CN107058206A - A kind of acclimation method of the methane-oxidizing bacteria heterotroph mixed bacterial of utilization methane production poly beta-hydroxybutyrate - Google Patents
A kind of acclimation method of the methane-oxidizing bacteria heterotroph mixed bacterial of utilization methane production poly beta-hydroxybutyrate Download PDFInfo
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- CN107058206A CN107058206A CN201710194019.8A CN201710194019A CN107058206A CN 107058206 A CN107058206 A CN 107058206A CN 201710194019 A CN201710194019 A CN 201710194019A CN 107058206 A CN107058206 A CN 107058206A
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Abstract
The invention discloses a kind of acclimation method of the methane-oxidizing bacteria heterotroph mixed bacterial of utilization methane production poly beta-hydroxybutyrate, belong to biodegradable plastic synthesis and changing waste into resources utilizes technical field.It is raw material using the activated sludge rich in microorganism, using methane as carbon source, using nitrogen as nitrogen source, into culture medium add copper with improve MMO activity under conditions of, the acclimating of methane-oxidizing bacteria heterotroph mixed bacterial is carried out, it is final to obtain the stable efficient methane-oxidizing bacteria heterotroph mixed bacterial using methane production PHB.This method carries out PHB synthesis by substrate of methane using methane-oxidizing bacteria heterotroph mixed bacterial, the discharge of greenhouse gases is reduced while PHB production costs are reduced, and the PHB products obtained can be degraded to methane again when passing into disuse under anaerobic, realize recycling for substrate.
Description
Technical field
Technical field is utilized the invention belongs to biodegradable plastic synthesis and changing waste into resources, is related to a kind of from active dirty
The method that domestication synthesizes methane-oxidizing bacteria-heterotroph mixed bacterial of poly-beta-hydroxy-butyrate (PHB) using methane in mud.
Background technology
PHB is the carbon source stock that a kind of microorganism synthesizes under growth restrictive condition, with good material performance, biology
Compatibility and biodegradability, are that one kind has promising conventional petroleum chemical plastic substitute very much.
Traditional PHB fermentations are strain typically using pure bacterium, and bar of the carbon source as substrate is being commercialized using glucose, sucrose etc.
Carried out under part, high PHB production costs seriously limit PHB extensive use.It is substrate using the waste containing organic matter, with work
Property sludge be strain carry out PHB fermentation can both substantially reduce the substrate cost of PHB production processes, can save because safeguarding nothing again
Collarium border and the operating cost brought.
It can all be produced in the recovery process of fossil fuel (coal, oil and natural gas) and the anaerobic digestion process of debirs
Raw substantial amounts of methane, methane is as world's second largest greenhouse gases, and its global warming up trend highest can be 72 times of carbon dioxide.
It is that substrate carries out PHB productions using methane, can not only reduces PHB production cost but also the row of global warming gas can be reduced
Put.Research shows to make by substrate of the waste gas containing methane PHB production cost to reduce about 30-35% [1], and utilizes existing
Some refuse landfills and the methane synthesis PHB of anaerobic digester system release, then its yield can be instead of present plastics market
20-30% [2].
Methane-oxidizing bacteria under aerobic condition, first with methane monooxygenase (MMO) by methane oxidation be methanol, then
Methanol is oxidized to by a series of enzymatic reaction by formaldehyde, formic acid and carbon dioxide successively, and obtains itself in the process
Energy needed for vital movement.Methane-oxidizing bacteria can be divided into methane oxidation I types bacterium and methane oxidation II type bacterium, but only methane
Oxidation II type bacterium have PHB synthesis capabilities.Substantial amounts of heterotroph also has PHB synthesis capabilities, the methane-oxidizing bacteria in nature
Often coexisted with other heterotrophs, the metabolite that methane-oxidizing bacteria is discharged by methane oxidation process provides for other heterotrophs
Carbon source, and other heterotrophs metabolite poisonous by removing or the life that required nutriment stimulation methane-oxidizing bacteria is provided
It is long.
In methane-oxidizing bacteria-heterotroph mixed bacterial, when methane oxidation II types bacterium is dominant microflora, it can be more conducive to mix
The PHB synthesis capabilities of flora, and high MMO enzyme activity can promote methane to the conversion of other organic matters that can be utilized by heterotroph,
The PHB synthesis capabilities of methane-oxidizing bacteria-heterotroph mixed bacterial are further improved, by suitable acclimation conditions, can be enriched with
Go out stable methane-oxidizing bacteria-heterotroph mixed bacterial with high PHB synthesis capabilities.
[1]Listewnik H F,Wendlandt K D,Jechorek M,et al.Process design for
the microbial synthesis of poly-β-hydroxybutyrate(PHB)from natural gas[J]
.Engineering in Life Sciences,2007,7(3):278-282.
[2]Rostkowski K H,Criddle C S,Lepech M D.Cradle-to-gate life cycle
assessment for a cradle-to-cradle cycle:biogas-to-bioplastic(and back)[J]
.Environmental science&technology,2012,46(18):9822-9829.
The content of the invention
It is an object of the invention to provide methane-oxidizing bacteria-heterotroph mixed bacterial that a kind of efficient utilization methane produces PHB
Acclimation method, reduce PHB production costs while, reduce greenhouse gases discharge.
Technical scheme：
A kind of acclimation method of methane-oxidizing bacteria-heterotroph mixed bacterial of utilization methane production poly-beta-hydroxy-butyrate, step
It is rapid as follows：
Step 1：The activated sludge that will be enriched in microorganism is used as the original of acclimating methane-oxidizing bacteria-heterotroph mixed bacterial
Material；
Step 2：The preparation of culture medium
(1) the mineral salt NFMS culture mediums of not Nitrogen element are prepared, consisting of：0.272g/L KH2PO4,2.868g/L
Na2HPO4·H2O, 0.1g/L MgSO4·7H2O, 0.20g/L CaCl2·6H2O and 1mL/L trace element；
(2) trace element solution is prepared：250mg/L Na-EDTA, 500mg/L FeSO4·7H2O, 380mg/L Fe-
EDTA, 400mg/L ZnSO4·7H2O, 15mg/L H3BO3, 20mg/L MnCl2·4H2O, 260mg/L Na2MoO4·
2H2O, 10mg/L NiCl2·6H2O, 50mg/L CoCl2·6H2O；
(3) CuSO is prepared4·5H2O storing solutions；
(4) CuSO is added to NFMS culture mediums4·5H2O storing solutions, make CuSO4Molar concentration be 5-10 μm of ol/L, with
Improve the MMO activity of mixed bacterial；
Step 3：The acclimating of methane-oxidizing bacteria-heterotroph mixed bacterial
(1) the NFMS culture mediums that activated sludge and step 2 (4) are obtained are added into system, make system head space and liquid phase
Volume ratio is 2:1-5:1, system is sealed；
(2) headspace gas is changed, it is 10-20% oxygen, 25% methane gas and 55-65% to make the composition of headspace gas
Nitrogen；Using methane gas as sole carbon source, with nitrogen as domestication process only nitrogen source, carry out methane-oxidizing bacteria-heterotroph and mix
Close the acclimating of flora；To ensure sufficient gaseous substrate, a headspace gas was changed per 24-72 hours；
(3) bacterium solution being enriched to is inoculated into what new step 2 (4) was obtained per the 6-10 days volume ratios by 4-10%
In NFMS culture mediums, to ensure the nutriment of abundance.
Step 4：PHB building-up processes
(1) at the end of each circulation, collect thalline and carry out PHB synthesis.
(2) thalline and new that collection is added into system contain 5-10 μm of ol/L CuSO4NFMS culture, make system
The volume ratio of head space and liquid phase is 5:1-11:1, system is sealed；
(3) headspace gas is changed, the composition for making headspace gas is 50-75% methane and 25-50% oxygen, in no nitrogen source
Under conditions of carry out PHB synthesis.To ensure sufficient gaseous substrate, a headspace gas, about 72 hours were changed per 24-48 hours
Afterwards, PHB building-up process is completed.
Step 5：After culture after about 220-320 days, the domestication of methane-oxidizing bacteria-heterotroph mixed bacterial is completed,
Bacterium solution is white, and containing substantial amounts of small flco, and the PHB synthesis capabilities of mixed bacterial are stable.The methane-oxidizing bacteria of acquisition-
In heterotroph mixed bacterial, methane-oxidizing bacteria is made up of four kinds of methane oxidation II type bacterium, and containing substantial amounts of there is high PHB to close
Into the heterotrophic microorganism of ability.
The invention has the advantages that：The suitable enrichment condition of present invention selection, being tamed out from activated sludge has
Methane-oxidizing bacteria-heterotroph mixed bacterial of PHB abilities is efficiently synthesized using methane, is subtracted while PHB manufacturing costs are reduced
The discharge of few greenhouse gases, and the PHB products obtained can be degraded to methane again when passing into disuse under anaerobic,
Realize recycling for substrate.
Brief description of the drawings
Fig. 1 is the curve for synthesizing PHB using methane in different domestication stage methane-oxidizing bacteria-heterotroph mixed bacterials.
Embodiment
Below in conjunction with accompanying drawing and technical scheme, the embodiment of the present invention is further illustrated.
The domestication process of methane-oxidizing bacteria-heterotroph mixed bacterial is mainly carried out in two steps：First, appropriate condition pressure is selected
Power is tamed to activated sludge, is enriched with methane-oxidizing bacteria-heterotroph mixed bacterial；2nd, under conditions of no nitrogen source, utilize
The methane-oxidizing bacteria of enrichment-heterotroph mixed bacterial carries out PHB synthesis.
Embodiment 1
(1) second pond from Daliang City of Liaoning Province Fu Jia village municipal sewage plant collects activated sludge, dirty with the activity
Mud as domestication seed sludge.
(2) NFMS culture mediums are prepared, CuSO is added4Storing solution, makes CuSO4Concentration be 5-10 μm of ol/L.Then utilize
The pH of NMFS culture mediums is adjusted to 6.8 by 1mol/L sodium hydroxide solution.
(3) method by centrifuging and being resuspended, is washed and is diluted to seed sludge using above-mentioned NMFS culture mediums, made
Final sludge concentration is about 1500mg/L.
(4) seed sludge after the above-mentioned dilutions of 50mL is seeded in 300mL serum bottle, and uses rubber stopper and nut
Sealing.
(5) above-mentioned 300mL serum bottles and atmosphere containing seed sludge are made using the fine needle with filter, so
Afterwards using the method that gentle body is refilled is vacuumized, the composition for making serum bottle headspace gas is 10-20% oxygen, 25%
The nitrogen of methane gas and 55-65%.Constituted using the gas of the gas-chromatography periodic monitoring head space with thermal conductivity detector (TCD), and often
A headspace gas is changed within 24 hours, to ensure the gaseous substrate of abundance.
(6) the 300mL serum bottles containing seed sludge are placed on 30 DEG C, in 150r/min shaking table, carry out methane oxygen
Change the culture of bacterium-heterotroph mixed bacterial.Every 10 days, the bacterium solution being enriched to is inoculated into new containing 5-10 μm of ol/
LCuSO4NFMS culture in, inoculative proportion is 10%, so circulation 10 times.Then by inoculative proportion be reduced to 4% and every
Once it is inoculated with 6-7 days.
(7) after circulation terminates every time, centrifuged 5 minutes under conditions of 10000r/min using centrifuge and collect thalline, so
The thalline of collection is resuspended to containing 5 μm of ol/LCuSO afterwards4NFMS culture mediums in, make sludge concentration about 1266.3mg/L,
Carry out PHB synthesis.
(8) methane-oxidizing bacteria after above-mentioned resuspension-heterotroph mixed bacterial is seeded in the serum of 120mL a series of,
Each serum bottle contains 10mL bacterium solution, utilizes rubber stopper and screw cap closures.
(9) 120mL serum bottles and atmosphere containing bacterium solution are made using with the fine needle of filter, then using taking out
The method that the gentle body of vacuum is refilled, the composition for making serum bottle headspace gas is 25-50% oxygen and 50-75% first
Alkane.Constituted using the gas of the gas-chromatography periodic monitoring head space with thermal conductivity detector (TCD), and every 24 hours change a head space
Gas, to ensure the gaseous substrate of abundance.
(10) by the 120mL serum bottles of the methane-oxidizing bacteria containing enrichment-heterotroph mixed bacterial be placed on 30 DEG C,
In 150r/min shaking table, PHB synthesis is carried out.After 72 hours, the PHB synthesis of methane-oxidizing bacteria-heterotroph mixed bacterial
Process is completed, and periodically takes two 120mL serum bottles every time during this period, detects the change of the intracellular PHB contents of mixed bacterial.
(11) activated sludge is after the domestication of 220 days, and the ability for synthesizing PHB using methane starts stabilization, is tamed and dociled after 320 days
Change and complete, bacterium solution is presented white and contains substantial amounts of small flco.The methane-oxidizing bacteria of enrichment-heterotroph mixed bacterial is in domestication
PHB resultant curves after 100 days, 220 days and 320 days are as shown in Figure 1.
(12) after domestication terminates, Comparison of Microbial Community is carried out to methane-oxidizing bacteria-heterotroph mixed bacterial of enrichment, obtained
In the methane-oxidizing bacteria-heterotroph mixed bacterial obtained, methane-oxidizing bacteria is made up of four kinds of methane oxidation II type bacterium：
Methylocella, Methylosinus, Methylocystis and Methylocapsa.Heterotroph it is main by
Beijerinckia, Sphingomonas, Pseudomonas and Methylophilus are constituted.
Embodiment 2
Collect activated sludge from the second pond of Lingshui River, Dalian, Liaoning province municipal sewage plant, using the sludge as tame and docile
Change the seed sludge of methane-oxidizing bacteria-heterotroph mixed bacterial, other embodiment is same as Example 1.
Embodiment 3
Activated sludge is collected from the second pond of Daliang City of Liaoning Province spring Liuhe municipal sewage plant, kind is used as using the sludge
Sub- sludge carries out the domestication of methane-oxidizing bacteria-heterotroph mixed bacterial, and other embodiment is same as Example 1.
Claims (1)
1. a kind of acclimation method of methane-oxidizing bacteria-heterotroph mixed bacterial of utilization methane production poly-beta-hydroxy-butyrate, it is special
Levy and be, step is as follows：
Step 1：The activated sludge that will be enriched in microorganism is used as the raw material of acclimating methane-oxidizing bacteria-heterotroph mixed bacterial；
Step 2：The preparation of culture medium
(1) the mineral salt NFMS culture mediums of not Nitrogen element are prepared, consisting of：0.272g/L KH2PO4, 2.868g/L's
Na2HPO4·H2O, 0.1g/L MgSO4·7H2O, 0.20g/L CaCl2·6H2O and 1mL/L trace element；
(2) trace element solution is prepared：250mg/L Na-EDTA, 500mg/L FeSO4·7H2O, 380mg/L Fe-
EDTA, 400mg/L ZnSO4·7H2O, 15mg/L H3BO3, 20mg/L MnCl2·4H2O, 260mg/L Na2MoO4·
2H2O, 10mg/L NiCl2·6H2O, 50mg/L CoCl2·6H2O；
(3) CuSO is prepared4·5H2O storing solutions；
(4) CuSO is added to NFMS culture mediums4·5H2O storing solutions, make CuSO4Molar concentration be 5-10 μm of ol/L, to improve
The MMO activity of mixed bacterial；
Step 3：The acclimating of methane-oxidizing bacteria-heterotroph mixed bacterial
(1) the NFMS culture mediums that activated sludge and step 2 (4) are obtained are added into system, make the volume of system head space and liquid phase
Than for 2:1-5:1, system is sealed；
(2) headspace gas is changed, it is 10-20% oxygen, 25% methane gas and 55-65% nitrogen to make the composition of headspace gas
Gas；Using methane gas as sole carbon source, with nitrogen as domestication process only nitrogen source, methane-oxidizing bacteria-heterotroph Mixed Microbes are carried out
The acclimating of group；To ensure sufficient gaseous substrate, a headspace gas was changed per 24-72 hours；
(3) bacterium solution being enriched to is inoculated into the NFMS trainings that new step 2 (4) is obtained per the 6-10 days volume ratios by 4-10%
Support in base, to ensure the nutriment of abundance；
Step 4：PHB building-up processes
(1) at the end of each circulation, collect thalline and carry out PHB synthesis；
(2) the NFMS cultures that the thalline and new step 2 (4) of collection are obtained are added into system, make system head space and liquid phase
Volume ratio is 5:1-11:1, system is sealed；
(3) headspace gas is changed, the composition for making headspace gas is 50-75% methane and 25-50% oxygen, in the bar without nitrogen source
PHB synthesis is carried out under part；To ensure sufficient gaseous substrate, a headspace gas, after 72 hours, PHB were changed per 24-48 hours
Building-up process complete；
Step 5：After culture after 220-320 days, the domestication of methane-oxidizing bacteria-heterotroph mixed bacterial is completed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643562A (en) * | 2018-06-27 | 2020-01-03 | 郑州大学 | Culture method for improving capacity of methane oxidation mixed flora for synthesizing poly-beta-hydroxybutyrate |
| CN114790437A (en) * | 2022-04-29 | 2022-07-26 | 中国科学院成都生物研究所 | Hydroxide bacterium flora with specific function and screening method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513847A (en) * | 2014-12-25 | 2015-04-15 | 哈尔滨商业大学 | Method for biosynthesizing poly-beta-hydroxy butyric acid (PHB) by utilizing mixed methane-oxidizing bacteria |
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2017
- 2017-03-29 CN CN201710194019.8A patent/CN107058206A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513847A (en) * | 2014-12-25 | 2015-04-15 | 哈尔滨商业大学 | Method for biosynthesizing poly-beta-hydroxy butyric acid (PHB) by utilizing mixed methane-oxidizing bacteria |
Non-Patent Citations (2)
| Title |
|---|
| TINGTING ZHANG等: "Enrichments of methanotrophic-heterotrophic cultures with high poly-β-hydroxybutyrate(PHB) accumulation capacities", 《JOURNAL OF ENVIRONMENTAL SCIENCES》 * |
| 姜丹等: "开放条件下高聚β-羟基丁酸酯含量的甲烷氧化混合菌群的富集", 《生物技术通讯》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643562A (en) * | 2018-06-27 | 2020-01-03 | 郑州大学 | Culture method for improving capacity of methane oxidation mixed flora for synthesizing poly-beta-hydroxybutyrate |
| CN114790437A (en) * | 2022-04-29 | 2022-07-26 | 中国科学院成都生物研究所 | Hydroxide bacterium flora with specific function and screening method and application thereof |
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Application publication date: 20170818 |