CN110438020A

CN110438020A - One plant of efficient dephosphorization saccharomycete and its application in sanitary sewage disposal

Info

Publication number: CN110438020A
Application number: CN201910656327.7A
Authority: CN
Inventors: 邱立平; 胡孟飞; 杨炎明; 谢康; 刘贵彩; 孙绍芳; 厉鹏远; 高明昌; 邱琪; 程仁振
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-07-19
Filing date: 2019-07-19
Publication date: 2019-11-12

Abstract

The present invention relates to one plant of efficient dephosphorization saccharomycete and its applications in sanitary sewage disposal, belong to environmental microbiology field.The yeast strain is named asCandida rugosaBL3, belong to fold Candida, for bacterium colony in faint yellow flat wax-like, there is the connection of milky filiform in centre, single colonie is creamy white circle, 2 ~ 3mm, surface is smooth wax-like, opaque, it is sticky easily to provoke, there is strong wine flavour, be capable of forming round ascospore, pseudohypha can be formed but also forms fungal filament.The bacterial strain has the function of efficient dephosphorization, dissolves oxygen environment without strict control, and can be used for biologic process for treating sewage still without phosphorus phenomenon is released after cultivating 60 h.The bacterial strain optimum pH range is 5 ~ 7, and for growth temperature at 20~35 DEG C, optimum carbon source is glucose+sodium acetate mixed carbon source.Find to actual domestic wastewater processing: under aerobic condition, when pH value is 5, and temperature is 25 DEG C, which is 84.9% to the removal rate of phosphor in sewage, ammonia nitrogen removal frank 39.5%, COD removal rate up to 78.3%.

Description

One plant of efficient dephosphorization saccharomycete and its application in sanitary sewage disposal

Technical field

The invention belongs to field of environmental biotechnology, and in particular to one plant of efficient dephosphorization saccharomycete and its at sanitary sewage Application in reason.

Background technique

Phosphorus is a kind of microelement that all life (animal, plant, microorganism) are depended on for existence, is to constitute thallus core egg The important composition ingredient of the substances such as white, nucleic acid；In nature, phosphorus mainly exists in the form of phosphate, about 2/3rds Phosphorus ore is used for phosphate fertilizer, is also used to manufacture phosphoric acid, insecticide, washing powder etc.；But phosphorus is a kind of not recyclable money of one-way circulation Source, its consumption will cause serious loss, or even more valuable than water and petroleum；According to statistics, global rock phosphate in powder annual mining volume About 100,000,000 tons, it is up within 2035 the output value summit of global rock phosphate in powder, phosphor resource shortage predicament will be faced later；Meanwhile with The continuous development of industrial enterprise, a large amount of phosphorus-containing wastewaters are discharged into natural water body, destroy the self-purification capacity of water body, lead to serious water The generation of phenomena such as China, red tide destroys water body and water ecological environment；Therefore, synchronous to realize that waste water dephosphorization and phosphorus recycling become solution The breach of certainly current phosphor resource form.

Biological phosphate-eliminating method is low with cost of investment, without adding the advantages such as chemical agent, without secondary pollution, becomes and answers at present With widest waste water dephosphorization mode；In previous research, phosphorus removal bio is mostly polyP bacteria, using polyP bacteria in aerobic item The principle that phosphorus is excessively inhaled under part achievees the purpose that waste water dephosphorization by way of excess sludge is discharged；But polyP bacteria reacts item Part is complex, needs strict control aerobic/anaerobic alternate environment.

Saccharomycete be phosphorus geochemical cycle during one of most important biotic factor, but because saccharomycete in activity The ratio that sludge accounts for is smaller, therefore saccharomycete research is ignored for a long time in sewage disposal system；Saccharomycete is studied not With the phosphorus removal property under the conditions of environmental factor, optimizes reaction condition to promote saccharomycete dephosphorization efficiency and reach the mesh of colleges and universities' dephosphorization 's.

Summary of the invention

The purpose of the present invention is to provide one plant of efficient dephosphorization saccharomycete and its applications in sanitary sewage disposal.

For achieving the above object, the technical scheme adopted by the invention is as follows:

One plant of efficient dephosphorization saccharomycete, belongs to fold Candida, which isCandida rugosa BL3。

The colonial morphology of above-mentioned fold candidiasis BL3 are as follows: in faint yellow flat wax-like, centre on YPD culture medium There is the connection of milky filiform；Carry out single colonie culture to it, when single colonie formation is creamy white circle, 2 ~ 3 mm, and surface is smooth It is wax-like, it is opaque, it is sticky easily to provoke；Mature later period, bacterium colony intermediate recess, surrounding protuberance have strong wine flavour.

The gene sequence characteristic of the 26S rDNA of above-mentioned fold candidiasis BL3 are as follows: 26S rDNA gene order length For 528bp, 26S rDNA sequence and GenBank database are compared using BLAST analytic approach, find the bacterial strain WithCandida rugosaAffiliation it is closest, homology is up to 100%, the systematic growth of fold candidiasis BL3 Chadogram sees Fig. 1.

Processing result discovery is carried out to actual domestic wastewater using above-mentioned fold candidiasis BL3: in OD₆₀₀ =0.2± 0.02, it is 5 in pH, under the conditions of temperature is 25 DEG C, shaking table shakes under (220 r/min) aerobic conditions, bacterial strain pair of the present invention The removal rate of phosphorus is up to 84.9%, to the removal rate of ammonia nitrogen up to 39.5%, to the removal rate of COD up to 78.3%.

Beneficial effects of the present invention are as follows: the present invention divides from domestic sewage treatment process A/O alternative aeration biofilter Fold candidiasis BL3 is separated out, which is able to achieve efficient dephosphorization under aerobic condition, and after culture 60h, still existing without phosphorus is released As can be used as dephosphorization high-performance bio microbial inoculum applied in sanitary sewage disposal, there is good dirt-removing power to sewage, can have Effect improves the treatment effeciency and stability of domestic sewage treatment process.The present invention has widened people to fold candidiasis (Candida rugosa) application study thinking in terms of its function, and provide for the efficient dephosphorization of sanitary sewage useful Bacterium source and technology have stronger practical application value.

Detailed description of the invention

Fig. 1 isCandida rugosaBL3 phylogenetic tree；

Fig. 2 isCandida rugosa The variation relation figure of BL3 growth and phosphorus removal property and pH；

Fig. 3 isCandida rugosaThe variation relation figure of BL3 growth and phosphorus removal property and temperature；

Fig. 4 isCandida rugosaBL3 growth and phosphorus removal property and carbon source kind relational graph；

Fig. 5 isCandida rugosaThe growth curve chart of BL3；

Fig. 6 isCandida rugosaPhosphorus removal property figure of the BL3 in sanitary sewage.

Specific embodiment

For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention Content is not limited solely to following example.

Example 1: separation, screening and the identification of bacterial strain.

(1) material prepares:

1. acquiring granule filter material from the A/O of operational excellence alternating biofilm system, and bacteria suspension is made, using gradient dilution Dilution is seeded on saccharomycete screening and culturing medium by method, carries out the coating separation of saccharomycete；With oese picking variety classes Saccharomycete carry out scribing line purifying；Finally purified saccharomycete is preserved in -86 DEG C of ultra low temperature freezers；

2. culture medium

Saccharomycete screening and culturing medium (1L): 20 g peptones, 20 g glucose, 10 g yeast extracts, 15 ~ 20 g agar powders, 15 The streptomysin of ug/ml, the chloramphenicol of 60 mg/L, 121 DEG C of 15 min of sterilizing；

YPD solid medium (1L): 20 g peptones, 20 g glucose, 10g yeast extract, 15 ~ 20g agar powder, 121 DEG C of sterilizings 15min；

YPD fluid nutrient medium (1L): 20 g peptones, 20 g glucose, 10g yeast extract, 121 DEG C of sterilizing 15min；

Synthetic wastewater culture medium (1L): 0.3 g glucose, 0.15 g sodium acetate, 0.15 g peptone, 0.01g yeast powder, 0.045g KH₂PO₄、0.16 g NH₄Cl、0.05 g NaCl、MgCl₂ 0.01g、FeSO₄0.01g；

3. experimental instruments

Outstanding riel SHZ-82A gas bath constant temperature oscillator, electro-heating standing-temperature cultivator, Sanyo's full-automatic high-pressure autoclave, UV-1750 Ultraviolet-uisible spectrophotometer-Prism Optical Technology Co, pH meter etc., superclean bench, PCR instrument, electrophoresis apparatus and electricity Swimming slot, the ultraviolet visualizer of gel.

(2) screening of efficient dephosphorization bacterial strain:

1. the strain saved in ultra low temperature freezer scribing line is incubated on YPD culture medium, 3 d, picking are cultivated under the conditions of 28 DEG C Clearly, it is easy to the single colonie of picking, is inoculated in YPD fluid nutrient medium, 28 DEG C, cultivate 12 h under the conditions of 220 rpm, is made Seed liquor；

It takes 16 ml saccharomycete seed liquors to be placed in centrifuge tube with liquid-transfering gun, 20 DEG C, be centrifuged 8 min under the conditions of 5000 rpm, abandons Supernatant twice with aseptic water washing centrifugation tube wall abandons flushing liquor, adds sterile water in centrifuge tube again and shakes up, identical item Repeated centrifugation is primary under part, abandons supernatant, adds 1 ml sterile water in centrifuge tube with liquid-transfering gun, shakes up, pour into wastewater medium It is interior, it is inoculated with successfully；

2. the wastewater medium after being inoculated with successfully is cultivated 3 ~ 5 days in 28 DEG C, 220 rpm constant-temperature tables, every 4 hours into Its OD is surveyed in row sampling₆₀₀Value, and dephosphorization situation is calculated, its comprehensive growing state and Removal obtain one plant of efficient dephosphorization ferment Mother strains.

(3) strain characteristic and identification

1. colony morphology characteristic and physio-biochemical characteristics: efficient dephosphorization saccharomycete obtained above being named as BL3, to bacterium The colonial morphology of strain BL3 is examined, the colonial morphology: for bacterium colony in faint yellow flat wax-like, there is milky silk in centre Shape connection, single colonie are creamy white circle, and surface is smooth wax-like, opaque, sticky easily to provoke, and have strong wine flavour；Bacterial strain The main biochemical feature of BL3: having a little precipitating when Liquid Culture, can form collarium, forms round ascospore, and no production ester produces acid Phenomenon can form pseudohypha but also form fungal filament；2 ~ 3 mm of thallus size；

2. for the 26S rDNA Molecular Identification of bacterial strain BL3: being extracted according to Tiangeng pastoris genomic dna extracts kit specification Yeast genome simultaneously carries out PCR amplification, and rear raw work (Shanghai) Bioisystech Co., Ltd, commission China is sequenced, after sequencing The gene order that length to bacterial strain BL3 is 528；Using BLAST software, by the gene order measured and Genbank database Sequence carry out sequence analysis: with fold Candida (Candida rugosa) 100% similarity, thus it is speculated that the bacterium isCandida rugosa；Accession number is KY107668.1；Physiology and biochemistry and the Molecular Identification of comprehensive bacterial strain are as a result, the bacterial strain is ordered It is entitledCandida rugosaBL3,Candida rugosaThe phylogenetic evolution tree of BL3 sees Fig. 1；Specific gene order is such as Shown in lower:

1 GGCATATCAA TAAGCGGAGG AAAAGAAACC AACCGGGATT GCCTCAGTAA

51 CGGCGAGTGA AGCGGCAACA GCTCAAATTT GAAAGCCCGC GGGCGTTGTA

101 ATTTGCAGGC GGATGTTTTG GGGCGGGCGC TGTCTACGTT CCTTGGAACA

151 GGACGCCGCA GAGGGTGAGA GCCCCGTGCG ATGGCGCCTC CAACCGCGTA

201 AAACTCCGCC GACGAGTCGA GTTGTTTGGG AATGCAGCTC CAAGTGGGTG

251 GTAAATTCCA TCTAAAGCTA AATACTGGCG AGAGACCGAT AGCGAACAAG

301 TACAGTGATG GAAAGATGAA AAGCACTTTG AAAAGAGAGT GAAACAGCAC

351 GTGAAATTGT TGAAAGGGAA GGGTATGCGA TTAGCGGCCA GCAGGAGGTG

401 CCTTCTCGTG AAAAGGCCGT GCACCGTCTT CGGACACCGT GCGCGGAGAT

451 GGCGAGGGGG CGCCTGAGGT CTGCGACTCG AGGTTGCTGG CGTAATGATT

501 GCATACCACC CGTCTTGAAA CACGGACC

The present invention isolates saccharomycete BL3 from traditional domestic sewage treatment process A/O alternative aeration biofilter, discovery It has stronger dephosphorization ability, and without stringent dissolved oxygen conditions, dephosphorizing rate shows up to 84%, and without phosphorus is released under aerobic state As；The Strain Designation isCandida rugosaBL3, accession number KY107668.1；The present invention has widened people coupleCandida rugosaApplication study thinking in terms of its function, and useful bacterium source is provided for the high-efficient denitrification and dephosphorization of sanitary sewage And technology, there is stronger practical application value.

Bacterial strain in 2 present invention of exampleCandida rugosaThe culture of BL3.

(1) present invention in bacterial strain training systern

1. difference pH

Before wastewater medium sterilizing using hydrochloric acid adjust respectively with sodium hydroxide pH value be 3,4,5,6,7,8,9(3 it is parallel Sample), 16mL bacterial suspension inoculation is taken, OD is adjusted₆₀₀=0.2 ± 0.02,60 h of shaking table (28 DEG C, 220 r/min) shake culture are surveyed Determine OD₆₀₀And PO in supernatant₄ ^3-- P concentration calculates dephosphorizing rate, as a result sees Fig. 2；As shown in Figure 2, bacterial strain of the present invention pH be 3~ OD in 9 ranges₆₀₀, dephosphorizing rate gradually rise, when pH value be 3 when, the removal rate of phosphate of bacterial strain BL3 only has 40.6%；PH value When between 5 ~ 7, the removal rate of phosphate of bacterial strain BL3 is maintained at 75% or more, and peak is 83.8% when appearing in pH=5.Work as pH When reaching 9, the removal rate of phosphate of bacterial strain BL3 drops to 68.6%；It can be seen from the above result that the suitable dephosphorization pH of bacterial strain BL3 Range is 5 ~ 7, Optimal pH 5；

2. different temperatures

It takes 16 mL bacteria suspensions to be inoculated in 350 mL synthetic wastewater culture mediums respectively, adjusts OD₆₀₀=0.2 ± 0.02, exist respectively Under the conditions of 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 60 h of shaking table (220 r/min) shaken cultivation, measurement OD₆₀₀And PO in supernatant₄ ^3-The concentration of-P calculates dephosphorizing rate, as a result sees Fig. 3.From the figure 3, it may be seen that bacterial strain is in aerobic conditions Under, the OD within the scope of 10~35 DEG C₆₀₀, dephosphorizing rate gradually rise, and removal rate of phosphate peaks at 25 DEG C, removal Rate is 75.7%, and when temperature is greater than 25 DEG C, removal rate of phosphate is gradually decrease to 69.8%；This may be the temperature because excessively high Making the enzyme activity of certain synthesis poly-P particles in bacterial strain BL3 reduces；Bacterial strain of the present inventionCandida rugosaBL3's is suitable For suitable growth temperature between 20~35 DEG C, optimum growth temp is 25 DEG C；

3. different carbon source

350 are inoculated in for sole carbon source respectively with glucose, sodium acetate, starch, methanol, mixed carbon source (glucose+sodium acetate) The sanitary sewage culture medium of mL not carbonaceous sources adjusts OD₆₀₀Under the conditions of=0.2 ± 0.02,28 DEG C, shaking table (220 r/min) vibration 60 h of culture are swung, OD is measured₆₀₀And PO in supernatant₄ ^3-- P concentration calculates dephosphorizing rate, as a result sees Fig. 4；As shown in Figure 4, work as carbon source Bacterial strain of the present invention when for mixed carbon sourceCandida rugosaBL3 Removal is best, dephosphorizing rate 54.6%；Followed by Portugal Grape sugar, dephosphorizing rate 50.6%；Bacterial strain grows worst in the culture medium using starch as sole carbon source, and dephosphorizing rate is only 5.67%.

(2) present invention in bacterial strain growth characteristics

1 ring of pickingCandida rugosaBL3 is inoculated in the conical flask containing 150 ml YPD fluid nutrient mediums, in 28 DEG C, cultivate 3 d in 220 rpm constant-temperature tables, be sampled every 4 h, its viable count is surveyed in coating culture, as a result as shown in Figure 5； As shown in Figure 5, for the growth curve of bacterial strain BL3 than more typical, preceding 6 h is the laundering period, and 6-16 h is logarithmic phase, and 16-56 h is steady Periodically, decline phase is entered after 56 h.Viable count changes over time equation:

n = 14.68885 + 15.37056/ {1 + exp [(t- 0.23666)/ 0.2423]} R²=0.98998

N is clump count in above formula, and unit is 109 cfu；T is time, unit h.

Bacterial strain in 3 present invention of exampleCandida rugosaApplication of the BL3 in sewage treatment: it takes in the colleges and universities of Shandong Water station conditioning tank sewage (COD concentration is 400mg/L, and phosphorus concentration 11mg/L, ammonia nitrogen concentration is 44 mg/L), carries out waste water and removes Phosphorus test, the specific steps are as follows: take 350 ml actual sewages (sterilizing) in 500 ml conical flasks, adjust OD₆₀₀=0.2± 0.02, it is 5 in pH, under the conditions of temperature is 25 DEG C, shaking table shakes aerobic culture, and oscillation frequency is 220 r/min；Respectively at 0 H, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h take detection water sample, take supernatant after centrifugation, measure positive phosphorus in supernatant, The concentration variation of ammonia nitrogen, COD, is as a result shown in Fig. 6；Bacterial strain of the present invention removes phosphorus in 12 h in aerobic incubation as seen from Figure 6 Except rate is up to 84.9%, to the removal rate of ammonia nitrogen up to 39.5%, to the removal rate of COD up to 78.3%.

Claims

1. one plant of efficient dephosphorization saccharomycete, which is characterized in that be not necessarily to A/O alternate environment condition, under non-critical aerobic condition i.e. Efficient dephosphorization can be achieved；By the strain inoculated in sanitary sewage culture medium, to the removal rate of phosphorus up to 84.9% in 12 h, to ammonia The removal rate of nitrogen is up to 39.5%, to the removal rate of COD up to 78.3%.

2. efficient dephosphorization saccharomycete described in claim 1 belongs to fold Candida, is named asCandida rugosaBL3, Accession number is KY107668.1；For the bacterial strain in faint yellow flat wax-like on YPD solid medium, centre has milky Filamentous Connection；Carrying out single colonie culture to it, when single colonie formation, is creamy white circle, and 2-3mm, surface is smooth wax-like, and it is opaque, it glues It is thick easily to provoke；Mature later period bacterium colony intermediate recess, surrounding protuberance, there is strong wine flavour.

3. efficient dephosphorization yeast bacterial screening method described in claim 1: being adopted from the A/O of operational excellence alternating biofilm system Collect granule filter material, and bacteria suspension is made, dilution is seeded on saccharomycete screening and culturing medium using gradient dilution method, carries out ferment The coating of female bacterium separates；Scribing line is carried out with the different types of saccharomycete of oese picking to purify；Finally by purified saccharomycete It is preserved in -80 DEG C of ultra low temperature freezers.

4. efficient dephosphorization saccharomycete screening and culturing medium main component described in claim 1: 1000 mL deionized waters, 20 g albumen Peptone, 20 g glucose, 10 g yeast extracts, 15 ~ 20 g agar powders, 15 ug/ml streptomysins, 60 mg/L chloramphenicol.

5. efficient dephosphorization saccharomycete described in claim 1 is suitable for that dephosphorization pH range is 5 ~ 7, Optimal pH 5；Suitable growth temperature exists Between 20~35 DEG C, optimum growth temp is 25 DEG C.

6. efficient dephosphorization Yeast Growth optimum carbon source described in claim 1 be mixed carbon source (glucose sugar: sodium acetate=2:1 ~ 3:1).

7. 6 h are the laundering period before efficient dephosphorization saccharomycete described in claim 1,6 ~ 16 h are logarithmic phase, and 16 ~ 56 h are to stablize Phase, 56 h enter decline phase later；Viable count changes over time equation:

n = 14.68885 + 15.37056/{1 + exp[(t- 0.23666)/ 0.2423]} R²=0.98998

N is clump count in above formula, and unit is 109 cfu；T is time, unit h.

8. application of the efficient dephosphorization saccharomycete in sanitary sewage described in claim 1.

9. application method of the efficient dephosphorization saccharomycete in sanitary sewage described in claim 1, it is characterised in that:

1. carrying out bacterial strain activation using YPD fluid nutrient medium using preceding: one ring of picking is above-mentionedCandida rugosaBL3 inoculation It is 5 in pH in 150ml fresh liquid YPD medium, temperature cultivates 12 h under the conditions of being 28 DEG C, its supernatant is abandoned after centrifugation Sterile water is added in liquid, obtains bacteria suspension, and OD600 value is adjusted to 0.2 ± 0.02；

2. taking the bacterial suspension inoculation after activation in sanitary sewage to be processed, bacterial suspension inoculation amount is sewage volume 5%, is being shaken Bed setting temperature is 28 DEG C, and revolving speed is shaken cultivation under 220 r/min；

3. sampling in every 4 hours is primary, CODcr in reactor, ammonia nitrogen, the concentration variation of positive phosphorus are detected；Detection method are as follows: positive phosphorus, Ammonium molybdate spectrophotometric method；Ammonia nitrogen, nessler reagent ultraviolet spectrophotometry；CODcr, dichromate titration.