CN101886040A - Method for preparing hydrogen-producing and ethanol-producing microbial aggregate - Google Patents
Method for preparing hydrogen-producing and ethanol-producing microbial aggregate Download PDFInfo
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- CN101886040A CN101886040A CN2010102037820A CN201010203782A CN101886040A CN 101886040 A CN101886040 A CN 101886040A CN 2010102037820 A CN2010102037820 A CN 2010102037820A CN 201010203782 A CN201010203782 A CN 201010203782A CN 101886040 A CN101886040 A CN 101886040A
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Abstract
The invention relates to a method for preparing a hydrogen-producing and ethanol-producing microbial aggregate, which comprises the following steps of: preprocessing inoculated anaerobic sludge, and introducing synthetic organic waste water; and when the degradation rate of glucose in outflow water of a UASB reactor reaches over 95 percent, gradually increasing the concentration of an organic matter in inflow water and keeping the pH value of the outflow water to be between 4 and 4.5. In the invention, starting with the realization of a synchronous hydrogen-producing and ethanol-producing technique of the organic waste water, the diversity of microbes in the mature hydrogen-producing and ethanol-producing microbial aggregate is analyzed; a clone technique and a sequencing technique are combined for studying the functional group of the microbe and analyzing to obtain a population structure of the optimal hydrogen-producing and ethanol-producing microbes, so as to provide theory and technique guide for artificially constructing an anaerobic, high-efficiency biological hydrogen production system along with stable operation and accelerate the industrialization of organic waste water resource utilization.
Description
Technical field
The present invention relates to a kind of preparation method of microbial aggregate, be specifically related to a kind of preparation method of hydrogen-producing and ethanol-producing microbial aggregate.
Background technology
The raw material of producing at present bio-ethanol extensively adopts edible material from soybeans such as the sugar of sugarcane, corn and starch, and supply is limited and use cost is high and limited its application.The generation of hydrogen mainly is by chemical process, need to consume lot of energy and may cause environmental pollution, also is faced with how effectively to reduce cost and reduce pollution problem to environment.Treatment of Organic Wastewater adopts biological process to handle more, and traditional organic waste water biologic treating technique (anaerobism technology, aerobic technology) focuses on and organism is decomposed into carbonic acid gas and water and fails to realize its recycling.
Containing a large amount of carbohydrate in many organic waste waters such as carbohydrate factory effluent, starch production wastewater and the drinks factory effluent etc., is that microbial fermentation produces hydrogen and the desirable substrate of alcoholic acid.The research of microorganism hydrogen producing technology and production of fuel ethanol aspect is increasing, but is substrate with organic waste water, transforms and pathways metabolism control is produced the research of hydrogen and alcoholic acid synchronously and but rarely had report by specified microorganisms flora such as aggregate.In various hydrogen producing technologies, biological hydrogen production can carry out at normal temperatures and pressures because of its action condition gentleness, can utilize the reproducible energy, also can carry out the utilization of refuse, and it is low and pollute characteristics such as few and receive publicity (Nath and Das, 2004) to have energy consumption.Bio-hydrogen production technology mainly contains photosynthetic organism hydrogen generation and fermenting organism hydrogen manufacturing (Das andVeziroglu, 2001).The former has can utilize sun power, substrate conversion efficiency advantages of higher, but have that hydrogen-producing speed is slow, light energy conversion efficiency is low, shortcoming such as photoreactor design complexity and limited its practical application.Compare with the photosynthetic organism hydrogen generation technology, fermenting organism hydrogen manufacturing has hydrogen-producing speed soon, does not need luminous energy, reactor operation convenient and can utilize advantage such as multiple waste, thereby practicality is stronger, more possesses industrialization prospect (Das and Veziroglu, 2001; Nathand Das, 2004; Magnusson et al., 2009).2005, " organic waste water fermentation method bio-hydrogen production technology productivity demonstration project " started in International Technology city, Harbin, and a day hydrogen producing reaches 1200m
3, but present fermenting organism hydrogen manufacturing cost still higher (Das and Veziroglu, 2009).Be faced with a difficult problem that how effectively to reduce cost, improve hydrogen generation efficiency in this technology industrialization process, and this and hydrogen production by biological metabolic process are closely related.
Doing raw material product hydrogen with glucose is example, produces hydrogen by formic acid cracked approach, and the microorganism that produces hydrogen with this approach has Escherichia coli and Enterobacter sp..The suitableeest product hydrogen pH is about 7.0 (Shin et al., 2007).This shows that glucose finally is converted into increase, hydrogen, carbonic acid gas and the energy of organic acid, biomass by the fermentation of microorganism.The essence of various product hydrogen type difference is that control and the seed sludge microbial species group structure to fermentation factor is different in the fermenting process.The main factor of having reported that influences fermentation and hydrogen production has: and microbe inoculation, concentration of substrate and type, reactor structure, hydraulic detention time (hydraulic retention time, HRT), organic loading, C/N, C/P, redox potential, pH and temperature etc.These factors are optimized and control efficient (Yu and Mu, 2006 that can significantly improve fermenting organism hydrogen manufacturing; Wang and Wan W, 2009).The research that the product hydrogen condition that employing experimental design and statistical method ferment to the butyric acid type is optimized and controls is more, but the selection that great majority do not solve optimal condition between the multiple response, and the multifarious variation and the microbial function group that fail to disclose microbial population under the different condition.(infrared spectra IR) waits and can study (Koskinen et al., 2007) to population structure, diversity and the pathways metabolism of microorganism well for Modern Molecular Biotechnology (clone library), biochemical technology (Biolog method) and spectroscopic techniques.For the fermentation of butyric acid type, utilizing the synchronous hydrogen-producing and ethanol-producing of organic waste water to have bigger advantage is the handling problem again that does not have liquid product, and practicality is stronger.On the other hand, the key of efficient anaerobe hydrogen producing technology is how hydrogen dominant bacteria (Cuetos et al., 2007 are produced in enrichment; Ren et al., 2007).Its seed sludge mostly is mixing microorganisms colony, and existing fermenting bacteria, hydrogen-producing bacteria also have hydrogen consumption bacterium such as methanogen.If can not effectively suppress the activity of hydrogen consumption bacterium, the operation of anaerobism hydrogen generating system will be failed.Therefore need seed sludge is carried out effective pre-treatment, main method has heating, acid treatment, alkaline purification, oxygenation, organic solvent processing, freeze thawing treatment, ultrasonication etc. at present.The efficient of its anaerobe hydrogen manufacturing of various pre-treating processs is also different.Cheng and Hansen (2006) have compared the pre-treatment mode of 5 kinds of anaerobic sludges, think the acid treatment best results.But studies show that of Hu and Cheng adopts organic solvent such as chloroform to handle the activity that can suppress hydrogen consumption bacterium in the anaerobic sludge most effectively.Wang and Wei (2008) have also studied the quality of the pre-treating process of anaerobic sludge in 5, and the result shows that heating treatment method is best, and simple and practical.But heat treated makes the diversity of microorganism send out rapid variation, produces the hydrogen type and is easy to transform to the butyric acid type.And some relatively mild processing modes form a microbial species group structure (Lee et al., 2009) that is beneficial to product hydrogen as keeping by change pH in the anaerobic sludge.This shows, in the different research best anaerobic sludge pre-treatment also different, its reason is that different processing mode is different to the influence of microbial diversity and population structure.Be necessary different pre-treating processs is optimized design, optimize the pre-treating process that is beneficial to synchronous hydrogen-producing and ethanol-producing.
On the other hand, one of organic waste water anaerobe hydrogen producing technology prerequisite of moving towards industrialization is exactly that the generation of hydrogen needs continuously and stablize.Present stage, the high efficiency reactor of Continuous Flow fermenting organism hydrogen manufacturing mainly contains Continuous Flow agitator tank reactor (continuous stirred tank reactor, CSTR) and upflow anaerobic sludge blanket reactor (upflow anaerobic sludge blanket, UASB).Can form the microbial aggregate that settling property is good, methanogenesis activity is high among the UASB, guarantee that the anaerobic biological process efficiently, stably carry out, is even more ideal anaerobe hydrogen-manufacturing reactor (Wang and Wan, 2009).Also can form the good product hydrogen microbial aggregate of settling property in UASB, reactor can steady running (Yuand Mu, 2006) for 3 years.In like manner, realize the continual and steady operation of the synchronous hydrogen-producing and ethanol-producing UASB of organic waste water reactor, best mode is the product hydrogen microbial aggregate that obtains this fermented type fast, but the research of this respect but rarely has report.
Summary of the invention
At above the deficiencies in the prior art, the invention provides a kind of method of hydrogen-producing and ethanol-producing microbial aggregate of quick acquisition ethanol-type fermentation type.
The present invention is achieved by the following technical solutions:
A kind of preparation method of hydrogen-producing and ethanol-producing microbial aggregate, described method comprises the steps:
(1) pre-treatment of inoculation anaerobic sludge: the anaerobic sludge of getting inoculation, utilizing acid treatment (pH value≤4) to handle the back inserts in the UASB reactor, the VSS that makes mud is 6~7g/L, feed with glucose is the organic waste water of carbon source again, water inlet COD=4.5~5.5g/L when UASB starts, pH is 7.0, HRT=30h, 34.5~35.5 ℃ of temperature.
(2) the glucose degradation rate in the water outlet of UASB reactor reaches more than 95%, progressively improves organic concentration in the water inlet, by add a certain amount of NaHCO in water inlet
3Be stabilized in 4~4.5 to regulate water outlet pH, the COD of finally intaking reaches 9.5~10.5g/L, and HRT shortens to 18h.
In step (1), except that carbon source, also comprise following composition (mg/L): NH in the organic waste water
4HCO
3Be 2024; K
2HPO
4Be 800; CaCl
2Be 50; MgCl
2Be 100; FeCl
2Be 25; NaCl is 10; CoCl
2Be 5; MnCl
2Be 5; AlCl
3Be 2.5; (NH
4)
6Mo
7O
24Be 15; H
3BO
4Be 5; NiCl
2 Be 5; CuCl
2Be 5; ZnCl
2Be 5.
In step (1), use HCL to carry out acid treatment.
In step (1), the VSS of mud is 6.5g/L.
In step (1), COD=5g/L when UASB starts.
In step (2), the COD=10g/L of finally intaking.
Beneficial effect of the present invention is: the present invention starts with from realizing the synchronous hydrogen-producing and ethanol-producing technology of organic waste water, carries out at seed sludge on the basis of pre-treatment microbial diversity being analyzed; In conjunction with clone and sequencing technologies the microbial function group is studied, analyze the population structure of best hydrogen-producing and ethanol-producing microbial.By the method for batch experiment, the processing condition of synchronous hydrogen-producing and ethanol-producing are optimized, to be used for the control of UASB reactor start-up and operational conditions.
In the UASB reactor, control and optimization (pH by operational condition, HRT, concentration of substrate etc.), induce the formation of synchronous hydrogen-producing and ethanol-producing microbial aggregate, analyze the variation of microbial species group structure in the aggregate forming process, pathways metabolism, and its physico-chemical property carried out systematic study, obtain the top condition that aggregate formed and stablized hydrogen-producing and ethanol-producing, obtain the mechanism that synchronous hydrogen-producing and ethanol-producing microbial aggregate forms, solve have liquid product in the traditional biological hydrogen producing technology utilize a difficult problem again, the biological hydrogen production system that moves for artificial constructed anaerobism efficient stable provides theory and technology to instruct, and accelerates the industrialization process of organic waste water recycling.
Description of drawings
Fig. 1 is the hydrogen-producing and ethanol-producing microbial aggregate light micrograph;
Fig. 2 is the phylogenetic tree based on the dominant population of 16S rRNA sequence;
Fig. 3 assembles stereoscan photograph for hydrogen-producing and ethanol-producing microbial.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further details.
1, the pre-treatment of seed sludge
Get mud in the anaerobic reactor of treatment of Citric Acid Industrial Wastewater, fully grind after for some time, cross behind 60 mesh sieves standby through laboratory culture.The UASB reactor adopts synthetic glass to make.Working volume is 3L, reaction zone 2.2L wherein, and settling region 0.8L, triphase separator partial volume are 2.6L; Get the standby mud of certain volume and handle 24h through 1M HCl, pH=3.5 handles the back and inserts in the UASB reactor, and the VSS (volatile solid in the active sludge) of final mud is about 6~7g/L, and optimum value is 6.5g/L.Synthetic waste water is adopted in water inlet, and carbon source is a glucose, and the COD (chemical oxygen demand (COD)) when UASB starts is 4.5~5.5g/L, and optimum value is 5.0g/L, and other composition is (mg/L): NH
4HCO
32024; K
2HPO
4800; CaCl
250; MgCl
2100; FeCl
225; NaCl 10; CoCl
25; MnCl
25; AlCl
32.5; (NH
4)
6Mo
7O
2415; H
3BO
45; NiCl
25; CuCl
25; ZnCl
25, water inlet pH is 7.0, HRT (hydraulic detention time)=30h, 35 ± 0.5 ℃ of temperature.
2, the formation of microbial aggregate
Glucose degradation rate in the water outlet of UASB reactor reaches more than 95%, progressively improves organic concentration in the water inlet, by add a certain amount of NaHCO in water inlet
3To regulate the water outlet pH in the UASB reactor, maintain 4~4.5.Final water inlet COD=9.5~10.5g/L, optimum value is 10.0g/L, HRT progressively shortens to 18h.The qualitative and quantitative analysis of the biomass of mud, dissolved organic matter in residual sugar amount and COD or TOC, the reactor in the content of regular sampling analysis hydrogen, the water outlet; The forming process of microbial aggregate in the observing response device regularly adopts the optical microphotograph imaging to analyze continuously.
3, microbial aggregate analysis of physical and chemical property
The settling property, hydrophobicity, perviousness, Zeta potential, the extracellular polymer substrate concentration that divide division hydrogen aggregate.Analysis by physico-chemical property, research is in the stability of producing aggregate under the hydrogen condition change, the result shows that the hydrogen-producing and ethanol-producing microbial aggregate structure is closely knit, the profile rule, as shown in Figure 1, aggregate forms the hydrophobicity increase that cell shows, extracellular polymeric is abundant, reactor is stable, and OLR is 12kg (m
3D)
-1With 16.8kg (m
3D)
-1Under the condition, it is maximum respectively that hydrogen-producing speed and alcohol concn reach, and is 2.89m
3(m
3D)
-1And 2840mgL
-1
4, produce the monitoring that the hydrogen biological community structure changes:
(1) sample collection
Regularly from the UASB reactor, collect mud, standby in-20 ℃ of preservations.When the growth for the treatment of hydrogen-generating granular sludge in the UASB reactor reaches stationary phase, all mud samples of collecting are thawed (comprising the seed sludge sample), the sterilized water washing, disperse the back to be used for the extraction of DNA.
(2) extraction and purification of DNA
With in the damping fluid of above-mentioned a certain amount of mud sample adding certain volume (containing Proteinase K), after the vibration, add 20% SDS, 65 ℃ of temperature are bathed 2h.Add isopyknic chloroform/primary isoamyl alcohol mixing, centrifugal collection supernatant liquor; Add isopyknic phenol/chloroform/primary isoamyl alcohol mixing, centrifugal, supernatant liquor is changed in the another centrifuge tube, add 0.6 times of volume Virahol, precipitate, abandon supernatant liquor, 70% ice ethanol cleans twice, aseptic wind is done, and obtains the crude extract of total DNA with TE damping fluid dissolution precipitation, places-20 ℃ of preservations.Adopt DNA glue to reclaim test kit the genomic dna crude extract is carried out purifying.
(3) pcr amplification of genomic dna
Amplimer: the 16S rDNA gene V3 district with most of bacteriums and Archimycetes has specific primer to F357 and R518.PCR reaction system: the template of 2 μ L, the forward and reverse primer of 2.5 μ L, 10 * PCR buffer of 1 μ LdNTP, 5 μ L, the MgCl of 1 μ L
2, 0.5 μ LTaqDNA polysaccharase and 34.5 μ L distilled waters.The pre-sex change 5min of PCR reaction conditions: 94oC, preceding 20 circulations are 94 ℃ of 1min, 65~55 ℃ of 1min and 72 ℃ of 3min (wherein each circulation back renaturation temperature descends 0.5 ℃), 10 circulations in back are 94 ℃ of 1min, 55 ℃ of 1min and 72 ℃ of 3min extend 7min, 4 ℃ of preservations down at 72 ℃ at last.The product of PCR reaction detects with 2% agarose gel electrophoresis.
(4) DG-DGGE electrophoresis
Adopt gradient mixing device, the polyacrylamide gel of preparation 10%, denaturing agent concentration from 35% to 55%.Offset plate is put into the electrophoresis chamber that electrophoretic buffer is housed, add pcr amplification sample 5 μ L.The electrophoresis poststaining carries out glue figure scanning, obtains the finger printing of DG-DGGE.Collection of illustrative plates adopts related software assessing with the population relative populations microbial diversity in the sample.
(5) clone and order-checking
The main band of choosing on the DG-DGGE collection of illustrative plates increases again, method is same as above, with PCR product cloning test kit amplified production is connected with carrier, connect product by the heat shock method, be transformed in the competent intestinal bacteria, select positive colony to check order, institute's calling sequence is sent to the GenBank database after treatment, adopt BLAST that contained sequence in target sequence and the gene pool is analyzed, obtain the nearest sequence of homology.Adopt DNAMAN (V5.2.2 version) to set up the phyletic evolution tree at last.
The result shows: UASB operational process microflora diversity constantly changes, and anaerobic hydrogen-generating granular sludge forms gradually, and its structure is closely knit, and microorganism is formed based on bacillus, is the key of anti-organic loading impact of UASB reactor and highly effective hydrogen yield.The interior microbial diversity of system for producing hydrogen keeps stable during stationary phase, indicates the formation of ripe hydrogen-producing and ethanol-producing microbial aggregate.Main microbial population has Clostridium sp.HPB-16 in phyletic evolution tree analysis (as shown in Figure 2) the back announcement hydrogen-producing and ethanol-producing aggregate, Clostridium sp.HPB-46, Clostridium sp.HPB-2, Clostridium sp.HPB-4, Oxalobacteraceae bacterium QD1, with can not culturing bacterium (Uncultured bacterium clone HPR93, UnculturedOlsenella sp.clone J27 and Uncultured bacterium clone SR_FBR_E5) form.
The stereoscan photograph of hydrogen-producing and ethanol-producing microbial aggregate shows the middle microorganism of seed sludge (shown in Fig. 3 (a)) based on thread fungus, short texture; Microbial aggregate is then based on bacillus, and structure is closely knit, and duct (shown in Fig. 3 (b, c and d)) clearly arranged, and these ducts help exchange of substance.After microbial aggregate forms, good because of its settling property, can in reactor, keep higher biomass, thereby the stability and the capacity of resisting impact load of UASB fermentation and hydrogen production system are significantly strengthened.
Claims (6)
1. the preparation method of a hydrogen-producing and ethanol-producing microbial aggregate is characterized in that described method comprises the steps:
(1) pre-treatment of inoculation anaerobic sludge: the anaerobic sludge of getting inoculation, make pH value≤4 after utilizing acid treatment, insert in the UASB reactor, the VSS that makes mud is 6~7g/L, feeding with glucose is the organic waste water of carbon source again, COD=4.5~5.5g/L when UASB starts, and water inlet pH is 7.0, HRT=30h, 34.5~35.5 ℃ of temperature.
(2) the glucose degradation rate in the water outlet of UASB reactor reaches more than 95%, progressively improves organic concentration in the water inlet, by add a certain amount of NaHCO in water inlet
3To regulate water outlet pH is 4~4.5, and guarantees final water inlet COD=9.5~10.5g/L, and HRT shortens to 18h.
2. preparation method according to claim 1 is characterized in that in step (1),, also can comprise following composition (mg/L): NH in the organic waste water
4HCO
3Be 2024; K
2HPO
4Be 800; CaCl
2Be 50; MgCl
2Be 100; FeCl
2Be 25; NaCl is 10; CoCl
2Be 5; MnCl
2Be 5; AlCl
3Be 2.5; (NH
4)
6Mo
7O
24Be 15; H
3BO
4Be 5; NiCl
2Be 5; CuCl
2Be 5; ZnCl
2Be 5.
3. preparation method according to claim 1 is characterized in that in step (1), uses HCL to carry out acid treatment.
4. preparation method according to claim 1 is characterized in that in step (1), the VSS of mud is 6.5g/L.
5. preparation method according to claim 1 is characterized in that in step (1), COD=5g/L when UASB starts.
6. preparation method according to claim 1 is characterized in that in step (2) COD=10g/L of finally intaking.
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CN102286379A (en) * | 2011-07-28 | 2011-12-21 | 中山大学 | Method for extracting pannonibacter phragmitetus from sludge |
CN102747106A (en) * | 2012-07-02 | 2012-10-24 | 同济大学 | Method for increasing protein bio-hydrogen production |
FR3000965A1 (en) * | 2013-01-14 | 2014-07-18 | Inst Nat De Recherche En Sciences Et Technologies Pour L'environnement Et L'agriculture Irstea | PROCESS FOR PRODUCING ETHANOL FROM ORGANIC WASTE AND INSTALLATION FOR CARRYING OUT SAID METHOD |
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CN1928099A (en) * | 2006-09-05 | 2007-03-14 | 江南大学 | Method of transforming abandoned biomass to acetic acid by two-phase coupling process of hydrogen-producing acid-producing and hydrogen-consuming hydrogen-producing |
CN101492696A (en) * | 2008-01-26 | 2009-07-29 | 聂艳秋 | High-efficiency method for producing hydrogen gas and methyl hydride with mix fermentation of sewage sludge and garbage |
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CN1928099A (en) * | 2006-09-05 | 2007-03-14 | 江南大学 | Method of transforming abandoned biomass to acetic acid by two-phase coupling process of hydrogen-producing acid-producing and hydrogen-consuming hydrogen-producing |
CN101492696A (en) * | 2008-01-26 | 2009-07-29 | 聂艳秋 | High-efficiency method for producing hydrogen gas and methyl hydride with mix fermentation of sewage sludge and garbage |
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《太阳能学报》 20100430 秦智等 生物制氢反应器产氢产乙酸菌群对挥发酸的转化 407-411 1-6 第31卷, 第4期 2 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102286379A (en) * | 2011-07-28 | 2011-12-21 | 中山大学 | Method for extracting pannonibacter phragmitetus from sludge |
CN102286379B (en) * | 2011-07-28 | 2014-05-28 | 中山大学 | Method for extracting pannonibacter phragmitetus from sludge |
CN102747106A (en) * | 2012-07-02 | 2012-10-24 | 同济大学 | Method for increasing protein bio-hydrogen production |
FR3000965A1 (en) * | 2013-01-14 | 2014-07-18 | Inst Nat De Recherche En Sciences Et Technologies Pour L'environnement Et L'agriculture Irstea | PROCESS FOR PRODUCING ETHANOL FROM ORGANIC WASTE AND INSTALLATION FOR CARRYING OUT SAID METHOD |
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