CN106582258B - A method of hydrogen sulfide is removed using heterotrophic microorganism - Google Patents
A method of hydrogen sulfide is removed using heterotrophic microorganism Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
- B01D53/84—Biological processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/46—Removing components of defined structure
- B01D53/48—Sulfur compounds
- B01D53/52—Hydrogen sulfide
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a kind of method using heterotrophic microorganism removal hydrogen sulfide, it is that specific heterotrophic microorganism is added in the device of removal hydrogen sulfide, aerobic reaction is carried out in the case where setting reaction system, is realized to the H in device reaction system2S、HS‑Or/and S2‑Removal；Wherein the heterotrophic microorganism refers to that the energy sulfur oxide compound containing thioquinones oxidoreducing enzyme (sulfide:quinone oxidoreductase) gene and persulfide dioxygenase (persulfid dioxygenase) gene in its genome generates a kind of heterotroph of zeroth order sulphur and thiosulfate or sulfate.The heterotrophicy bacteria that the present invention filters out has the ability of efficient oxidation external source hydrogen sulfide, and have the characteristics that growth is rapid, toxigenic capacity is cheap, can form larger biomass, it efficiently solves and is limited in the prior art using the condition that autotrophic bacterium removal hydrogen sulfide is subject to, there is very big application potential in the industrial production.
Description
Technical field
The present invention relates to a kind of method of biological removal sulfide more particularly to it is a kind of using heterotrophic microorganism go it is vulcanisation
Object (H2S、HS-Or/and S2-) method.
Background technique
Hydrogen sulfide is the toxic gas of irritant smell, be in industrial processes (such as sewage treatment plant, animal husbandry with
And composting plant etc.) the primary waste gases ingredient that is widely present.Currently, the minimizing technology of hydrogen sulfide pernicious gas is broadly divided into physics
Method, chemical method and bioanalysis, wherein biological method removal hydrogen sulfide has irreplaceable excellent compared to chemical method and physical method
Gesture, application prospect are relatively broad.
Microorganism plays the role of the oxidative absorption of hydrogen sulfide vital during biological removal hydrogen sulfide.Mesh
The preceding inoculation method for microorganism in aerobic hydrogen sulfide biological removal reactor mainly has training and artificial inoculation method.It tames and dociles
Feeding method refers to that nutrient solution is first added in a device before being passed through the gas containing hydrogen sulfide keeps the microorganism in device naturally raw
Long, the microorganism that then passing to the gas containing hydrogen sulfide makes part that can play the role of hydrogen sulfide removal is worked, by such as
This is tamed for a long time, and the microbes that can remove hydrogen sulfide are enriched with, and cannot remove the microbes quilt of hydrogen sulfide
It eliminates, it can be seen that this method has blindness to the selection of microorganism.Artificial inoculation method is divided to two kinds again, and one is inoculations to have
There is the activated sludge of hydrogen sulfide removal ability, this method actually has same blindness with above-mentioned training；It is another
It is the pure culture microorganism that inoculation can remove hydrogen sulfide, refer mainly to early detection can use hydrogen sulfide as electron donor
Energy is generated for the chemautotrophy and photoautotrophic microorganism of own growth, that is, is commonly referred to as Thiobacillus
(Thiobacillus), solfataricus genus (Sulfolobus) or green sulphur bacteria (Chlorobium).However, such autotrophy is thin
Bacterium is compared to heterotrophicy bacteria slow growth, it is difficult to larger biomass is formed, and its growth usually requires the environment of slant acidity, and
Acid condition is but unfavorable for the absorption and oxidation of hydrogen sulfide.Through retrieving, gone down using heterotrophic microorganism in aerobic conditions vulcanisation
The method of hydrogen yet there are no registration.
Summary of the invention
For the blindness problem of training in the prior art and the limitation of autotrophic bacteria removal hydrogen sulfide application, this hair
Bright purpose is to provide a kind of utilization heterotrophic microorganism removal hydrogen sulfide (H2S、HS-Or/and S2-) method.
The method of the present invention for removing sulphide removal using heterotrophic microorganism is that life is added in the device for removing sulphide removal
The heterotrophic microorganism of long stationary phase carries out aerobic reaction in the case where setting reaction system, realizes to the H in device reaction system2S、
HS-Or/and S2-Removal；
It is characterized by:
The heterotrophic microorganism, which refers to, contains thioquinones oxidoreducing enzyme (sulfide:quinone in its genome
Oxidoreductase, sqr) gene and sulphur dioxygenase (persulfid dioxygenase, pdo) gene a kind of heterotrophism
Microorganism；
The reaction system is: concentration is 4- (2- the ethoxy) -1- piperazine ethanesulfonic acid (HEPES:4- (2- of 50 ± 5mM
Hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, or have buffer capacity at neutral ph
Power and the buffer harmless to the heterotrophic microorganism；The sulphur for being also wherein 10 μM~1000 μM added with concentration in the reaction system
Change sodium, and the heterotrophic microorganism bacterial turbidity OD contained in the reaction system600nm=0.5~10；
The aerobic reaction condition is: 10 DEG C~40 DEG C of temperature, pH 6~9,200 ± 50r.p.m. concussion；
H in described device reaction system2S、HS-Or/and S2-After removal, according to the difference of heterotrophic microorganism bacterial strain used
Its product is sulphur gastral cavity sulphur and thiosulfate or sulfate.It that is: is that will be converted to sulphur after Oxidation of Hydrogen Sulfide using heterotrophic microorganism
Thiosulfate and sulphur gastral cavity sulphur, or completely it is oxidized to sulfate.
In the above-mentioned method using heterotrophic microorganism removal hydrogen sulfide: the heterotrophic microorganism is preferably Gluconobacter oxydans
Bacillus (Gluconobacter oxydans) 621H, pseudomonasputida (Pseudomonas putida) S16, verdigris are false single
Born of the same parents bacterium (Pseudomonas aeruginosa) PAO1, Ralstonia bacterium (Cupriavidus necator) JMP134 or ocean
Green onion burkholderia (Burkholderia cepacia) ATCC 25416.
Further, the cultural method of the heterotrophic microorganism is: 30 ± 2 DEG C in defined medium, 200 ±
10r.p.m. shake culture 24 ± 4 hours to the bacterial growth period reaches stationary phase；
Wherein: the defined medium formula is: industrial fermentation glucose 30g/L, industrial fermentation Dried Corn Steep Liquor Powder
15g/L。
In the above-mentioned method using heterotrophic microorganism removal hydrogen sulfide, the reaction system is preferably: concentration is 50mM's
4- (2- ethoxy) -1- piperazine ethanesulfonic acid buffer, the vulcanization for being also wherein 20 μM~800 μM added with concentration in the reaction system
Sodium, and the heterotrophic microorganism bacterial turbidity OD contained in the reaction system600nm=1~8.
In the above-mentioned method using heterotrophic microorganism removal hydrogen sulfide, the aerobic reaction condition is preferably: 20 DEG C of temperature
~37 DEG C, pH 7~8,120 ± 20r.p.m. concussion.
In the above-mentioned method using heterotrophic microorganism removal hydrogen sulfide, the pseudomonasputida (Pseudomonas
Putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Ralstonia bacterium (Cupriavidus
Necator) JMP134 or 25416 bacterial strain of Burkholderia cepacia (Burkholderia cepacia) ATCC are aerobic anti-
The ability of oxidation of sulfureted hydrogen in system is answered to enhance with the induction of external source addition hydrogen sulfide, polysulfide or elemental sulfur.
Present invention applicant has found that a series of heterotrophic microorganism containing Oxidation of Hydrogen Sulfide system can efficiently rapidly
Oxidation external source hydrogen sulfide, depending on bacterium difference, product is sulphur gastral cavity sulphur and thiosulfate or is sulfate.Experiment confirms this
A little heterotrophic microorganisms have growth rapid compared to autotrophic microbe, and biomass is big, and culture facilitates economic feature.And its unit
Dry weight oxidation rate is suitable with the autotrophic bacteria sulfur oxide compound rate reported before.Based on this, the present invention provides a kind of benefits
With the method for heterotrophic microorganism removal hydrogen sulfide, this method very good solution deficiency of hydrogen sulfide removal in the prior art,
There is very big application potential in industrial production.
To sum up, the invention has the following advantages over the prior art:
1. the present invention is to have according to the heterotrophicy bacteria that bacterial genomes information searching contains oxidation of sulfureted hydrogen key gene
The screening process of purpose, the heterotrophicy bacteria filtered out has the very efficient ability of oxidation external source hydrogen sulfide, compared to having reported
Autotrophic bacteria [the Immobilized-Cell and Sulfur-Settling Free-Cell Recycle in road
Reactors.Biotechnology Progress, 1991.7 (6): p.495-500.], wherein Gluconobacter oxvdans
(G.oxydans) oxidability of 621H is higher, and other bacterium oxidabilities are therewith suitable (Fig. 2).
2. a series of heterotrophicy bacterias that the present invention filters out can be according to the oxidation product of the different control sulfide of bacterial strain
It for thiosulfate and sulphur gastral cavity sulphur, or is sulfate.
3. the ability for heterotrophicy bacteria its oxidation of sulfureted hydrogen that the present invention filters out has inductive effect.I.e. when there are sulphur for external source
When changing hydrogen, inducing its oxidation of sulfureted hydrogen related gene more to express enhances the ability of its sulfur oxide compound.
4. heterotrophicy bacteria growth is rapidly, culture is convenient, and toxigenic capacity is low.
5. the bacterium that the present invention filters out has quite high tolerance, when external source concentration of hydrogen sulfide reaches 1mM, still
It can efficiently remove.
Detailed description of the invention
Fig. 1 Gluconobacter oxvdans (Gluconobacter oxydans) 621H, pseudomonasputida
(Pseudomonas putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Ralstonia bacterium
(Cupriavidus pinatubonensis) JMP134 or Burkholderia cepacia (Burkholderia cepacia)
25,416 5 plants of bacteriums of ATCC have apparent oxidation external source hydrogen sulfide ability.
Fig. 2 Gluconobacter oxvdans (Gluconobacter oxydans) 621H, pseudomonasputida
(Pseudomonas putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Ralstonia bacterium
(Cupriavidus pinatubonensis) JMP134 or Burkholderia cepacia (Burkholderia cepacia)
25,416 5 plants of bacterial oxidation external source hydrogen sulfide rates of ATCC.(per cell dry weight, oxidized per unit time external source hydrogen sulfide
Amount.)
Fig. 3 hydrogen sulfide, polysulfide and elemental sulfur are to Ralstonia bacterium (Cupriavidus pinatubonensis)
JMP134 (A), pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 (B), Burkholderia cepacia
(Burkholderia cepacia) ATCC 2541 (C) or pseudomonasputida (Pseudomonas putida) S16 (D) oxygen
Change the inductive effect of hydrogen sulfide.
Fig. 4 Gluconobacter oxvdans (Gluconobacter oxydans) 621H, pseudomonasputida
(Pseudomonas putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Ralstonia bacterium
(Cupriavidus pinatubonensis) JMP134 or Burkholderia cepacia (Burkholderia cepacia)
The final product of 25,416 5 plants of bacterial oxidation hydrogen sulfide of ATCC.
Specific embodiment
Here is specific implementation example of the invention.It should be pointed out that these embodiments are only exemplary, not
Any restrictions are constituted to the scope of the present invention.The details and form of embodiment are carried out under thinking and range of the invention
Modification and replacement are fallen within the protection scope of the present invention.
Gluconobacter oxvdans (Gluconobacter oxydans) 621H, pseudomonasputida (Pseudomonas
Putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 or Burkholderia cepacia
(Burkholderia cepacia) ATCC 25416 is purchased from American Type Culture Collection Center (American Type
Culture Collection), Ralstonia bacterium (Cupriavidus pinatubonensis) JMP134 purchased from it is logical send (on
Sea) Biotechnology Co., Ltd.
The culture medium prescription of heterotrophic microorganism: industrial fermentation glucose, 30g/L；Industrial fermentation Dried Corn Steep Liquor Powder,
15g/L。
HEPES buffer solution group becomes (1 liter of distilled water): 4- (2- ethoxy) -1- piperazine ethanesulfonic acid (HEPES:4- (2-
Hydroxyethyl) -1-piperazineethanesulfonic acid) 13g, diethylene triamine pentacetic acid (DTPA) (DTPA:
Diethylenetriaminepentaacetic acid) 0.02g, NaOH adjusting pH to 7.4.
The detection method of hydrogen sulfide are as follows: take 1ml sample, 80ul is added and mixes double ammonia reagents, in being placed at room temperature for after mixing
20min measures OD670nmUnder absorbance measured again after diluting sample with deionized water if absorbance is more than 1.Mixing is double
The formula of ammonia reagent such as the following table 1:
Table 1: two different formulas are selected according to the predicted concentration of hydrogen sulfide.
The preparation of polysulfide: 13mg sulphur powder and 70mg vulcanized sodium are added in the distilled water for using argon gas deoxygenation in advance, close
Envelope, pH value are adjusted with hydrochloric acid to 9.3.
Acetone magister of sulfur: by excessive sulphur powder adding into acetone, sufficiently dissolution is shaken, saturated solution is containing about 20mM sulphur.
The detection method of sulphur gastral cavity sulphur are as follows: add 1% boric acid of 0.55ml into 1.5ml EP pipe, 1min is heated in boiling water, is added
Enter 0.25ml sample, 0.2ml 0.1M potassium cyanide heating 1min. is added and cools down at room temperature, 0.1ml nitric acid ferron is added, at once
Mixing is then centrifuged removal if there is cell, measures OD460nm, blank control is using deionized water.
Sulphite, sulfate and thiosulfate use ion chromatography (ICS-1100system；Dionex it) detects, tool
Concrete conditions in the establishment of a specific crime are as follows: flow rate of mobile phase: 1ml/min, column temperature: 30 DEG C, suppressor type: ASRS_4mm, suppressor electric current: 55mA, pressure
Power range: the appearance time of 200-3000psi.KOH=20mM, sulphite, sulfate and thiosulfate is respectively 7.4 points
Clock, 7.9 minutes, 25.7 minutes.
1: five plant of heterotrophicy bacteria oxidation of sulfureted hydrogen rate research of embodiment.
By five plants of heterotrophicy bacterias (Gluconobacter oxvdans (Gluconobacter oxydans) 621H, stench vacation monospore
Bacterium (Pseudomonas putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Ralstonia
Bacterium (Cupriavidus necator) JMP134 or Burkholderia cepacia (Burkholderia cepacia) ATCC
25416) 30 degrees Celsius the culture of 200rpm shake culture 20 hours or more, bacterial turbidity is greater than OD600nmWhen=4, it is collected by centrifugation
Cell, and clean one time with pure water, then by the heterotroph cell dissolution handled well in pH=7.4, contain 50 μM of divinyls
4- (2- ethoxy) -1- piperazine second sulphur of pentaacetic acid (DTPA:diethylenetriaminepentaacetic acid)
In sour (HEPES:4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, and adjust thin
Bacterium turbidity is OD600=2.In application, cultured bacterium solution can also be used to directly or after dilution handle sulfide.
Heterotroph cell suspension after 10mL is adjusted is added in 50mL centrifuge tube, and the end that Fresh is added immediately is dense
Degree is 200 μM of vulcanized sodium starting reactions, and entire reaction condition was 30 DEG C of slight concussions (100 ± 10r.p.m.), and at 10 minutes,
30 minutes, the residual concentration of hydrogen sulfide in 60 minutes three point in time sampling measurement systems.
Cell is collected by centrifugation again after the reaction was completed, is cleaned in pure water three times, removes supernatant, it is dry to be then transferred to freezing
It is freeze-dried 12 hours or more in dry machine, measures dry cell weight.It is calculated by the pace of change and dry cell weight of concentration of hydrogen sulfide
Every plant of bacterium per cell dry weight out, unit time aoxidize the rate of external source hydrogen sulfide.
It is detected through above method, the above-mentioned five plants heterotrophicy bacterias with Oxidation of Hydrogen Sulfide system all have apparent efficient oxygen
The ability of outside the pale of civilization source hydrogen sulfide, is detailed in Fig. 1.
It is computed, they aoxidize the rate of external source hydrogen sulfide in 6 to 50 μm of olmin-1G (dry cell weight)-1Between,
It is detailed in Fig. 2.
Embodiment 2: the inductive effect research of hydrogen sulfide.
By Ralstonia bacterium (Cupriavidus pinatubonensis) JMP134, pseudomonas aeruginosa
(Pseudomonas aeruginosa) PAO1, Burkholderia cepacia (Burkholderia cepacia) ATCC 2541,
Or pseudomonasputida (Pseudomonas putida) S16 bacterium by method culture in embodiment 1 to logarithmic growth early period when
(OD600=0.5) final concentration of 20 μM of Na, are additionally added in the medium2S, polysulfide or acetone sulphur continue culture induction
1 hour, then collect thallus by method in embodiment 1, measure Oxidation of Hydrogen Sulfide rate, and with the control group ratio without induction
Compared with, as the result is shown hydrogen sulfide, polysulfide and acetone sulphur can Induction of bacterial and enhance the ability of its oxidation of sulfureted hydrogen,
It is detailed in Fig. 3.
Embodiment 3: Oxidation of Hydrogen Sulfide Study on product.
According to the condition in embodiment 1, by five plants of heterotrophicy bacteria (Gluconobacter oxvdans (Gluconobacter
Oxydans) 621H, pseudomonasputida (Pseudomonas putida) S16, pseudomonas aeruginosa (Pseudomonas
Aeruginosa) PAO1, Ralstonia bacterium (Cupriavidus necator) JMP134 or Burkholderia cepacia
(Burkholderia cepacia) ATCC 25416) culture 20 hours or more, when bacterial turbidity is greater than OD600When=4, centrifugation
Collect cell, and clean one time with pure water, then by cell dissolution after processing in pH=7.4, contain 50 μM of diethylenetriamines
4- (2- ethoxy) -1- piperazine ethanesulfonic acid of pentaacetic acid (DTPA:diethylenetriaminepentaacetic acid)
In (HEPES:4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, and adjust bacterium
Turbidity OD600=8.
Heterotroph cell suspension after 10mL is adjusted is added in 50mL centrifuge tube, and the end that Fresh is added immediately is dense
Degree is 800 μM of vulcanized sodium starting reactions, and entire reaction condition was 30 DEG C of slight concussions (100r.p.m.), and at 30 minutes, 60 points
Clock, the residual concentration of hydrogen sulfide in 120 minutes three point in time sampling measurement systems, while testing sulphur gastral cavity sulphur, sulphite, sulphur
The production of hydrochlorate and thiosulfate.
As the result is shown in six hours C.necator JMP134 by Oxidation of Hydrogen Sulfide at sulfate, and stench vacation monospore
Bacterium (P.putida) S16, pseudomonas aeruginosa (P.aeruginosa) PAO1, Burkholderia cepacia (B.cepacia)
The final product of ATCC 25416 and Gluconobacter oxvdans (G.oxydans) 621H oxidation of sulfureted hydrogen is sulphur gastral cavity sulphur and thio
Sulfate (see Fig. 4).
Claims (2)
1. a kind of method for removing sulphide removal using heterotrophic microorganism is that growth stationary phase is added in the device for removing sulphide removal
Genome in contain thioquinones oxidoreducing enzyme (sqr) gene and sulphur dioxygenase (pdo) gene heterotrophic microorganism, setting
Aerobic reaction is carried out under reaction system, is realized to sulfide H in device2S、HS-Or/and S2-Removal；Wherein, the reactant
System is: concentration is 4- (2- ethoxy) -1- piperazine ethanesulfonic acid buffer of 50 ± 5mM, or has buffer capacity at neutral ph
Power and the buffer harmless to the heterotrophic microorganism；The sulphur for being also wherein 10 μM~1000 μM added with concentration in the reaction system
Change sodium, and the heterotrophic microorganism bacterial turbidity OD contained in the reaction system600nm=0.5~10；The aerobic reaction condition is:
10 DEG C~40 DEG C of temperature, pH 6~9,200 ± 50r.p.m. concussion；
It is characterized by:
Contain the heterotrophic microorganism of thioquinones oxidoreducing enzyme (sqr) gene and sulphur dioxygenase (pdo) gene in the genome
Refer to Gluconobacter oxvdans (Gluconobacter oxydans) 621H, pseudomonasputida (Pseudomonas
Putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Ralstonia bacterium (Cupriavidus
Necator) JMP134 or Burkholderia cepacia (Burkholderia cepacia) ATCC 25416；
Contain thioquinones oxidoreducing enzyme (sqr) gene and sulphur dioxygenase (pdo) gene in the genome of the growth stationary phase
The culture preparation method of heterotrophic microorganism be: containing industrial fermentation with glucose 30g/L and industrial fermentation corn pulp
30 DEG C of aerobic condition cultures are to stationary phase in the culture medium of dry powder 15g/L；
The removal to sulfide in device, which refers to, will be converted to thiosulfate and sulfane sulphur or complete after sulfide oxidation
Complete is oxidized to sulfate.
2. the method for removing sulphide removal using heterotrophic microorganism according to claim 1, it is characterised in that: the stench is false single
Spore bacterium (Pseudomonas putida) S16, pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Rolls are logical
Salmonella (Cupriavidus necator) JMP134 or Burkholderia cepacia (Burkholderia cepacia) ATCC
The ability of 25416 bacterial strains sulfur oxide compound in aerobic reaction system is with external source addition sulfide, polysulfide or elemental sulfur
Induction and enhance.
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