CN101886040B - 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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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 sugar and the starch of sugarcane, corn, and supply is limited and use cost is high and limited its application.The generation of hydrogen mainly is through chemical process, need to consume lot of energy and possibly cause environmental pollution, also is faced with how effectively to reduce cost and reduce the 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 that organism is decomposed into carbonic acid gas fails realization to its recycling with water.
Containing a large amount of glucide 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 mikrobe 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 hydrogen synchronously and but rarely had report with alcoholic acid research through specified microorganisms flora such as aggregate.In various hydrogen producing technologies, biological hydrogen production is gentle because of its action condition, can carry out at normal temperatures and pressures, 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 has shortcomings such as hydrogen-producing speed is slow, light energy conversion efficiency is low, photoreactor complex design and limited its practical application.With the photosynthetic organism hydrogen generation compared with techniques; Fermenting organism hydrogen manufacturing has hydrogen-producing speed soon, does not need luminous energy, reactor drum 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 " the International Technology city started in 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 through formic acid cracked approach, and the mikrobe that produces hydrogen with this approach has Escherichia coli and Enterobacter sp..The righttest 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, living weight through the fermentation of mikrobe.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 crowd that fail to disclose microbial population under the different condition.(ir spectra IR) waits and can study (Koskinen et al., 2007) to population structure, variety and the pathways metabolism of mikrobe 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).Mostly its seed sludge is mixing microorganisms colony, and existing fermenting bacterial, 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, s.t., 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 s.t. best results.But showing, the research of Hu and Cheng adopt 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 variety of mikrobe 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 through 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 the different pretreatment method 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 even flow fermenting organism hydrogen manufacturing mainly contains even 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).In UASB, also can form the good product hydrogen microbial aggregate of settling property, reactor drum 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 drum, 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
To the deficiency of above 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 realizes through following technical scheme:
A kind of preparation method of hydrogen-producing and ethanol-producing microbial aggregate, said method comprises the steps:
(1) pre-treatment of inoculation anaerobic sludge: the anaerobic sludge of getting inoculation; Utilize s.t. (pH value≤4) to handle the back and insert in the UASB reactor drum, the VSS that makes mud is 6～7g/L, and feeding 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 drum reaches more than 95%, progressively improves organic concentration in the water inlet, through in water inlet, adding a certain amount of NaHCO
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 s.t..
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 crowd is studied, analyze the population structure of best hydrogen-producing and ethanol-producing microbial.Through 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 drum; Control and optimization (pH, HRT, concentration of substrate etc.) through operational condition; Induce the formation of synchronous hydrogen-producing and ethanol-producing microbial aggregate; Analyze variation, the pathways metabolism of microbial species group structure in the aggregate forming process, and its physico-chemical property is 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, for the biological hydrogen production system of artificial constructed anaerobism efficient stable operation provides theory and technology to instruct, accelerate 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 accompanying drawing and embodiment the present invention is explained 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 subsequent use through laboratory culture.The UASB reactor drum adopts synthetic glass to process.Working volume is 3L, reaction zone 2.2L wherein, and settling region 0.8L, triphase separator partial volume are 2.6L; Get the subsequent use mud of certain volume and handle 24h through 1M HCl, pH=3.5 handles the back and inserts in the UASB reactor drum, 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 (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 drum reaches more than 95%, progressively improves organic concentration in the water inlet, through in water inlet, adding a certain amount of NaHCO
3To regulate the water outlet pH in the UASB reactor drum, 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 living weight of mud, dissolved organic matter in residual sugar amount and COD or TOC, the reactor drum 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 through physico-chemical property; Research is in the stability of producing aggregate under the hydrogen condition change, and 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 drum 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 drum, collect mud, subsequent use in-20 ℃ of preservations.When the growth of treating hydrogen-generating granular sludge in the UASB reactor drum reaches stationary phase, all are collected that good mud sample thaws (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; Add isopyknic phenol/chloroform/primary isoamyl alcohol mixing, centrifugal; Supernatant is changed in the another centrifuge tube, add 0.6 times of volume Virahol, precipitate, abandon supernatant; 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, 1 μ LdNTP, 10 * PCR buffer of 5 μ L, the MgCl of 1 μ L
2, 0.5 μ LTaqDNA polysaccharase and 34.5 μ L distilled waters.The preparatory 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 below 7min at 72 ℃ at last, 4 ℃ of preservations.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, and method is connected amplified production with PCR product cloning test kit with the above with carrier; Connect product through 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 DB after treatment; Adopt BLAST that contained sequence in target sequence and the gene pool is compared analysis, 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 variety constantly changes, and anaerobic hydrogen-generating granular sludge forms gradually, and its structure is closely knit, and it is main that mikrobe is formed with bacillus, is the key of anti-organic loading impact of UASB reactor drum 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 and can not culturing bacterium (Uncultured bacterium clone HPR93, UnculturedOlsenella sp.clone J27 and Uncultured bacterium clone SR_FBR_E5) forming.
The stereoscan photograph of hydrogen-producing and ethanol-producing microbial aggregate shows that mikrobe is main with thread fungus in the seed sludge (shown in Fig. 3 (a)), short texture; Microbial aggregate is main with bacillus then, 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 drum, keep higher living weight, thereby the stability and the capacity of resisting impact load of UASB fermentation and hydrogen production system are significantly strengthened.
Claims (5)
1. the preparation method of a hydrogen-producing and ethanol-producing microbial aggregate is characterized in that said method comprises the steps:
(1) pre-treatment of inoculation anaerobic sludge: the anaerobic sludge of getting inoculation; Described anaerobic sludge is the mud in the anaerobic reactor of treatment of Citric Acid Industrial Wastewater; Make pH value≤4 after utilizing s.t., insert in the UASB reactor drum, the VSS that makes mud is 6～7g/L; Feeding with glucose is the organic waste water of carbon source again, and other composition of said organic waste water is: NH
4HCO
3Be 2024mg/L; K
2HPO
4Be 800mg/L; CaCl
2Be 50mg/L; MgCl
2Be 100mg/L; FeCl
2Be 25mg/L; NaCl is 10mg/L; CoCl
2Be 5mg/L; MnCl
2Be 5mg/L; AlCl
3Be 2.5mg/L; (NH
4)
6Mo
7O
24Be 15mg/L; H
3BO
4Be 5mg/L; NiCl
2Be 5mg/L; CuCl
2Be 5mg/L; ZnCl
2Be 5mg/L; COD=4.5～5.5g/L when UASB starts, 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 drum reaches more than 95%, progressively improves organic concentration in the water inlet, through in water inlet, adding a certain amount of NaHCO
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), uses HCL to carry out s.t..
3. preparation method according to claim 1 is characterized in that in step (1), the VSS of mud is 6.5g/L.
4. preparation method according to claim 1 is characterized in that in step (1), COD=5g/L when UASB starts.
5. preparation method according to claim 1 is characterized in that in step (2) COD=10g/L of finally intaking.
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| 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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| 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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