CN104694453A - Method for screening and enriching halophilic poly PHA (phytohemagglutinin) mixed bacteria by using glucose as substrate - Google Patents
Method for screening and enriching halophilic poly PHA (phytohemagglutinin) mixed bacteria by using glucose as substrate Download PDFInfo
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- CN104694453A CN104694453A CN201510101347.XA CN201510101347A CN104694453A CN 104694453 A CN104694453 A CN 104694453A CN 201510101347 A CN201510101347 A CN 201510101347A CN 104694453 A CN104694453 A CN 104694453A
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Abstract
The invention relates to a method for screening and enriching halophilic poly PHA (phytohemagglutinin) mixed bacteria by using glucose as a substrate, belonging to the technical field of PHA production. The method comprises the following steps: acquiring entrance sediment, inoculating into a SBR (sequencing batch reactor), and fully filling with a substrate water solution using glucose as the main organic substance; starting stirring and aeration devices of the reactor, charging air at uniform speed to control the dissolved oxygen in the substrate solution at 2-4 mg/L, and continuously operating for 10-12 hours, wherein the pH value is 6.5-7.5 and is not regulated in the operation process, and the temperature of the reactor is controlled at 30+/-0.1 DEG C; shutting down the stirring device and aeration device, and standing for 10-60 minutes; eliminating the supernate and 1/10 of sludge in the reactor; and repeating like this until the maximum mass percent of PHA in the microbe cells reaches 20% above and is kept stable for 5-10 continuous periods, thereby completing screening. The method is simple to implement, and can easily implement engineering operation. The screened PHA accumulative halophilic mixed bacteria can be used for PHA production in the fermentation process by using acetic acid, glucose and starch, and have high PHA accumulation capacity.
Description
Technical field
The present invention relates to a kind of method that selective enrichment has the mixed bacterial of polyhydroxyalkanoate (PHA) accumulation ability, especially with glucose be substrate with natural estuario bed mud for inoculum selective enrichment under high salt condition has the method for PHA accumulation ability addicted to salt mixed bacterium.The method produces the first step of PHA as two-step approach, can produce PHA provide flora for subsequent fermentation, reduces PHA production cost.The method belongs to biological production PHA technical field.
Background technology
At present, be that the plastics of raw material production have been widely used in industry-by-industry with oil.But topsoil, water pollutions that waste plastic " white pollution " that bring and garbage-surrounded city phenomenon and Plastics Combustion produce seriously have impact on the living environment of people.Therefore, biodegradable plastic receives as the substitute of the plastics taking oil as raw material production and pays close attention to widely.Polyhydroxyalkanoate (PHA) is the intracellular polymer that a quasi-microorganism accumulates carbon source formation in vivo.PHA has good physical property, biocompatibility and biodegradability, is considered to the following important substance substituting chemical plastic.At present, industrial production PHA is mainly with sterile fermentation, and its high cost limits its widespread use.Substrate cost, thin intracellular PHA content and PHA extracting method are the main factors affecting biological production PHA cost.For this reason, use inexpensive fermentation substrate can be passed through, improve thin intracellular PHA content, minimizing or cancel sterilizing program, simplify the production cost that modes such as extracting PHA program reduces PHA.Halophilic microorganism is the class peculiar microorganism of growth in hypersaline environment, has special cellularstructure, physiological function, gene and adaptation mechanism.Research has had been found that a lot of halophilic bacterium has high PHA accumulation ability.Hypersaline environment prevents the growth of other non-halophilic microorganisms, and bacterial classification keeps not needing sterilizing program, can carry out PHA production in an open-ended fashion; In addition, because halophilic microorganism cell interior has high osmotic pressure, once enter the hypotonic environment of fresh water will cause rapidly breaking of cell walls, PHA release is fast realized.Do not make with medicament broken wall reclaim PHA and reduce PHA cost; Finally, halophilic microorganism can utilize multiple organic waste fermentative production PHA, reduces the substrate cost that PHA produces.In summary it can be seen, halophilic bacterium is produced PHA on cost, is had larger advantage.
At present, biological production PHA generally adopts two-step approach.Namely the first step realizes the selective enrichment to PHA accumulation flora, and second step utilizes these fermentable substrates to produce PHA.In two-step process, the flora of the poly-PHA ability of the first step enrichment height is most critical one step.Can this step produce the output of PHA using directly affecting subsequent fermentation and use cheap waste to produce PHA as substrate.The way selection population adopting dynamically production (ADF) using acetic acid as substrate at present to the mode that the selective enrichment of PHA accumulation microorganism is general.But the population screened by this way can only use acetic acid to produce PHA as substrate when second step ferments, cannot use other substrates.Acetic acid is a kind of expensive substrate.If be that substrate produces PHA with acetic acid, in PHA production cost, the cost of acetic acid just contribute to 40%.For this reason, two-step approach produce PHA technique in, set up a kind of system of selection of PHA accumulation flora in the first step, realize second step utilize low cost substrate even waste fermentation PHA be very important.Such method can reduce the production cost of PHA greatly.
Summary of the invention
The object of the present invention is to provide a kind of take glucose as substrate with natural estuario bed mud for inoculum selective enrichment accumulation PHA is addicted to the method for salt mixed bacterium.The method improves PHA output most possibly, reduces PHA production cost.
Take glucose as the method that substrate screens addicted to salt PHA accumulation mixed bacterium, mainly comprise the following steps:
Step 1, gather estuario bed mud and be inoculated in known sbr reactor device, the mud mixed liquid of inoculation accounts for the 1/4-1/5 of SBR cumulative volume;
Step 2, substrate solution is filled: contain in the every aqueous solution of this substrate: 0.5-2.0g glucose in reactor, other elements that microorganism growth is required, other elements adopting following material to provide microorganism growth necessary in aqueous solution as every in substrate: 0.11g NH
4cl, 30.0g NaCl, 45.0mg MgCl
26H
2o, 113.3mg NH
4cl, 26.0mg KH
2pO
4, 7.6mg KCl, 3.0mg FeSO
47H
2o, 1.5mgZnSO
47H
2o, 8.0mg CaCl
22H
2o, 1.2mg MnSO
4h
2o;
Step 3, intermittent mode is adopted to run sbr reactor device, start stirring transposition and the aerating apparatus of sbr reactor device, stir speed (S.S.) is 60-120r/min, in reactor, at the uniform velocity inject air, and in control substrate solution, dissolved oxygen is at 2-4mg/L, continuous operation 10-12h, pH scope is 6.5-7.5, does not regulate pH in operational process, reactor with temperature controlling instruments control temperature at 30 ± 0.1 DEG C;
Step 4, closes whipping appts and aerating apparatus, leaves standstill 10min-60min;
Step 5, gets rid of the supernatant liquor in sbr reactor device, retentive activity mud;
Step 6, gets rid of 1/10 of the mud in sbr reactor device;
Step 7, step 2-step 6 is a cycle, repeating step 2-step 6, until in the cycle in microorganism cells PHA biggest quality percentage composition reach more than 20% and continuously the 5-10 cycle keeps stablizing, complete screening.
The hardware system that the present invention is based on is known sbr reactor device.Described natural inoculum is estuario bed mud, it is directly inoculated in reactor.
The present invention has the following advantages:
(1) the method adopts glucose to be substrate domestication flora, and glucose is cheap and easy to get, can reduce substrate cost when flora is selected;
(2) screening is all higher addicted to the content and productive rate that salt mixing microorganisms is carrying out its thin intracellular PHA in second step fermentative production;
(3) screen addicted to salt mixing microorganisms at the PHA carrying out in second step fermentative production to produce for substrate with the glucose of cheapness for PHBV multipolymer, this multipolymer increases the commercial exploitation in its downstream;
(4) screening is being carried out using acetic acid, glucose or even the larger molecular organics such as starch, Mierocrystalline cellulose as fermentation substrate in second step fermentative production addicted to salt mixing microorganisms, realizes the utilization to some low price substrates;
(5) the method screening is addicted to salt mixing microorganisms carrying out not needing sterilizing in two-step approach PHA production, can carry out open production.
Accompanying drawing explanation
Fig. 1: take glucose as the method picture frame of substrate screening addicted to salt PHA accumulation mixed bacterium.
Embodiment
Illustrate that the present invention is described in further detail with embodiment below in conjunction with accompanying drawing, but the present invention is not limited to following examples.
Embodiment 1
Take useful volume as the sbr reactor device of 2L, utilize the screening that the inventive method is carried out addicted to salt PHA accumulation bacterium, embodiment:
Step 1, at collection river mouth, river mouth, the Tanghe River, Qinhuangdao, Hebei province bed mud, sampling position is positioned at, 600 meters, the Bohai Sea.After removing the grains of sand, obtain 0.4L bed mud, be inoculated in sbr reactor device.In sbr reactor device, inject substrate aqueous solution to 2L, now inoculum concentration is 0.102g/L;
Step 2, fills the substrate solution based on glucose in reactor, and the proportioning of this substrate solution comprises: often liter of substrate aqueous solution contains: 0.87g glucose, 0.11g NH
4cl, 30.0g NaCl, 45.0mgMgCl
26H
2o, 113.3mg NH
4cl, 26.0mg KH
2pO
4, 7.6mg KCl, 3.0mg FeSO
47H
2o, 1.5mg ZnSO
47H
2o, 8.0mg CaCl
22H
2o, 1.2mg MnSO
4h
2o, surplus is water;
Step 3, intermittent mode is adopted to run reactor, start stirring transposition and the aerating apparatus of reactor, stir speed (S.S.) is 100r/min, in reactor, at the uniform velocity inject air, and in control substrate solution, dissolved oxygen is at 3mg/L, continuous operation 12h, pH is 6.8, does not regulate pH in operational process, reactor with temperature controlling instruments control temperature at 30 ± 0.1 DEG C;
Step 4, closes whipping appts and aerating apparatus, leaves standstill 30min;
Step 5, gets rid of the supernatant liquor in reactor, retentive activity mud;
Step 6, gets rid of 1/10 of the mud in sbr reactor device;
Step 7, step 2-step 6 is a cycle, repeating step 2-step 6.After 100 cycles, in the cycle, in microorganism cells, PHA biggest quality percentage composition reaches 20% and continuous 10 cycles keep stable, completes screening.
The all chemical reagent used in substrate in the present embodiment are chemical pure level.
Fermentative production PHA is carried out with the acetic acid of 1500mg/LCOD, glucose, starch for substrate respectively addicted to salt mixed bacterium according to above-mentioned steps screening, the thin intracellular PHA mass percent obtained is respectively 44.7%, 27.6%, 12.0%, and the PHA volume productive rate of acquisition is 0.48g/L, 0.33g/L, 0.15g/L.
Comparative example 1
Take useful volume as the sbr reactor device of 2L, utilize sodium acetate to be substrate, screening that the method identical with above-mentioned operation scheme is carried out addicted to salt PHA accumulation bacterium, embodiment:
Step 1, at collection river mouth, river mouth, the Tanghe River, Qinhuangdao, Hebei province bed mud, sampling position is positioned at, 600 meters, the Bohai Sea.After removing the grains of sand, obtain 0.4L bed mud, be inoculated in sbr reactor device.In sbr reactor device, inject substrate aqueous solution to 2L, now inoculum concentration is 0.102g/L;
Step 2, fills the substrate solution based on sodium acetate in reactor, and the proportioning of this substrate solution comprises: often liter of substrate aqueous solution contains: 1.18g sodium acetate, 0.11g NH
4cl, 30.0g NaCl, 45.0mgMgCl
26H
2o, 113.3mg NH
4cl, 26.0mg KH
2pO
4, 7.6mg KCl, 3.0mg FeSO
47H
2o, 1.5mg ZnSO
47H
2o, 8.0mg CaCl
22H
2o, 1.2mg MnSO
4h
2o, surplus is water;
Step 3, intermittent mode is adopted to run reactor, start stirring transposition and the aerating apparatus of reactor, stir speed (S.S.) is 100r/min, in reactor, at the uniform velocity inject air, and in control substrate solution, dissolved oxygen is at 3mg/L, continuous operation 12h, pH is 6.8, does not regulate pH in operational process, reactor with temperature controlling instruments control temperature at 30 ± 0.1 DEG C;
Step 4, closes whipping appts and aerating apparatus, leaves standstill 30min;
Step 5, gets rid of the supernatant liquor in reactor, retentive activity mud;
Step 6, gets rid of 1/10 of the mud in sbr reactor device;
Step 7, step 2-step 6 is a cycle, repeating step 2-step 6.After 100 cycles, in the cycle, in microorganism cells, PHA biggest quality percentage composition reaches 20% and continuous 10 cycles keep stable, completes screening.
The all chemical reagent used in substrate in the present embodiment are chemical pure level.
Fermentative production PHA is carried out with the acetic acid of 1500mg/LCOD, glucose, starch for substrate respectively addicted to salt mixed bacterium according to above-mentioned steps screening, the thin intracellular PHA mass percent obtained is respectively 38.1%, 8.7%, 8.5%, and the PHA volume productive rate of acquisition is 0.38g/L, 0.11g/L, 0.11g/L.By this result and embodiment 1 more known, the glucose that the inferior COD of identical operation scheme measures carries out the flora fermentative production PHA tamed ability as substrate is better than sodium acetate domestication flora, can save cost 20%, 67%, 27% with acetic acid, glucose, starch respectively for substrate carries out fermentative production PHA.
Comparative example 2
Take useful volume as the sbr reactor device of 2L, utilize starch to be substrate, screening that the method identical with above-mentioned operation scheme is carried out addicted to salt PHA accumulation bacterium, embodiment:
Step 1, at collection river mouth, river mouth, the Tanghe River, Qinhuangdao, Hebei province bed mud, sampling position is positioned at, 600 meters, the Bohai Sea.After removing the grains of sand, obtain 0.4L bed mud, be inoculated in sbr reactor device.In sbr reactor device, inject substrate aqueous solution to 2L, now inoculum concentration is 0.102g/L;
Step 2, fills the substrate solution based on starch in reactor, and the proportioning of this substrate solution comprises: often liter of substrate aqueous solution contains: 0.78g starch, 0.11g NH
4cl, 30.0g NaCl, 45.0mgMgCl
26H
2o, 113.3mg NH
4cl, 26.0mg KH
2pO
4, 7.6mg KCl, 3.0mg FeSO
47H
2o, 1.5mg ZnSO
47H
2o, 8.0mg CaCl
22H
2o, 1.2mg MnSO
4h
2o, surplus is water;
Step 3, intermittent mode is adopted to run reactor, start stirring transposition and the aerating apparatus of reactor, stir speed (S.S.) is 100r/min, in reactor, at the uniform velocity inject air, and in control substrate solution, dissolved oxygen is at 3mg/L, continuous operation 12h, pH is 6.8, does not regulate pH in operational process, reactor with temperature controlling instruments control temperature at 30 ± 0.1 DEG C;
Step 4, closes whipping appts and aerating apparatus, leaves standstill 30min;
Step 5, gets rid of the supernatant liquor in reactor, retentive activity mud;
Step 6, gets rid of 1/10 of the mud in sbr reactor device;
Step 7, step 2-step 6 is a cycle, repeating step 2-step 6.After 100 cycles, in the cycle, in microorganism cells, PHA biggest quality percentage composition reaches 20% and continuous 10 cycles keep stable, completes screening.
The all chemical reagent used in substrate in the present embodiment are chemical pure level.
Fermentative production PHA is carried out with the acetic acid of 1500mg/LCOD, glucose, starch for substrate respectively addicted to salt mixed bacterium according to above-mentioned steps screening, the thin intracellular PHA mass percent obtained is respectively 26.7%, 16.3%, 13.2%, and the PHA volume productive rate of acquisition is 0.33g/L, 0.17g/L, 0.15g/L.By this result and embodiment 1 more known, the glucose that the inferior COD of identical operation scheme measures carries out the flora fermentative production PHA tamed ability as substrate is better than starch domestication flora, can save cost 31%, 48% with acetic acid, glucose respectively for substrate carries out fermentative production PHA.
Claims (3)
1. addicted to salt mixed bacterium with glucose be substrate screening PHA accumulation addicted to the method for salt mixed bacterium, it is characterized in that, mainly comprise the following steps:
Step 1, gather estuario bed mud and be inoculated in sbr reactor device, the mud mixed liquid of inoculation accounts for the 1/4-1/5 of cumulative volume;
Step 2, filling with glucose in reactor is main substrate solution, and the proportioning of this substrate solution comprises: often liter of substrate aqueous solution contains: 2.0-0.5g glucose, other elements that microorganism growth is required;
Step 3, intermittent mode is adopted to run reactor, start stirring transposition and the aerating apparatus of reactor, stir speed (S.S.) is 60-120r/min, in reactor, at the uniform velocity inject air, and in control substrate solution, dissolved oxygen is at 2-4mg/L, continuous operation 10-12h, pH scope is 6.5-7.5, does not regulate pH in operational process, reactor with temperature controlling instruments control temperature at 30 ± 0.1 DEG C;
Step 4, closes whipping appts and aerating apparatus, leaves standstill 10min-60min;
Step 5, gets rid of the supernatant liquor in reactor, retentive activity mud;
Step 6, gets rid of 1/10 of the mud in sbr reactor device;
Step 7, step 2-step 6 is a cycle, repeating step 2-step 6, until in the cycle in microorganism cells PHA biggest quality percentage composition reach more than 20% and continuously the 5-10 cycle keeps stablizing, complete screening.
2. according to the method for claim 1, it is characterized in that, other elements adopting following material to provide microorganism growth necessary in the every aqueous solution of substrate in step 2: 0.11g NH
4cl, 30.0g NaCl, 45.0mgMgCl
26H
2o, 113.3mg NH
4cl, 26.0mg KH
2pO
4, 7.6mg KCl, 3.0mgFeSO
47H
2o, 1.5mg ZnSO
47H
2o, 8.0mg CaCl
22H
2o, 1.2mg MnSO
4h
2o.
3. adopt the method for claim 1 or 2 screening PHA accumulation to carry out fermentative production PHA addicted to salt mixed bacterium, it is characterized in that, use glucose, starch or Mierocrystalline cellulose as fermentation substrate.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1225338A (en) * | 1998-12-28 | 1999-08-11 | 华南理工大学 | Bio-floculation wastewater treatment utilizing synthesized micro-organism/polyester |
| CN103865960A (en) * | 2014-03-13 | 2014-06-18 | 北京工业大学 | Method for taking glucose as substrate to continuously produce polyhydroxylbutyrate valerate (PHBV) in one step by using halophilic mixed bacteria |
| CN104031906A (en) * | 2014-06-27 | 2014-09-10 | 天津大学 | Screening and domestication method of bacteria colony for producing polyhydroxyalkanoate by using xylose |
| CN104178544A (en) * | 2014-08-21 | 2014-12-03 | 东北林业大学 | Method for synthesizing poly-beta-hydroxybutyrate from yeast and bacterial community by using activated sludge |
-
2015
- 2015-03-08 CN CN201510101347.XA patent/CN104694453A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1225338A (en) * | 1998-12-28 | 1999-08-11 | 华南理工大学 | Bio-floculation wastewater treatment utilizing synthesized micro-organism/polyester |
| CN103865960A (en) * | 2014-03-13 | 2014-06-18 | 北京工业大学 | Method for taking glucose as substrate to continuously produce polyhydroxylbutyrate valerate (PHBV) in one step by using halophilic mixed bacteria |
| CN104031906A (en) * | 2014-06-27 | 2014-09-10 | 天津大学 | Screening and domestication method of bacteria colony for producing polyhydroxyalkanoate by using xylose |
| CN104178544A (en) * | 2014-08-21 | 2014-12-03 | 东北林业大学 | Method for synthesizing poly-beta-hydroxybutyrate from yeast and bacterial community by using activated sludge |
Non-Patent Citations (1)
| Title |
|---|
| 俞苗新等: "活性污泥法合成可生物降解塑料的工程化可行性探讨", 《环境污染与防治》 * |
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Application publication date: 20150610 |