CN104877920B - Candida krusei LSA and its application in degradation of ammonia nitrogen - Google Patents
Candida krusei LSA and its application in degradation of ammonia nitrogen Download PDFInfo
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Abstract
Application the invention discloses candida krusei LSA and its in degradation of ammonia nitrogen.Candida krusei LSA provided by the invention, preservation registration number are CGMCC NO.10562.The present invention also protects the culture of candida krusei LSA.The present invention also protects the bacteria suspension of candida krusei LSA.The present invention also protects a kind of product, and active ingredient is candida krusei LSA, the culture or the bacteria suspension；The function of the product is following (a) or (b) or (c) or (d)：(a) degradation of ammonia nitrogen；(b) ammonia nitrogen in degradation water；(c) ammonia nitrogen in degradation excrement；(d) the ammonia emission of excrement is reduced.Candida krusei provided by the invention can be applied to following aspect：(1) quality problem of ammonia and nitrogen pollution is improved；(2) it is used in poultry house, improves environmental sanitation in house；(3) feed addictive as chicken uses.
Description
Technical field
The invention belongs to microbial technology fields, and in particular to one plant of candida krusei and the Cruise vacation silk
Application of the yeast in degradation of ammonia nitrogen.
Background technology
The waste water and excrement generated during livestock and poultry cultivation can generate substantial amounts of foul gas during storage, wherein with
The harm of ammonia is the most apparent, and the lighter induces the generation of the animal respiratory diseases such as poultry, pig, causes animal stress reaction, weight
Person, which can cause, to be poisoned to death.Substantial amounts of ammonia emission promotes the formation of lung particulate matter (PM2.5) simultaneously, and is diffused into air
In with some acid substance reactions, form ammonium salt aerosol, cause ecosystem eutrophication and acidifying.In addition, containing highly concentrated
If the livestock breeding wastewater of degree ammonia nitrogen is dealt with improperly, it can also cause the eutrophication of water body.Therefore, aquaculture is administered to produce
The discharge of ammonia and ammonia nitrogen is of great significance to protection human ecological environment, improvement animal health and welfare in journey.
Research shows that the generation of ammonia nitrogen and ammonia in livestock breeding wastewater and excrement is largely dependent upon wherein micro- life
The metabolic activity of object makes it possible through the activity control ammonia nitrogen of Regulate Environment microbiologic population or the generation of ammonia.And show
The measure of some control farm ammonia pollution is mostly using physical method (as strengthened ventilation in house, building fecaluria separation facility
Deng), operating cost is high and because without discharge of the elimination ammonia nitrogen and ammonia to surrounding enviroment at all, can still cause secondary pollution,
And the defects of above method can then be overcome using microbial process, therefore have in terms of livestock-raising ammonia and ammonia and nitrogen pollution is administered
There is higher application value.
The content of the invention
Application the object of the present invention is to provide candida krusei LSA and its in degradation of ammonia nitrogen.
Candida krusei (Candida krusei) LSA provided by the invention, in the preservation of February 11 in 2015
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, (abbreviation CGMCC, address are：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC NO.10562.Cruise vacation silk
Yeast (Candida krusei) LSA CGMCC NO.10562, abbreviation candida krusei LSA.
The present invention also protects the culture of candida krusei LSA.
The present invention also protects the bacteria suspension of candida krusei LSA.The bacteria suspension specifically can be by using sterile physiological
Brine is resuspended thalline and obtains.Bacteria concentration in the bacteria suspension concretely 8.0 × 108cfu/mL-9.0×108cfu/mL。
The present invention also protects candida krusei LSA, the culture or the bacteria suspension answering in product is prepared
With；The function of the product is following (a) or (b) or (c) or (d)：(a) degradation of ammonia nitrogen；(b) ammonia nitrogen in degradation water；(c) drop
Solve the ammonia nitrogen in excrement；(d) the ammonia emission of excrement is reduced.The excrement can be feces of livestock and poultry, such as chicken manure.
The present invention also protects a kind of product, and active ingredient is candida krusei LSA, the culture or the bacterium
Suspension；The function of the product is following (a) or (b) or (c) or (d)：(a) degradation of ammonia nitrogen；(b) ammonia nitrogen in degradation water；(c)
Ammonia nitrogen in degradation excrement；(d) the ammonia emission of excrement is reduced.The excrement can be feces of livestock and poultry, such as chicken manure.
The present invention also protects candida krusei LSA, the culture or the bacteria suspension answering in degradation of ammonia nitrogen
With.The degradation of ammonia nitrogen concretely ammonia nitrogen in degradation water or the ammonia nitrogen in degradation excrement.The excrement can be feces of livestock and poultry,
Such as chicken manure.In the application, temperature can be 20-40 DEG C, concretely 30 DEG C.In the application, pH value can be 3.0-7.0,
Concretely 7.0.In the application, carbon source of the glucose needed for as candida krusei LSA growths specifically may be employed.
In the application, carbon-nitrogen mass ratio needed for candida krusei LSA growths can be 10-40, concretely 20.
The present invention also protects candida krusei LSA, the culture or the bacteria suspension reducing the ammonia of excrement
Application in emission.The excrement can be feces of livestock and poultry, such as chicken manure.
The present invention also protects a kind of method of degradation of ammonia nitrogen, includes the following steps：By candida krusei LSA, described
Culture or the bacteria suspension are mixed with the sample containing ammonia nitrogen, realize degradation of ammonia nitrogen.
The present invention also protects a kind of method for the ammonia emission for reducing excrement, includes the following steps：By Cruise vacation silk
Yeast LSA, the culture or the bacteria suspension are applied to excrement, so as to reduce the ammonia emission of excrement.The excrement can
For feces of livestock and poultry, such as chicken manure.
The present invention also protects a kind of method for reducing the ammonia concentration in poultry house, includes the following steps：Cruise is false
Silk yeast LSA, the culture or the bacteria suspension are applied in poultry house, so as to reduce the ammonia concentration in poultry house.
Candida krusei provided by the invention, cultural method is simple, the speed of growth is fast, is resistant to the ammonia of high concentration
Nitrogen, environmental suitability are strong, safe.Candida krusei provided by the invention can be applied to following aspect：(1) due to it
With very strong ammonia nitrogen degradation ability, available for the quality problem for improving ammonia and nitrogen pollution, suitable for the waste water containing ammonia nitrogen in high density
It administers；(2) since it can reduce the emission of excrement ammonia, available in poultry house, environmental sanitation in house is improved；(3) it is given birth to
Elongate member therefore can use as the feed addictive of chicken close to the microenvironment of chicken gastrointestinal tract, can improve nitrogen in feed
Utilization rate, the thalline survived in animal wastes can be further reduced the emission of ammonia again, improve environment in house so as to reach
Effect.The present invention has preferable application prospect.
Description of the drawings
Fig. 1 is the bacterium colony picture of candida krusei LSA.
Fig. 2 is the electron microscopic picture of candida krusei LSA.
Fig. 3 is the graph of candida krusei LSA degrading high concentration ammonia nitrogens.
The influence that Fig. 4 is temperature (A) and pH (B) grows candida krusei LSA.
The influence of Fig. 5 different carbon sources (A) and C/N (B) to candida krusei LSA ammonia nitrogen removal franks.
Fig. 6 is the control effect figure that candida krusei LSA generates poultry manure ammonia.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is conventional method unless otherwise specified.Test material used in following embodiments is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Average.In following embodiment, the water for being used to prepare each culture medium is deionized water.OD is used in following embodiment600nmIt is worth table
Levy thalli growth amount.C/N, that is, carbon-nitrogen mass ratio, also known as carbon-nitrogen ratio.
YPD fluid nutrient mediums：Yeast extract 10g, peptone 20g, glucose 20g, water 1000mL, natural pH.
Basal medium：Carbon source, (NH4)2S04、NaCl 1.0g、K2HP04·2H200.5g, MgS04·7H200.25g, it is micro-
Secondary element solution 1.0ml, water 1000mL adjust pH7.2-7.4.(NH4)2S04For nitrogen source.
Trace element solution：EDTA 10.0g、ZnSO41.2g、CaCl21.5g、MnCl2·4H2O 1.0g、FeSO4·
H2O2.0g、(NH4)6Mo7O24·4H2O 1.0g、CuSO4·5H2O 1g、CoCl2·6H2O 1.0g, water 1000mL adjust pH
7.2-7.4。
Ammonia nitrogen (NH4 +- N) content assay method use salicylic acid-hypochlorite photometry (GB7481-1987).
Embodiment 1, candida krusei LSA isolating and purifying and identify
First, candida krusei LSA is isolated and purified
1st, the enrichment of ammonia nitrogen degradation bacterium
20g chicken manure samples are taken, is inoculated in the conical flask equipped with 100ml sterile purified waters and (sterilizing glass is added in bottle
Pearl), 30 DEG C, 150r/min vibration 30min zoogloea is made fully to break up, stand 20min.Supernatant 5ml is taken, is inoculated in and is equipped with
In the conical flask of 100ml enriched mediums, 30 DEG C, 150r/min shaken cultivations 2 days take 1ml culture solutions to be inoculated in again new
In enriched medium, so continuously it was enriched with for 10 generations repeatedly.
2nd, screening, separation and the purifying of ammonia nitrogen degradation bacterium
Enrichment culture liquid 1ml is drawn, through 10-1After gradient dilution, 200 μ l is taken to be coated on separation tablet, 30 DEG C of cultures 24
~72h.Start the growing state of observation bacterium afterwards for 24 hours, selection growth is fast, bacterium colony is big, the bacterium colony more than quantity is carried out using trilinear method
Purifying.After purifying for 3 generations, 4 DEG C of inclined-planes are carried out respectively and are preserved.
Strains tested after isolating and purifying is inoculated in activation medium, 30 DEG C of shaken cultivation 18-24h.Then press
10% inoculum concentration is inoculated in sterile centrifugation tube, and 4000r/min centrifugation 10min abandon supernatant, with 0.86% sterile physiological salt
Three times, bacteria suspension finally is made with the sterile saline of same volume in water washing, is inoculated in 50ml screening and culturing mediums, 30 DEG C,
150r/min shaken cultivations for 24 hours, measure ammonia-nitrogen content, filter out one plant of best bacterial strain of degradation effect, be named as LSA.
2nd, the identification of strain
1st, morphologic observation
By LSA inoculations on YPD solid medium tablets, colony morphology characteristic is observed.The bacterium colony of LSA bacterial strains shines
Piece is shown in Fig. 1, and electromicroscopic photograph is shown in Fig. 2.LSA bacterial strains are in dotted, white, opaque, the smooth of the edge, and protrusion, matt, bacterial strain is ovum
Circular, easily accumulation.
2nd, biochemical identification
Identify that card carries out Physiology and biochemistry mirror to LSA bacterial strains using biological Mei Liai (biomerieux) Vitek32-YST
Fixed, qualification result is Candida krusei.
3rd, molecular biology identification
The 18S rDNA of LSA bacterial strains are expanded, PCR product after purification are sequenced, sequencing result is such as sequence table
Shown in sequence 1.
Comprehensive morphological identification, biochemical identification and molecular biology identification as a result, LSA bacterial strains belong to candida krusei
(Candida krusei)。
Candida krusei (Candida krusei) LSA, was preserved in China Microbiological bacterium on 2 11st, 2015
(abbreviation CGMCC, address are kind preservation administration committee common micro-organisms center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC NO.10562.Candida krusei (Candida
Krusei) LSA CGMCC NO.10562, abbreviation candida krusei LSA.
The research of embodiment 2, candida krusei LSA degradation of ammonia nitrogen functions
First, candida krusei LSA studies the degradation characteristic of ammonia nitrogen in high density
1st, culture medium first is prepared
Culture medium first：Using glucose as sole carbon source, carbon source and (NH are adjusted on the basis of basal medium4)2S04Concentration, make ammonia nitrogen concentration be 1200mg/L, C/N 10, pH 7.0.
2nd, picking candida krusei LSA monoclonals are inoculated in 100mL YPD fluid nutrient mediums, 30 DEG C, 150r/
Min shaken cultivations 24-36h (makes the cell concentration in cultivating system reach 8.0-9.0 × 108Cfu/mL), 4000rpm is centrifuged
10min collects thalline, is washed 3 times with sterile saline.
3rd, the thalline that step 2 is taken to obtain, is resuspended with sterile saline, obtains bacteria concentration as 8.0 × 108The bacterium of cfu/mL
Suspension.
4th, by the bacterial suspension inoculation that 0.1mL steps 3 obtain to 80mL culture medium first, 30 DEG C, 150r/min shaken cultivations, often
Sampling once, measures the OD of the ammonia nitrogen concentration and cultivating system in cultivating system when 12 is small600nmValue.
The result is shown in Fig. 3.When 0-24 is small after inoculation, candida krusei LSA fast-growths peak after 36h.Therewith
It is corresponding, ammonia nitrogen concentration in the cultivating system rapid decrease in 0-36h, after 36h ammonia nitrogen concentration downward trend gradually slow down.
The result shows that：Candida krusei LSA can adapt to new environment quickly, rapidly enter exponential phase, Preliminary degradation ammonia
The ability of nitrogen is strong；The ammonia nitrogen of high concentration does not generate inhibitory action to the growth of bacterial strain, when ammonia nitrogen concentration is 1000mg/L, bacterium
Body content maximum can reach OD600nm=2.96.
2nd, the influence of temperature and pH to strain growth
1st, picking candida krusei LSA monoclonals are inoculated in 100mL YPD fluid nutrient mediums, 30 DEG C, 150r/
Min shaken cultivations 24-36h (makes the cell concentration in cultivating system reach 8.0-9.0 × 108Cfu/mL), 4000rpm is centrifuged
10min collects thalline, is washed 3 times with sterile saline.
2nd, the thalline that step 1 is taken to obtain, is resuspended with sterile saline, obtains bacteria concentration as 8.0 × 108The bacterium of cfu/mL
Suspension.
3rd, by the bacterial suspension inoculation that 0.8ml steps 2 obtain to 80mL YPD fluid nutrient mediums (pH7.0), at a certain temperature
150r/min shaken cultivations for 24 hours, then detect OD600nmValue.The cultivation temperature of use be respectively 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C or
40℃.The result is shown in Fig. 4 A.Within the temperature range of 20-40 DEG C, the growth of candida krusei LSA is not subject to significant shadow
It rings.
4th, by the bacterial suspension inoculation that 0.8ml steps 2 obtain to 80ml YPD fluid nutrient mediums (pH 3.0,4.0,5.0,
6.0th, 7.0,8.0 or 9.0), 30 DEG C, 150r/min shaken cultivations for 24 hours, then detect OD600nmValue.The result is shown in Fig. 4 B.PH3-7's
In the range of, candida krusei LSA can keep good growth conditions.Under alkaline environment, candida krusei LSA's
Growth is then subject to certain inhibitory action.Candida krusei LSA is conveniently grown under slant acidity environment, and is sent out in growth
Organic acid is generated during ferment.
The result shows that candida krusei LSA has stronger environmental resistance, suitable temperature and pH growth items
Part is just adapted with the environment in henhouse, beneficial to its growth, has the potentiality applied in house.Candida krusei LSA
It is grown in the case where pH is 3 strong acid environment and at a temperature of 40 DEG C, this environment is close to the microenvironment in chicken gastrointestinal tract, therefore, Crewe
This Candida LSA has the potentiality as feed addictive simultaneously.
3rd, the influence of carbon source and C/N to bacterial strain ammonia nitrogen degradation performance
1st, influence of the carbon source to bacterial strain ammonia nitrogen degradation performance
(1) culture medium second is prepared
Culture medium second：Carbon source and (NH are adjusted on the basis of basal medium4)2S04Concentration, make the ammonia nitrogen concentration be
300mg/L, C/N 10, pH 7.0.Starch, mannitol, sodium citrate, glucose, sodium acetate or sodium acid carbonate is respectively adopted
As sole carbon source.
(2) picking candida krusei LSA monoclonals are inoculated in 100mL YPD fluid nutrient mediums, 30 DEG C, 150r/
Min shaken cultivations 24-36h (makes the cell concentration in cultivating system reach 8.0-9.0 × 108Cfu/mL), 4000rpm is centrifuged
10min collects thalline, is washed 3 times with sterile saline.
(3) thalline that step (2) is taken to obtain, is resuspended with sterile saline, obtains bacteria concentration as 8.0 × 108cfu/mL
Bacteria suspension.
(4) by the bacterial suspension inoculation that 0.8ml steps (3) obtain to 80mL culture medium second, 30 DEG C, 150r/min shaken cultivations
For 24 hours, then detect the ammonia nitrogen concentration in cultivating system and calculate ammonia nitrogen degradation rate.
Ammonia nitrogen degradation rate=(ammonia nitrogen after when ammonia nitrogen concentration-culture 24 in initial time cultivating system is small in system is dense
Degree) ammonia nitrogen concentration * 100% in/initial time cultivating system.
Ammonia nitrogen concentration/1.01 in ammonia nitrogen concentration=culture medium second in initial time cultivating system.
The result is shown in Fig. 5 A.When glucose is carbon source, the ability of candida krusei LSA ammonium oxidations is most strong, is secondly
Sodium acetate, it is impossible to utilize inorganic carbon.
2nd, influences of the C/N to bacterial strain ammonia nitrogen degradation performance
(1) culture medium third is prepared
Culture medium third：Using glucose as sole carbon source, glucose and (NH are adjusted on the basis of basal medium4)2S04Concentration, make ammonia nitrogen concentration be 300mg/L, C/N 5,10,20 or 40, pH 7.0.
(2) picking candida krusei LSA monoclonals are inoculated in 100mL YPD fluid nutrient mediums, 30 DEG C, 150r/
Min shaken cultivations 24-36h (makes the cell concentration in cultivating system reach 8.0-9.0 × 108Cfu/mL), 4000rpm is centrifuged
10min collects thalline, is washed 3 times with sterile saline.
(3) thalline that step (2) is taken to obtain, is resuspended with sterile saline, obtains bacteria concentration as 8.0 × 108cfu/mL
Bacteria suspension.
(4) by the bacterial suspension inoculation that 0.8ml steps (3) obtain to 80mL culture mediums third, 30 DEG C, 150r/min shaken cultivations
For 24 hours, then detect the ammonia nitrogen concentration in cultivating system and calculate ammonia nitrogen degradation rate.
The result is shown in Fig. 5 B.Work as C/N<When 20, with the rise of C/N, rise trend is presented in ammonia nitrogen degradation rate.It is 20 in C/N
When, interior ammonia nitrogen degradation rate reaches highest for 24 hours, is 82%.When C/N is 5, ammonia nitrogen degradation rate is substantially less than other each groups, shows carbon
Source deficiency can directly affect the growth metabolism of candida krusei LSA, ammonia nitrogen degradation rate is caused to decline.When C/N is up to 40,
Candida krusei LSA still has good degradation property.
The influence of embodiment 3, candida krusei LSA to excrement ammonia emission
1st, picking candida krusei LSA monoclonals are inoculated in 100mL YPD fluid nutrient mediums, 30 DEG C, 150r/
Min shaken cultivations 24-36h (makes the cell concentration in cultivating system reach 8.0-9.0 × 108Cfu/mL), 4000rpm is centrifuged
10min collects thalline, is washed 3 times with sterile saline.
2nd, the thalline that step 1 is taken to obtain, is resuspended with sterile saline, obtains bacteria concentration as 8.0 × 108The bacterium of cfu/mL
Suspension.
3rd, a large beaker (1L) is taken, a small beaker is put into large beaker and (20ml absorbing liquids are housed in small beaker；It inhales
Receipts liquid is 0.01mol/L sulfuric acid solutions), it is put into 100g fresh chicken manures sample in large beaker and bacterium that 10ml steps 2 obtain is hanged
Liquid (using isometric sterile saline as the blank control of bacteria suspension) and abundant mixing are then big with 6 layers of preservative film sealing
Beaker, incubated at room temperature, respectively at for 24 hours, 48h, 72h and 96h be detected (detection project：Measure the ammonia-nitrogen content in absorbing liquid
And obtain ammonia emission result.
The result is shown in Fig. 6 (ordinate of Fig. 6 be absorbing liquid in ammonia nitrogen concentration, mg/L).Compared with blank control group, add in
The ammonia emission of the test group of bacteria suspension significantly reduces, i.e. candida krusei LSA can be colonized and play in chicken manure
Deodorization.
Claims (12)
- Candida krusei 1. (Candida krusei) LSA, deposit number is CGMCC NO.10562.
- 2. the culture of candida krusei described in claim 1.
- 3. the bacteria suspension of candida krusei described in claim 1.
- 4. candida krusei described in claim 1, culture described in claim 2 or bacteria suspension described in claim 3 exist Prepare the application in product；The function of the product is following (a) or (b)：(a) degradation of ammonia nitrogen；(b) ammonia for reducing excrement dissipates Hair amount.
- 5. application according to claim 4, which is characterized in that the degradation of ammonia nitrogen is ammonia nitrogen or degradation excrement in degradation water Just the ammonia nitrogen in.
- 6. a kind of product, active ingredient be candida krusei described in claim 1, culture described in claim 2 or Bacteria suspension described in claim 3；The function of the product is following (a) or (b)：(a) degradation of ammonia nitrogen；(b) ammonia of excrement is reduced Gas emission.
- 7. product according to claim 6, which is characterized in that the degradation of ammonia nitrogen is ammonia nitrogen or degradation excrement in degradation water Just the ammonia nitrogen in.
- 8. candida krusei described in claim 1, culture described in claim 2 or bacteria suspension described in claim 3 exist Application in degradation of ammonia nitrogen.
- 9. candida krusei described in claim 1, culture described in claim 2 or bacteria suspension described in claim 3 exist Reduce the application in the ammonia emission of excrement.
- 10. a kind of method of degradation of ammonia nitrogen, includes the following steps：By candida krusei, claim described in claim 1 Bacteria suspension described in 2 cultures or claim 3 is mixed with the sample containing ammonia nitrogen, realizes degradation of ammonia nitrogen.
- 11. a kind of method for the ammonia emission for reducing excrement, includes the following steps：By Cruise vacation silk described in claim 1 Bacteria suspension described in culture described in yeast, claim 2 or claim 3 is applied to excrement, so as to which the ammonia for reducing excrement dissipates Hair amount.
- 12. a kind of method for reducing the ammonia concentration in poultry house, includes the following steps：Cruise described in claim 1 is false Bacteria suspension described in culture described in silk yeast, claim 2 or claim 3 is applied in poultry house, so as to reduce poultry house Interior ammonia concentration.
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| CN102796668A (en) * | 2012-08-14 | 2012-11-28 | 叶城县金秋实业生物肥有限公司 | Preparation method of fermentation microbial inoculum for reinforcing and promoting organic fertilizer compost |
| CN103911310A (en) * | 2013-01-07 | 2014-07-09 | 鹤壁市人元生物技术发展有限公司 | Organic material decomposing agent and its preparation method |
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| CN102796668A (en) * | 2012-08-14 | 2012-11-28 | 叶城县金秋实业生物肥有限公司 | Preparation method of fermentation microbial inoculum for reinforcing and promoting organic fertilizer compost |
| CN103911310A (en) * | 2013-01-07 | 2014-07-09 | 鹤壁市人元生物技术发展有限公司 | Organic material decomposing agent and its preparation method |
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