CN105368911A - Method for synthesizing polyhydroxyalkanoate with kitchen waste anaerobic fermentation liquid as substrate - Google Patents
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Abstract
The invention provides a method for synthesizing polyhydroxyalkanoate with kitchen waste anaerobic fermentation liquid as a substrate. The method comprises the following steps: 1, conducting pretreatment on the kitchen waste anaerobic fermentation liquid with lipase and amylase; 2, inoculating functional bacteria into the kitchen waste anaerobic fermentation liquid obtained after pretreatment in step 1, and conducting synthesis of the polyhydroxyalkanoate. The method is not influenced by the high concentration of salt in the kitchen waste anaerobic fermentation liquid, and microorganisms can grow normally in the high-salt environment and can synthesize PHAs efficiently. By means of the method, no extra carbon source is needed to be added, the polyhydroxyalkanoate can be produced industrially.
Description
Technical field
The present invention relates to a kind of method of the synthesizing polyhydroxyalkanoateby that ferments, the polyhydroxyalkanoate production method that to be specifically related to anaerobic fermentation of kitchen waste liquid be fermenting substrate.
Background technology
Along with the food safety such as " sewer oil " and " rubbish pig " and problem of environmental pollution highlight day by day, China pays much attention to changing food waste work for the treatment of in recent years.The anaerobically fermenting process of changing food waste, because taking into account resource utilization and energy feature, has become the mainstream technology of current China's mass-producing changing food waste process engineering (being greater than 200 ton per days).The further process of the fermented liquid (natural pond liquid) that changing food waste anaerobic biological treatment produces and recycling are the gordian techniquies urgently broken through at present.
The waste water produced in changing food waste treating processes and fermented liquid are used as a kind for the treatment of high-concentration saline organic wastewater process, and not only processing cost is high, and causes the waste of organism in fermented liquid, nutrition resource.And using restaurant-kitchen garbage degreased wastewater as the recycling approach producing edible fungi liquid strain, still there is food safety hidden danger.When changing food waste is carried out fermentative processing, the anaerobic fermentation of kitchen waste liquid produced is a kind of unmanageable high concentrated organic wastewater, on the one hand it contains the organic pollutant such as high concentration COD, ammonia nitrogen, colourity is high, turbidity is high and with stench, its chemical constitution is based on protein, carbohydrate, animal tallow on the other hand, has certain recycling and is worth.Although anaerobic fermentation of kitchen waste liquid is close with traditional natural pond liquid composition, salinity is too high becomes the Main Bottleneck limiting its recycling.Its direct returning to farmland, generally up to 1.0-1.5%, is utilized the salinization problem that there is soil salinity accumulation and bring by the salinity in fermented liquid.At present in anaerobically fermenting process complete set technology, generally changing food waste fermented liquid is equal to percolate high pollution treatment of Organic Wastewater, adopt membrane technology route, its processing cost high (generally at 15-30 yuan/ton), accounts for more than 30% of changing food waste processing cost.
Patent CN101235400B discloses a kind of method utilizing active sludge to produce polyhydroxyalkanoate, the method utilizes the residual active sludge produced in sewage treatment process to be raw material, specifically disclose and use active sludge to be hydrolyzed acidifying, obtain organic acid, use the engineering bacteria fermentation that the organic acid utilization be separated contains fadD and fadE gene to produce PHAs.The residual active sludge that the method uses is by being produced in sewage treatment process, and its saltiness is low, has no adverse effect to the normal growth of microorganism and metabolic process; And this project bacterium produces through artificial recombination, therefore the method adaptive surface is little.
It take excess sludge fermentation broth as the method for substrate synthesizing polyhydroxyalkanoateby that patent application CN102505025A discloses a kind of, the method uses excess sludge to carry out anaerobically fermenting, be separated the voltaile fatty acid fermented liquid produced, this voltaile fatty acid fermented liquid and the active sludge through aerobic aeration, continuous domestication 18 ~ 22 cycles (22 ~ 24 hours each cycles), adding sodium acetate is in addition additional carbon, carries out the synthesis of polyhydroxyalkanoate.The residual active sludge that the method uses is similarly in sewage treatment process and produces, and its saltiness is low; This external demand adds carbon source in addition to produce polyhydroxyalkanoate.
Patent application CN103074272A discloses a kind of Methylotrophic genus bacillus (Bacillusmethylotrophicus) three torch-04 and application thereof, the liquid nutrient medium of this Methylotrophic genus bacillus comprises: sucrose 0.5 ~ 2%, yeast extract paste 0.5 ~ 2%, potassium primary phosphate 0.05 ~ 0.02%, magnesium sulfate 0.01 ~ 0.10%, sodium-chlor 0.01 ~ 0.10%, calcium carbonate 0.01 ~ 0.10%, pH5-9.It is sodium-chlor 0.01 ~ 0.10% that this Methylotrophic genus bacillus is suitable for cultivating saltiness, and discloses this bacterium for the control to plant-bacterial-wilt, anthrax.It is sodium-chlor 0.01 ~ 0.10% that this patent application shows that this Methylotrophic genus bacillus is suitable for cultivating saltiness, illustrates that in Methylotrophic genus bacillus suitable growth environment, saltiness is low, and does not disclose it in prior art and have and produce PHAs function.
Summary of the invention
For solving the deficiencies in the prior art, the invention provides and a kind ofly use microorganism with anaerobic fermentation of kitchen waste liquid for substrate produces the method for polyhydroxyalkanoate, the method is by the impact of high salt concentration in anaerobic fermentation of kitchen waste liquid, and microorganism efficiently can not only can synthesize PHAs at hypersaline environment normal growth.And the inventive method can realize industrially producing polyhydroxyalkanoate without the need to additionally adding carbon source.
Main purpose of the present invention is to provide a kind of with the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, and described method comprises: (1) use lipase and amylase carry out the pre-treatment to anaerobic fermentation of kitchen waste liquid; And (2) carry out inoculating the synthesis that function yeast carries out polyhydroxyalkanoate in pretreated anaerobic fermentation of kitchen waste liquid in described step (1).
Term " changing food waste " refers to food garbage and is in the general name of rubbish.Food garbage refers to the course of processing waste of the diet residuum in restaurant, restaurant, unit dining room etc. and the fruits and vegetables, meat, grease, millet cake etc. in rear kitchen.Rubbish from cooking refers to the biodegradable organic waste such as fruits and vegetables and garbage material, leftovers leftovers, melon and fruit peel abandoned in family daily life.
Term " anaerobically fermenting " refers to that refuse is under anaerobic stabilized by the Metabolic activity of microorganism, simultaneously with methane and CO
2produce.Anaerobic fermented liquid refers to the remaining liq that organic waste produces after anaerobically fermenting.
Term " anaerobic fermentation of kitchen waste liquid " refers to the waste water produced after changing food waste carries out anaerobically fermenting process.
Term " polyhydroxyalkanoate " (PHAs) is the NEW PLASTIC polyester that a class has many premium propertiess such as biodegradability, biocompatibility, piezoelectricity, can be used as the ideal substitute of chemosynthesis plastics, be with a wide range of applications in various fields such as biomedicine, pharmacy, electronics.PHAs is born of the same parents' self-energy of synthesizing under nutritional imbalance condition of microorganism and the shelf stability material of carbon source, and be extensively present in bacterial body, its molecular weight mostly is 50,000 to 1, and 000,000 dalton (Da), monomer whose is all R configuration.
As one embodiment of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, in described step (1), pretreatment condition is: pH6.0 ~ 7.0, temperature 35 DEG C, lipase concentration 0.1g/L, Acarbose concentrations 0.01g/L.
As a kind of preferred implementation of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, the pre-treatment of described changing food waste fermented liquid refers to and the pH value of fermented liquid is adjusted to 6.0 ~ 7.0, be heated to 35 DEG C and after preheating 20min, add lipase by 0.1g/L and stir 30min; Add amylase by 0.01g/L again and stir 30min, completing the pre-treatment of fermented liquid.
As one embodiment of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, in described step (2), function yeast comprises Methylotrophic genus bacillus (Bacillusmethylotrophicus), extends Halomonas (Halomonaselongata), Alcaligenes faecalis (Alcaligenesfaecalissubspfaecalis), Alcaligenes (Alcaligenesaquatilis).
Methylotrophic genus bacillus, elongation Halomonas, Alcaligenes faecalis, Alcaligenes all can business obtain.
As one embodiment of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, in described step (2), biosynthesizing polyhydroxyalkanoate condition is temperature 25 ~ 45 DEG C, pH7.0 ~ 7.5 condition bottom fermentation cultivates 72 ~ 144h.
As one embodiment of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, in described step (2), biosynthesizing polyhydroxyalkanoate fermentation condition is anaerobism, aerobic or natural ventilation.
As a kind of preferred implementation of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, in described step (2), biosynthesizing polyhydroxyalkanoate condition is: use Methylotrophic genus bacillus to cultivate 108h as function yeast at temperature 35 DEG C, pH7.0 ~ 7.5 condition bottom fermentation.
As a kind of preferred implementation of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, in described step (2), biosynthesizing polyhydroxyalkanoate condition is: use and extend Halomonas and cultivate 72h as function yeast at temperature 35 DEG C, good oxygen condition pH7.0 ~ 7.5 condition bottom fermentation.
As one embodiment of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, described method also comprises the step of the polyhydroxyalkanoate of synthesis in described step (2) being carried out to purifying: fermented liquid is centrifugal, dry to centrifugal pellet frozen, add chloroform broken wall, insulation is filtered, and filtrate is dried, and obtains the polyhydroxyalkanoate of purifying.
As a kind of preferred implementation of the present invention, of the present invention with in the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, the step that the described polyhydroxyalkanoate to synthesis in described step (2) carries out purifying comprises: by gained fermented liquid centrifugal 5 ~ 15min under 5000 ~ 10000rpm, by throw out lyophilize, then chloroform broken wall is added, chloroform addition is that every 0.12g freeze drying cell adds 10 ~ 15ml chloroform, and 12 ~ 36h is incubated at 55 ~ 65 DEG C, filter, gained filtrate is dried naturally, obtains PHAs of the present invention.
As one embodiment of the present invention, the pH value described in aforesaid method is regulated and can be controlled by interpolation dilute sulphuric acid or dilute hydrochloric acid.
As one embodiment of the present invention, the temperature described in aforesaid method is by controlling as the well heater in reactor and temperature regulator.
Preparation method of the present invention has following beneficial effect: (1) method of the present invention with anaerobic fermentation of kitchen waste process after the fermented liquid that produces be raw material, belong to utilization of waste material, low raw-material cost, greatly reduce the production cost of PHAs.(2) method of the present invention is under anaerobic fermentation of kitchen waste liquid saltiness high (generally up to 1.0-1.5%) is unfavorable for the condition of function yeast normal growth and production, still can synthesize PHAs efficiently, realize organism in changing food waste fermented liquid to reclaim and decrement, reduce the subsequent disposal difficulty of fermented liquid, there is obvious environmental benefit and economic benefit.(3) method of the present invention is without the need to additionally adding carbon source, can realize industry produces PHAs.
Accompanying drawing explanation
Fig. 1 is the reacting flow chart of most preferred embodiment of the present invention.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1 Methylotrophic genus bacillus (Bacillusmethylotrophicus) is with anaerobic fermentation of kitchen waste liquid synthesis PHAs
1.1 anaerobic fermentation of kitchen waste liquid pre-treatment
The pH value of fermented liquid being regulated uses sulfuric acid to be adjusted to 6.0 ~ 7.0, uses heater heats to 35 DEG C and after preheating 20min, adds lipase and stir 30min by 0.1g/L; Add amylase by 0.01g/L again and stir 30min, completing the pre-treatment of fermented liquid.
1.2 inoculate with Methylotrophic genus bacillus the synthesis that pretreated anaerobic fermentation of kitchen waste liquid carries out PHA
The present embodiment for function yeast with Methylotrophic genus bacillus, regulates the condition of building-up process to carry out the synthesis of PHA, temperature range is 25 ~ 45 DEG C, pH is that 7.0 ~ 7.5 condition bottom fermentations cultivate 72 ~ 144h.Biosynthetic process can be the state of anaerobism, the aerobic or obstructed oxygen of reaction system.PHA concentration in differential responses condition and reaction secondary fermentation liquid is as shown in table 1.
Table 1 Methylotrophic genus bacillus carries out the PHAs concentration of condition and the production of fermenting
| Test number | A: time (h) | B: temperature (DEG C) | C: aeration condition | PHA(g/L) |
| 1 | 72 | 35 | -1 | 0.234 |
| 2 | 144 | 25 | 0 | 0.128 |
| 3 | 108 | 45 | -1 | 0.374 |
| 4 | 108 | 35 | 0 | 0.950 |
| 5 | 72 | 45 | 0 | 0.192 |
| 6 | 108 | 25 | 1 | 0.296 |
| 7 | 144 | 35 | 1 | 0.634 |
| 8 | 108 | 35 | 0 | 0.926 |
| 9 | 108 | 45 | 1 | 0.396 |
| 10 | 108 | 35 | 0 | 0.938 |
| 11 | 72 | 35 | 1 | 0.464 |
| 12 | 72 | 25 | 0 | 0.150 |
| 13 | 108 | 25 | -1 | 0.304 |
| 14 | 108 | 35 | 0 | 0.898 |
| 15 | 144 | 35 | -1 | 0.502 |
| 16 | 108 | 35 | 0 | 0.938 |
Wherein, aeration condition 0 is natural ventilation, and aeration condition 1 as well oxygen condition, aeration condition-1 is anaerobic state.
From upper table result, use Methylotrophic genus bacillus efficiently can synthesize PHAs in preparation method of the present invention, on the fermentation condition of optimum, productive rate can reach nearly 1g/L, has the practicality in industry.
Embodiment 2 extends Halomonas (Halomonaselongata) with anaerobic fermentation of kitchen waste liquid synthesis synthesis PHB
1.1 anaerobic fermentation of kitchen waste liquid pre-treatment
The pH value of fermented liquid being regulated uses sulfuric acid to be adjusted to 6.0 ~ 7.0, uses heater heats to 35 DEG C and after preheating 20min, adds lipase and stir 30min by 0.1g/L; Add amylase by 0.01g/L again and stir 30min, completing the pre-treatment of fermented liquid.
1.2 inoculate to extend Halomonas the synthesis that pretreated anaerobic fermentation of kitchen waste liquid carries out PHB
The present embodiment, to extend Halomonas for function yeast, regulates the condition of building-up process to carry out the synthesis of PHB, temperature range is 25 ~ 45 DEG C, pH is 7.0 ~ 7.5 condition bottom fermentations cultivation 72 ~ 144h.Biosynthetic process can be the state of anaerobism, the aerobic or obstructed oxygen of reaction system.PHB concentration in differential responses condition and reaction secondary fermentation liquid is as shown in table 2.
Table 2 extends the PHB concentration of condition that Halomonas carries out fermenting and production
| Test number | A: time (h) | B: temperature (DEG C) | C: aeration condition | PHB(g) |
| 1 | 72 | 35 | -1 | 0.084 |
| 2 | 108 | 35 | 0 | 0.06 |
| 3 | 108 | 35 | 0 | 0.052 |
| 4 | 108 | 35 | 0 | 0.058 |
| 5 | 72 | 35 | 1 | 0.114 |
| 6 | 72 | 25 | 0 | 0.062 |
| 7 | 108 | 35 | 0 | 0.054 |
| 8 | 72 | 35 | 1 | 0.135 |
| 9 | 108 | 35 | 0 | 0.058 |
Wherein, aeration condition 0 is natural ventilation, and aeration condition 1 as well oxygen condition, aeration condition-1 is anaerobic state.
From upper table result, use elongation Halomonas efficiently can synthesize PHB in preparation method of the present invention, on the fermentation condition of optimum, productive rate can reach nearly 0.114g/L, has the practicality in industry.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1., with the method for anaerobic fermentation of kitchen waste liquid for substrate synthesizing polyhydroxyalkanoateby, described method comprises:
(1) use lipase and amylase carry out the pre-treatment to anaerobic fermentation of kitchen waste liquid; And
(2) carry out inoculating the synthesis that function yeast carries out polyhydroxyalkanoate in pretreated anaerobic fermentation of kitchen waste liquid in described step (1).
2. method according to claim 1, wherein, in described step (1), pretreatment condition is: pH6.0 ~ 7.0, temperature 35 DEG C, lipase concentration 0.1g/L, Acarbose concentrations 0.01g/L.
3. method according to claim 1, wherein, in described step (2), function yeast comprises Methylotrophic genus bacillus (Bacillusmethylotrophicus), extends Halomonas (Halomonaselongata), Alcaligenes faecalis (Alcaligenesfaecalissubspfaecalis), Alcaligenes (Alcaligenesaquatilis).
4. method according to claim 1, wherein, in described step (2), biosynthesizing polyhydroxyalkanoate condition is temperature 25 ~ 45 DEG C, pH7.0 ~ 7.5 condition bottom fermentation cultivation 72 ~ 144h.
5. method according to claim 1, wherein, in described step (2), biosynthesizing polyhydroxyalkanoate fermentation condition is anaerobism, aerobic or natural ventilation.
6. method according to claim 1, wherein, in described step (2), biosynthesizing polyhydroxyalkanoate condition is: use Methylotrophic genus bacillus to cultivate 108h as function yeast at temperature 35 DEG C, natural ventilation, pH7.0 ~ 7.5 condition bottom fermentation.
7. method according to claim 1, wherein, in described step (2), biosynthesizing polyhydroxyalkanoate condition is: use and extend Halomonas as function yeast at temperature 35 DEG C, good oxygen condition pH7.0 ~ 7.5 condition bottom fermentation cultivation 72h.
8. method according to claim 1, wherein, described method also comprises the step of the polyhydroxyalkanoate of synthesis in described step (2) being carried out to purifying: fermented liquid is centrifugal, dry to centrifugal pellet frozen, add chloroform broken wall, insulation is filtered, and filtrate is dried, and obtains the polyhydroxyalkanoate of purifying.
9. method according to claim 8, wherein, described purification step comprises: by gained fermented liquid centrifugal 5 ~ 15min under 5000 ~ 10000rpm, by throw out lyophilize, then add chloroform broken wall, chloroform addition is that every 0.12g freeze drying cell adds 10 ~ 15ml chloroform, and 12 ~ 36h is incubated at 55 ~ 65 DEG C, filter, gained filtrate is dried naturally, obtains PHAs of the present invention.
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Cited By (2)
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| CN111705010A (en) * | 2020-02-19 | 2020-09-25 | 东北林业大学 | Application of halomonas in synthesis of biodegradable mulching film by using straws and kitchen waste grease |
| CN114921503A (en) * | 2022-06-22 | 2022-08-19 | 北京化工大学 | Method for synthesizing polyhydroxyalkanoate and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111705010A (en) * | 2020-02-19 | 2020-09-25 | 东北林业大学 | Application of halomonas in synthesis of biodegradable mulching film by using straws and kitchen waste grease |
| CN111705010B (en) * | 2020-02-19 | 2024-02-09 | 东北林业大学 | Application of halomonas in synthesizing biodegradable mulching film by utilizing straw and kitchen waste grease |
| CN114921503A (en) * | 2022-06-22 | 2022-08-19 | 北京化工大学 | Method for synthesizing polyhydroxyalkanoate and application thereof |
| CN114921503B (en) * | 2022-06-22 | 2023-10-20 | 北京化工大学 | Method for synthesizing polyhydroxyalkanoate and application thereof |
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