CN100529093C - Method for producing alcohol and feed by utilizing seaweed chemical waste material - Google Patents
Method for producing alcohol and feed by utilizing seaweed chemical waste material Download PDFInfo
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- CN100529093C CN100529093C CNB2007100142464A CN200710014246A CN100529093C CN 100529093 C CN100529093 C CN 100529093C CN B2007100142464 A CNB2007100142464 A CN B2007100142464A CN 200710014246 A CN200710014246 A CN 200710014246A CN 100529093 C CN100529093 C CN 100529093C
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Abstract
The invention discloses a method for producing alcohol and feed by alga chemical waste, i.e. using the cellulose-containing waste residues as raw materials formed in the course of producing sodium alginate, mannitol, I or trehalose glue products in the alga chemical industry, making enzymatic hydrolysis by cellulase and anaerobic fermentation by brewing yeast or double fermentation by cellulase producing bacteria and brewing yeast, then making solid-liquid separation and obtaining alcohol and feed. And the invention provides economic, environmental protection and effective industrial technique for waste residues and waste liquid in the alga chemical industry, able to turn waste into wealth and provide important technical support for full and high value utilization of alga resources and development of alga circulating economy.
Description
Technical field
The invention belongs to the oceanic resources biological technical field, relate in particular to a kind of method of utilizing seaweed chemical waste material to produce ethanol and protein feed by the bio-transformation of enzyme and microorganism.
Background technology
Marine alga mainly refers to nature or the algae propagated artificially in the ocean such as brown alga (sea-tangle, bulk kelp, sea accumulate, bladder wrack, wakame, Sargassum fusiforme, sargassun, rope algae, laver etc.).China Shandong, Fujian, zhejiang and other places have the base of propagating artificially of different scales.South America and North America be coastal a large amount of wild abundant algae resources such as bulk kelp.
Marine alga is an important chemical material, can be used to produce Chemicals such as sodium alginate, N.F,USP MANNITOL, iodine, fucoidin.China is after 20th century brown alga kind cultural techniques such as the fifties large-scale promotion sea-tangle, having set up to produce iodine, N.F,USP MANNITOL and sodium alginate (practise and claim " old three samples ") around marine algae resource is the seaweed industry system of main products, has increased product innovations such as fucoidin in recent years again.Yet, along with being on the increase of the quantity of utilizing marine alga production chemical product and kind, the quantity of the waste material that forms after the marine alga process chemical process is also increasing, and some area is also disorderly abandoned because of these residue waste materials or is lacked effective treatment process and utilize approach to cause environmental pollution, therefore, the processing of seaweed chemical waste material is to need the urgent problem that solves in the marine resource chemistry manufacture field with comprehensive utilization.
Summary of the invention
An environmental pressure that the waste material that faces at seaweed industry causes and the difficult problem that comprehensive utilization need solve the purpose of this invention is to provide a kind of technology of using enzyme engineering and microbial technique processing seaweed chemical waste material production ethanol and feed.
Technological method of the present invention comprises three kinds of basic modes: a kind of method be earlier with cellulase preparation with the hydrolysis of seaweed industry waste material, insert yeast saccharomyces cerevisiae then and ferment.Second kind is the cascade fermentation method of two kinds of microorganisms.That is, produce cellulase with the cellulase producing bacteria fermentation, the seaweed industry of hydrolysis simultaneously waste residue carries out the S. cervisiae anaerobically fermenting then.The third method is the mixed fermentation of two kinds of microorganisms.That is, cellulase producing bacteria and S. cervisiae are carried out mixed culture earlier, change anaerobically fermenting then over to.
The method of utilizing seaweed chemical waste material to produce ethanol and feed of the present invention, specifically form by following step:
(1) seaweed chemical waste material is selected: described waste material is that marine alga chemical industry is produced the waste residue that contains cellulose components that forms in sodium alginate, N.F,USP MANNITOL, iodine or the marine alga carbohydrate gum product process; Wherein: described marine alga is that sea-tangle, sargassun, bulk kelp, sea accumulate, one of bladder wrack, wakame, Sargassum fusiforme, rope algae, laver, and described waste residue is that sea-tangle, sargassun, bulk kelp, sea accumulate, one of bladder wrack, wakame, Sargassum fusiforme, rope algae, laver waste residue or its arbitrary proportion mix;
(2) selection of zymin: zymin is commercially availablely cellulose degradation can be the monose that can be utilized by yeast saccharomyces cerevisiae and cellulase, the hemicellulase of oligosaccharides;
(3) bacterial classification is selected: select cellulase producing bacteria for use: viride (Trichoderma viride) AF93252, and Wuhan University China typical culture collection center provides, and preserving number is CCTCC NO.AF93252; The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, commercially available;
(4) cellulase liquid fermented bacterium preparation: with the described cellulase producing bacteria bacterial strain of step (3), under aseptic condition, through the test tube slant, shake bottle, first class seed pot, secondary seed jar amplification culture step by step, make the liquid fermenting bacterial classification that the bacterium number reaches OD value 1.0～1.2 * 10;
Wherein:
The described fermention medium that shakes bottle, first class seed pot, the use of secondary seed jar is to contain by weight percentage: Mandel ' the s mineral nutrition liquid of 0.1% peptone, 1.2% carbon source, pH5～6;
Described slant medium is that above-mentioned Mandel ' s mineral nutrition adds 2% agar powder;
Described shake-flask culture condition is 30 ℃～32 ℃ of temperature, pH5～6, shaking speed 180～220r/min, incubation time 48～60 hours; First class seed pot, secondary seed jar culture condition are 28 ℃～32 ℃ of temperature, pH4.5～6, tank pressure 0.5Kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.8～1.0, mixing speed 120～200r/min, aerobic cultivation 48～60 hours;
(5) seaweed chemical waste material enzymatic hydrolysis: getting the described waste residue of step (1), to add water to slag concentration be 20%～35% by weight percentage, regulates pH to 4.5～6 with dilute acid soln; With the weight percent meter of total amount, the commercially available cellulase that adds 10%～30% amount is or/and the hemicellulose zymin, and 28 ℃～32 ℃ reactions 24～26 hours are standby;
(6) produce ethanol with fermentation by saccharomyces cerevisiae: use the yeast minimum medium, 30 ℃～37 ℃ of temperature, pH4～6, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; Set by step (5) preparation hydrolysis seaweed chemical waste material liquid integrating meter, insert its volume percent and be 10%～15% fermentation by saccharomyces cerevisiae bacterium liquid or 0.1%～0.5% commercially available Angel Yeast particle and carry out the anaerobism sealed fermenting, leavening temperature is 28 ℃～32 ℃, and the pH value is 4～6; Fermentation time 48～58 hours reaches volume percent 8% when above when detecting the karusen ethanol concn, and fermentation stops;
Perhaps (7) seaweed chemical waste material fermentation: getting the described waste residue of step (1) and the described fermention medium of step (4) is that 1: 5～8 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 70～80%, again with the weight percent meter of charge amount, add the described liquid fermenting bacterial classification of 10%～15% step (4), with 28 ℃～32 ℃ of culture temperature, pH4.5～6, tank pressure 0.6～0.8kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.8～1.0, the condition of mixing speed 120～200r/min, aerobic fermentation is after 48～130 hours, and is standby;
(8) anaerobically fermenting is produced ethanol: use the yeast minimum medium, and 30 ℃～37 ℃ of temperature, pH4～6, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; The fermented liq integrating meter of (7) preparation set by step inserts its volume percent and is 10%～15% fermentation by saccharomyces cerevisiae bacterium liquid or 0.1%～0.5% commercially available Angel Yeast particle and carries out the anaerobism sealed fermenting, and leavening temperature is 28 ℃～32 ℃, and the pH value is 4～6; Fermentation time 48～58 hours reaches volume percent 8% when above when detecting the karusen ethanol concn, and fermentation stops;
Perhaps (9) seaweed chemical waste material mixed fermentation: getting the described waste residue of step (1) and the described fermention medium of step (4) is that 1: 5～8 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 70～80%, again with the weight percent meter of charge amount, it is described with yeast minimum medium activatory fermentation by saccharomyces cerevisiae bacterium liquid to add described liquid fermenting bacterial classification of 10%～15% step (4) and 5%～10% step (6), with 28 ℃～32 ℃ of culture temperature, pH4.5～6, tank pressure 0.6～0.8kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.8～1.0, the condition of mixing speed 120～200r/min, aerobic fermentation 48～130 hours, stop then the ventilation, change the anaerobism sealed fermenting stage over to, leavening temperature is 28 ℃～32 ℃, the pH value is 4～6; Fermentation time 48～58 hours reaches volume percent 8% when above when detecting the karusen ethanol concn, and fermentation stops;
(10) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is in two ways as animal-feed: 1. wet feed is needed by raising object and purpose, mix with finished product feedstuff or fodder additives; Be directly used in feeding animals; 2. wet feed is dry at normal temperatures, pulverize the back and be used for feed processing with protein-based additive form.
The above-mentioned utilization in the method that seaweed chemical waste material produces ethanol and feed, preferred implementation is:
The described temperature of step (4) to (9) is 30 ℃.
The described pH of step (4) to (9) is 5.0～6.
Step step (4), (7) or (9) described air flow are with fermentating liquid volume m
3/ volume of air m
3Minute count 1:0.9.
The described aerobic fermentation time of step (7) or (9) is 100-130 hour.
Enzyme engineering technology of the present invention is meant the utilisation technology of zymins such as cellulase, hemicellulase.Described microbial technique is meant the microorganism of cellulase-producing and utilizes the microorganism of materials such as generation ethanol such as yeast.Described seaweed chemical waste residue and liquid is meant that the algae that utilizes nature or propagate artificially (sea-tangle, bulk kelp, sea accumulate, bladder wrack, wakame, Sargassum fusiforme, sargassun, rope algae, laver etc.) is formed solid of raw material production marine chemical industry product and Liquid wastes.The described energy is meant the ethanol homenergic material that forms by biotransformations such as enzymatic hydrolysis and fermentations.Described feed is meant that with the seaweed chemical waste residue and liquid be microorganism and the meta-bolites thereof of raw material through the nutritive ingredients such as rich in proteins of bio-transformation formation.
The ethanol equal energy source material that adopts method of the present invention to produce can be used for fields such as medicine, food, chemical industry, traffic after concentrating; " green " high-quality feed of aquacultures such as poultry, domestic animal, aquatic products can be used as or be used to prepare to the rich in proteins of producing and the feed of microbial metabolites.
In order to understand the effect of essence of the present invention and conversion technology of the present invention better, the applicant uses the marine alga waste residue and liquid of how tame factory, and comprehensive utilization enzyme engineering technology and microbial technique have carried out bio-transformation, production capacity and the experiment of product single cell protein to the seaweed chemical waste residue.The result shows: conversion technology of the present invention can effectively utilize different seaweed industry refuses, produces ethanol and single cell protein.
The present invention provides economy, environmental protection, effective industrial technology for the comprehensive utilization of seaweed chemical waste residue and liquid, can solve the environmental pollution problem that the seaweed chemical waste residue and liquid causes effectively, turn waste into wealth, be the abundant and higher value application of marine algae resource, for development marine alga recycling economy provides the important techniques support.
Embodiment
Embodiment 1: sea-tangle waste residue, cascade fermentation.
Utilize seaweed chemical waste material to produce the method for ethanol and feed, form by following step:
(1) seaweed chemical waste material is selected: select the sea-tangle waste residue, according to the water content difference of waste residue, adding an amount of water, to make slag concentration be 30% suspension by weight percentage, to 6, standby with dilute acid soln adjusting pH to 5.
(2) bacterial classification is selected: select cellulase producing bacteria for use: viride (Trichoderma viride) AF93252 Wuhan University China typical culture collection center provides, and preserving number is CCTCC NO.AF93252; The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, buy from the supermarket.
(3) liquid fermenting strain preparation: with the described cellulase producing bacteria bacterial strain of step (2), under aseptic condition, through the test tube slant, shake bottle, first class seed pot, secondary seed jar amplification culture step by step, make the liquid fermenting bacterial classification that the bacterium number reaches OD value 1.0～1.2 * 10;
Wherein:
The fermention medium that shakes bottle, first class seed pot, the use of secondary seed jar is to contain by weight percentage: Mandel ' the s mineral nutrition liquid of 0.1% peptone, 1.2% carbon source, pH6;
Used slant medium is that above-mentioned Mandel ' s mineral nutrition adds 2% agar powder;
Used shake-flask culture condition is 30 ℃ of temperature, pH6, shaking speed 200r/min, incubation time 48-50 hour; First class seed pot, secondary seed jar culture condition are 30 ℃ of temperature, pH6, tank pressure 0.5Kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.9, mixing speed 150r/min, aerobic cultivation 48-50 hour;
(4) seaweed chemical waste material fermentation: getting the described waste residue of step (1) and the described fermention medium of step (3) is that 1: 7 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 80%, again with the weight percent meter of charge amount, add the described liquid fermenting bacterial classification of 14% step (3), with 30 ℃ of culture temperature, pH6, tank pressure 0.7～0.8kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.9, the condition of mixing speed 150r/min, aerobic fermentation is standby after 120 hours;
(5) anaerobically fermenting is produced ethanol: with 33 ℃ of yeast minimum medium temperature, and pH4.8, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; The fermented liq integrating meter of (4) preparation set by step inserts its volume percent and is 14% fermentation by saccharomyces cerevisiae bacterium liquid or 0.3%～0.5% commercially available yeast particles and carries out the anaerobism sealed fermenting, and leavening temperature is 30 ℃, and the pH value is 4.8; Fermentation time 48-58 hour, reach volume percent 8% when above when detecting the karusen ethanol concn, fermentation stops;
(6) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is as follows as animal-feed: wet feed is needed by raising object and purpose, mix with finished product feedstuff or fodder additives; Be directly used in feeding animals.
Embodiment 2: sargassun waste residue, cascade fermentation
Utilize seaweed chemical waste material to produce the method for ethanol and feed, form by following step:
(1) seaweed chemical waste material is selected: select the sargassun waste residue, according to the water content difference of waste residue, adding an amount of water, to make slag concentration be 25% suspension by weight percentage, with dilute acid soln adjusting pH to 5.5, standby.
(2) bacterial classification is selected: viride (Trichoderma viride) AF93252, and Wuhan University China typical culture collection center (CCTCC) provides [preserving number] AF93252; The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, buy from the supermarket.
(3) liquid fermenting strain preparation: with the described cellulase producing bacteria bacterial strain of step (2), under aseptic condition, through the test tube slant, shake bottle, first class seed pot, secondary seed jar amplification culture step by step, make the liquid fermenting bacterial classification that the bacterium number reaches OD value 1.0～1.2 * 10;
Wherein:
The fermention medium that shakes bottle, first class seed pot, the use of secondary seed jar is to contain by weight percentage: Mandel ' the s mineral nutrition liquid of 0.1% peptone, 1.2% carbon source, pH5.5;
Used slant medium is that above-mentioned Mandel ' s mineral nutrition adds 2% agar powder;
Used shake-flask culture condition is 32 ℃ of temperature, pH5.5, shaking speed 220r/min, incubation time 54-60 hour; First class seed pot, secondary seed jar culture condition are 32 ℃ of temperature, pH5.5, tank pressure 0.5Kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:1.0, mixing speed 200r/min, aerobic cultivation 55-60 hour;
(4) seaweed chemical waste material fermentation: getting the described waste residue of step (1) and the described fermention medium of step (3) is that 1: 8 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 75%, again with the weight percent meter of charge amount, add the described liquid fermenting bacterial classification of 15% step (3), with 32 ℃ of culture temperature, pH5.5, tank pressure 0.6～0.7kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:1.0, the condition of mixing speed 200r/min, standby after aerobic fermentation 78-100 hour;
(5) anaerobically fermenting is produced ethanol: with 35 ℃-37 ℃ of yeast minimum medium temperature, and pH5.5, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; The fermented liq integrating meter of (4) preparation set by step inserts its volume percent and is 15% fermentation by saccharomyces cerevisiae bacterium liquid or 0.2%～0.4% commercially available yeast particles and carries out the anaerobism sealed fermenting, and leavening temperature is 32 ℃, and the pH value is 5.5; Fermentation time 48-58 hour, reach volume percent 8% when above when detecting the karusen ethanol concn, fermentation stops;
(6) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is as follows as animal-feed: wet feed is dry at normal temperatures, and pulverize the back and be used for feed processing with protein-based additive form.
Embodiment 3: sea-tangle raw material, cascade fermentation
Utilize seaweed chemical waste material to produce the method for ethanol and feed, form by following step:
(1) seaweed chemical waste material is selected: select dried sea-tangle to pulverize, with about 1: 40 ratio, adding an amount of water, to make slag concentration be 35% suspension by weight percentage, with dilute acid soln adjusting pH to 5, standby.
(2) bacterial classification is selected: viride (Trichoderma viride) AF93252 Wuhan University China typical culture collection center (CCTCC) provides [preserving number] AF93252; The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, buy from the supermarket.
(3) liquid fermenting strain preparation: with the described cellulase producing bacteria bacterial strain of step (2), under aseptic condition, through the test tube slant, shake bottle, first class seed pot, secondary seed jar amplification culture step by step, make the liquid fermenting bacterial classification that the bacterium number reaches OD value 1.0～1.2 * 10;
Wherein:
The fermention medium that shakes bottle, first class seed pot, the use of secondary seed jar is to contain by weight percentage: Mandel ' the s mineral nutrition liquid of 0.1% peptone, 1.2% carbon source, pH5;
Used slant medium is that above-mentioned Mandel ' s mineral nutrition adds 2% agar powder;
Used shake-flask culture condition is 31 ℃ of temperature, pH5, shaking speed 180r/min, incubation time 48 hours; First class seed pot, secondary seed jar culture condition are 31 ℃ of temperature, pH5, tank pressure 0.5Kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.8, mixing speed 120r/min, aerobic cultivation 48 hours;
(4) seaweed chemical waste material fermentation: getting the described waste residue of step (1) and the described fermention medium of step (3) is that 1: 5 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 70%, again with the weight percent meter of charge amount, add the described liquid fermenting bacterial classification of 10% step (3), with 31 ℃ of culture temperature, pH5, tank pressure 0.7～0.8kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.8, the condition of mixing speed 120r/min, standby after aerobic fermentation 48-60 hour;
(5) anaerobically fermenting is produced ethanol: with 30 ℃-32 ℃ of yeast minimum medium temperature, and pH4, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; The fermented liq integrating meter of (4) preparation set by step inserts its volume percent and is 10% fermentation by saccharomyces cerevisiae bacterium liquid or 0.1%～0.3% commercially available yeast particles and carries out the anaerobism sealed fermenting, and leavening temperature is 31 ℃, and the pH value is 4; Fermentation time 48-58 hour, reach volume percent 8% when above when detecting the karusen ethanol concn, fermentation stops;
(6) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is in two ways as animal-feed: 1. wet feed is needed by raising object and purpose, mix with finished product feedstuff or fodder additives; Be directly used in feeding animals; 2. wet feed is dry at normal temperatures, pulverize the back and be used for feed processing with protein-based additive form.
Embodiment 4: bulk kelp waste residue, cascade fermentation
Utilize seaweed chemical waste material to produce the method for ethanol and feed, form by following step:
(1) seaweed chemical waste material is selected: select the bulk kelp waste residue, according to the water content difference of waste residue, adding an amount of water, to make slag concentration be 20% suspension by weight percentage, with dilute acid soln adjusting pH to 5.5, standby.
(2) bacterial classification is selected: select cellulase producing bacteria for use: viride (Trichoderma viride) AF93252 Wuhan University China typical culture collection center (CCTCC) provides [preserving number] AF93252; The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, buy from the supermarket.
(3) liquid fermenting strain preparation: with the described cellulase producing bacteria bacterial strain of step (2), under aseptic condition, through the test tube slant, shake bottle, first class seed pot, secondary seed jar amplification culture step by step, make the liquid fermenting bacterial classification that the bacterium number reaches OD value 1.0～1.2 * 10;
Wherein:
The fermention medium that shakes bottle, first class seed pot, the use of secondary seed jar is to contain by weight percentage: Mandel ' the s mineral nutrition liquid of 0.1% peptone, 1.2% carbon source, pH5.5;
Used slant medium is that above-mentioned Mandel ' s mineral nutrition adds 2% agar powder;
Used shake-flask culture condition is 30 ℃ of temperature, pH5.5, shaking speed 200r/min, incubation time 50-56 hour; First class seed pot, secondary seed jar culture condition are 30 ℃ of temperature, pH5.5, tank pressure 0.5Kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:1.0, mixing speed 200r/min, aerobic cultivation 50-56 hour;
(4) seaweed chemical waste material fermentation: getting the described waste residue of step (1) and the described fermention medium of step (3) is that 1: 6 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 75%, again with the weight percent meter of charge amount, add the described liquid fermenting bacterial classification of 13% step (3), with 30 ℃ of culture temperature, pH5.5, tank pressure 0.7kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.9, the condition of mixing speed 180r/min, standby after aerobic fermentation 98-120 hour;
(5) anaerobically fermenting is produced ethanol: with 35 ℃-36 ℃ of yeast minimum medium temperature, and pH5.5, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; The fermented liq integrating meter of (4) preparation set by step inserts its volume percent and is 15% fermentation by saccharomyces cerevisiae bacterium liquid or 0.2%～0.4% commercially available yeast particles and carries out the anaerobism sealed fermenting, and leavening temperature is 30 ℃, and the pH value is 5.5; Fermentation time 48-58 hour, reach volume percent 8% when above when detecting the karusen ethanol concn, fermentation stops;
(6) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is as follows as animal-feed: wet feed is dry at normal temperatures, and pulverize the back and be used for feed processing with protein-based additive form.
Embodiment 5: enzymatic hydrolysis, anaerobically fermenting
Utilize seaweed chemical waste material to produce the method for ethanol and feed, form by following step:
(1) zymin and bacterial classification are selected: cellulase preparation is available from the grand mcroorganism engineering limited liability company in Yishui, Shandong Province.
The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, buy from the supermarket.
(2) seaweed chemical waste material enzymatic hydrolysis: select dry sea-tangle waste residue, adding suitable quantity of water to slag concentration is 35% by weight percentage, regulates pH to 5 to 6, with the weight percent meter of total amount with dilute acid soln, the cellulase preparation of adding 30%, 30 ℃～32 ℃ were reacted 24 hours.
(3) anaerobically fermenting is produced ethanol: with 33 ℃ of yeast minimum medium temperature, and pH4.8, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; Will be set by step the marine alga waste residue enzyme hydrolyzate of (2) preparation to insert its volume percent be that 14% fermentation by saccharomyces cerevisiae bacterium liquid or 0.3%～0.5% commercially available Angel Yeast particle carry out the anaerobism sealed fermenting, leavening temperature is 30 ℃, the pH value is 4.8; Fermentation time 48-58 hour, reach volume percent 8% when above when detecting the karusen ethanol concn, fermentation stops;
(4) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is as follows as animal-feed: wet feed is needed by raising object and purpose, mix with finished product feedstuff or fodder additives; Be directly used in feeding animals.
Embodiment 6: sea-tangle waste residue, mixed fermentation.
Utilize seaweed chemical waste material to produce the method for ethanol and feed, form by following step:
(1) seaweed chemical waste material is selected: select the sea-tangle waste residue, according to the water content difference of waste residue, adding an amount of water, to make slag concentration be 30% suspension by weight percentage, to 6, standby with dilute acid soln adjusting pH to 5.
(2) bacterial classification is selected: select cellulase producing bacteria for use: viride (Trichoderma viride) AF93252, and Wuhan University China typical culture collection center (CCTCC) provides [preserving number] AF93252; The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, buy from the supermarket.
(3) liquid fermenting strain preparation: with the described cellulase producing bacteria bacterial strain of step (2), under aseptic condition, through the test tube slant, shake bottle, first class seed pot, secondary seed jar amplification culture step by step, make the liquid fermenting bacterial classification that the bacterium number reaches OD value 1.0～1.2 * 10;
Wherein:
The fermention medium that shakes bottle, first class seed pot, the use of secondary seed jar is to contain by weight percentage: Mandel ' the s mineral nutrition liquid of 0.1% peptone, 1.2% carbon source, pH6;
Used slant medium is that above-mentioned Mandel ' s mineral nutrition adds 2% agar powder;
Used shake-flask culture condition is 30 ℃ of temperature, pH6, shaking speed 200r/min, incubation time 48-50 hour; First class seed pot, secondary seed jar culture condition are 30 ℃ of temperature, pH6, tank pressure 0.5Kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.9, mixing speed 150r/min, aerobic cultivation 48-50 hour;
(4) yeast saccharomyces cerevisiae bacterial classification preparation: use the yeast minimum medium, 30 ℃～37 ℃ of temperature, pH4～6, activation yeast saccharomyces cerevisiae bacterial classification (Angel Yeast), preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby;
(5) seaweed chemical waste material fermentation: getting the described waste residue of step (1) and the described fermention medium of step (3) is that 1: 7 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 75%, again with the weight percent meter of charge amount, it is described with yeast minimum medium activatory yeast saccharomyces cerevisiae bacterial classification to add described liquid fermenting bacterial classification of 14% step (3) and 5% step (4), with 30 ℃ of culture temperature, pH6, tank pressure 0.7～0.8kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1:0.9, the condition of mixing speed 150r/min, aerobic fermentation 100 hours.Stop ventilation then, change the anaerobism sealed fermenting stage over to, leavening temperature is 30 ℃, and the pH value is 4.8; Fermentation time 48-58 hour, reach volume percent 8% when above when detecting the karusen ethanol concn, fermentation stops;
(6) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is as follows as animal-feed: wet feed is needed by raising object and purpose, mix with finished product feedstuff or fodder additives; Be directly used in feeding animals.
Embodiment 7: enzymatic hydrolysis, anaerobically fermenting
Utilize seaweed chemical waste material to produce the method for ethanol and feed, form by following step:
(1) zymin and bacterial classification are selected: cellulase preparation is available from the grand mcroorganism engineering limited liability company in Yishui, Shandong Province.
The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, buy from the supermarket.
(2) seaweed chemical waste material enzymatic hydrolysis: select wakame, Sargassum fusiforme, rope algae, laver arbitrary proportion blended waste residue, adding suitable quantity of water to slag concentration is 30% by weight percentage, regulate pH to 5.5 with dilute acid soln, weight percent meter with total amount, the cellulase preparation of adding 25%, 30 ℃ were reacted 26 hours.
(3) anaerobically fermenting is produced ethanol: with 30 ℃ of yeast minimum medium temperature, and pH4.8, activation yeast saccharomyces cerevisiae bacterial classification (Angel Yeast), preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; Will be set by step the marine alga waste residue enzyme hydrolyzate of (2) preparation to insert its volume percent be that 12% fermentation by saccharomyces cerevisiae bacterium liquid carries out the anaerobism sealed fermenting, leavening temperature is 30 ℃, the pH value is 4.8; Fermentation time 54-58 hour, reach volume percent 8% when above when detecting the karusen ethanol concn, fermentation stops;
(4) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is as follows as animal-feed: wet feed is needed by raising object and purpose, mix with finished product feedstuff or fodder additives; Be directly used in feeding animals.
Claims (5)
1. method of utilizing seaweed chemical waste material to produce ethanol and feed, form by following step:
(1) seaweed chemical waste material is selected: described waste material is that marine alga chemical industry is produced the waste residue that contains cellulose components that forms in sodium alginate, N.F,USP MANNITOL, iodine or the marine alga carbohydrate gum product process; Wherein: described marine alga is that sea-tangle, sargassun, bulk kelp, sea accumulate, one of bladder wrack, wakame, Sargassum fusiforme, rope algae, laver, and described waste residue is that sea-tangle, sargassun, bulk kelp, sea accumulate, one of bladder wrack, wakame, Sargassum fusiforme, rope algae, laver waste residue or its arbitrary proportion mix;
(2) selection of zymin: zymin is commercially availablely cellulose degradation can be the monose that can be utilized by yeast saccharomyces cerevisiae and cellulase, the hemicellulase of oligosaccharides;
(3) bacterial classification is selected: select cellulase producing bacteria for use: viride (Trichoderma viride) AF93252, and Wuhan University China typical culture collection center provides, and preserving number is CCTCC NO.AF93252; The yeast saccharomyces cerevisiae bacterial classification: Angel Yeast, commercially available;
(4) cellulase liquid fermented bacterium preparation: with the described cellulase producing bacteria bacterial strain of step (3), under aseptic condition, through the test tube slant, shake bottle, first class seed pot, secondary seed jar amplification culture step by step, make the liquid fermenting bacterial classification that the bacterium number reaches 0D value 1.0～1.2 * 10;
Wherein:
The described fermention medium that shakes bottle, first class seed pot, the use of secondary seed jar is to contain by weight percentage: Mandel ' the s mineral nutrition liquid of 0.1% peptone, 1.2% carbon source, pH5～6;
Described slant medium is that above-mentioned Mandel ' s mineral nutrition adds 2% agar powder;
Described shake-flask culture condition is 30 ℃～32 ℃ of temperature, pH5～6, shaking speed 180～220r/min, incubation time 48～60 hours; First class seed pot, secondary seed jar culture condition are 28 ℃～32 ℃ of temperature, pH4.5～6, tank pressure 0.5Kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1: 0.8～1.0, mixing speed 120～200r/min, aerobic cultivation 48～60 hours;
(5) seaweed chemical waste material enzymatic hydrolysis: getting the described waste residue of step (1), to add water to slag concentration be 20%～35% by weight percentage, regulates pH to 4.5～6 with dilute acid soln; With the weight percent meter of total amount, the commercially available cellulase that adds 10%～30% amount is or/and the hemicellulose zymin, and 28 ℃～32 ℃ reactions 24～26 hours are standby;
(6) produce ethanol with fermentation by saccharomyces cerevisiae: use the yeast minimum medium, 30 ℃～37 ℃ of temperature, pH4～6, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; Set by step (5) preparation hydrolysis seaweed chemical waste material liquid integrating meter, insert its volume percent and be 10%～15% fermentation by saccharomyces cerevisiae bacterium liquid or 0.1%～0.5% commercially available Angel Yeast particle and carry out the anaerobism sealed fermenting, leavening temperature is 28 ℃～32 ℃, and the pH value is 4～6; Fermentation time 48～58 hours reaches volume percent 8% when above when detecting the karusen ethanol concn, and fermentation stops;
Perhaps (7) seaweed chemical waste material fermentation: getting the described waste residue of step (1) and the described fermention medium of step (4) is that 1: 5～8 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 70～80%, again with the weight percent meter of charge amount, add the described liquid fermenting bacterial classification of 10%～15% step (4), with 28 ℃～32 ℃ of culture temperature, pH4.5～6, tank pressure 0.6～0.8kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1: 0.8～1.0, the condition of mixing speed 120～200r/min, aerobic fermentation is after 48～130 hours, and is standby;
(8) anaerobically fermenting is produced ethanol: use the yeast minimum medium, and 30 ℃～37 ℃ of temperature, pH4～6, activation yeast saccharomyces cerevisiae bacterial classification, preparation mycetocyte number reaches the above fermentation by saccharomyces cerevisiae bacterium liquid of 100,000,000/ml, and is standby; The fermented liq integrating meter of (7) preparation set by step inserts its volume percent and is 10%～15% fermentation by saccharomyces cerevisiae bacterium liquid or 0.1%～0.5% commercially available Angel Yeast particle and carries out the anaerobism sealed fermenting, and leavening temperature is 28 ℃～32 ℃, and the pH value is 4～6; Fermentation time 48～58 hours reaches volume percent 8% when above when detecting the karusen ethanol concn, and fermentation stops;
Perhaps (9) seaweed chemical waste material mixed fermentation: getting the described waste residue of step (1) and the described fermention medium of step (4) is that 1: 5～8 amount is mixed with weight ratio, pack in the fermentor tank with the amount of coefficient 70～80%, again with the weight percent meter of charge amount, it is described with yeast minimum medium activatory fermentation by saccharomyces cerevisiae bacterium liquid to add described liquid fermenting bacterial classification of 10%～15% step (4) and 5%～10% step (6), with 28 ℃～32 ℃ of culture temperature, pH4.5～6, tank pressure 0.6～0.8kg/cm
2, air flow is with fermentating liquid volume m
3/ volume of air m
3Minute the meter 1: 0.8～1.0, the condition of mixing speed 120～200r/min, aerobic fermentation 48～130 hours, stop then the ventilation, change the anaerobism sealed fermenting stage over to, leavening temperature is 28 ℃～32 ℃, the pH value is 4～6; Fermentation time 48～58 hours reaches volume percent 8% when above when detecting the karusen ethanol concn, and fermentation stops;
(10) liquid-solid separation: adopt nylon cloth filtration or the centrifugal 10min method of 10000rpm to carry out the liquid-solid separation of fermented liquid; Supernatant liquor steams ethanol through adopting vacuum distillation method, reconcentration, concentration is ethanol more than 90%; The precipitated solid material is in two ways as animal-feed: 1. wet feed is needed by raising object and purpose, mix with finished product feedstuff or fodder additives; Be directly used in feeding animals; 2. wet feed is dry at normal temperatures, pulverize the back and be used for feed processing with protein-based additive form.
2. the method for utilizing seaweed chemical waste material to produce ethanol and feed as claimed in claim 1, it is characterized in that: the described temperature of step (4) to (9) is 30 ℃.
3. the method for utilizing seaweed chemical waste material to produce ethanol and feed as claimed in claim 1, it is characterized in that: the described pH of step (4) to (9) is 5.0～6.
4. the method for utilizing seaweed chemical waste material to produce ethanol and feed as claimed in claim 1, it is characterized in that: step step (4), (7) or (9) described air flow are with fermentating liquid volume m
3/ volume of air m
3Minute count 1: 0.9.
5. the method for utilizing seaweed chemical waste material to produce ethanol and feed as claimed in claim 1, it is characterized in that: the described aerobic fermentation time of step (7) or (9) is 100-130 hour.
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