1. a kind of biological deodorizing bacteria agent, it is characterized in that by the bacterium liquid adsorption of deamination and deodorization bacterial strain on bentonite, obtain after adding brown sugar simultaneously, described deamination deodorization bacterial strain is named after Enterobacter hormaecheiQDN01, It is preserved in China Center for Type Culture Collection, and its preservation number is CCTCC NO: M 2013670. 2. The biological deodorizing bacterial agent as claimed in claim 1, characterized in that the bacterial liquid containing the deaminated and deodorizing bacterial strain is adsorbed on bentonite to make 1.0× ¹⁰ bacterium preparations/g, and at the same time Add 1% commercially available brown sugar by mass ratio to obtain the deodorizing bacteria agent. 3. the application of the biological deodorizing bacterium agent described in claim 1 or 2 in biological deodorization. 4. The application according to claim 3, characterized in that said biological deodorization comprises removal of peculiar smell in livestock excrement and malodorous industrial waste water. 5. application as claimed in claim 4, is characterized in that described livestock manure is chicken manure. 6. A method for deamination and deodorization of chicken manure, characterized in that it may further comprise the steps: (1) Absorb the bacterial liquid containing the deaminated and deodorized bacterial strain on bentonite, and add brown sugar at the same time to obtain the biological deodorant bacterial agent, wherein, the deaminated and deodorized bacterial strain is named Enterobacter hormaechei QDN01, and is preserved in China Typical Culture Collection Center, its collection number is CCTCC NO: M 2013670; (2) Use starch to adjust the C/N ratio of chicken manure to 20-30:1; (3) Sprinkle the deodorant bacterium agent of step (1) evenly in the chicken manure after step (2), and sprinkle once every 3 days; (4) Evaluation of the deodorizing effect. 7. The method according to claim 6, characterized in that the bacterial solution containing the deaminated and deodorized bacterial strains is adsorbed on bentonite to make 1.0×10 ¹² /g bacterial preparations, and simultaneously 1 % of commercially available brown sugar to obtain a deodorant bacterium agent. 8. The method according to claim 6, characterized in that adopting starch to regulate the chicken manure C/N ratio is 25:1, and the deodorizing bacteria agent of step (1) is 200g bacteria agent/ ^m according to the usage amount Evenly shed In the chicken manure after step (2).