CN101130764B - Method and device for producing biological siliceous material with reactor culture sponge or its cell - Google Patents
Method and device for producing biological siliceous material with reactor culture sponge or its cell Download PDFInfo
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Abstract
The invention relates to a method and a device of reactor culture sponge or the cell production biological siliceous material. The reactor applies natural seawater, micro algae culture sponge of the normal sponge bait, wherein the adhesion base of the growth face which forms 45-135 degree with the horizontal direction, the sponge or the cell can grow along the growth surface, the natural seawater has silane coupling agent, the bone spicule is acquired with the phenomenon that the original skeleton is abandoned in the growth and motion process of the sponge structure, or the biological siliceous material is acquired by producing bone spicule in the exsomatize sponge cell culture system. The production method introduces silane coupling agent by constructing the sponge culture reactor, which proceeds with the biological synthesis of the siliceous material with the chemical modification in order to make the sponge or the sponge cell into 'factory' of specific biological siliceous material,and provides the probability of the application for synthesizing the sponge siliceous bone spicule.
Description
Technical field
The present invention relates to utilize biological method to obtain the method for biological siliceous material, is the method and the device of reactor culture sponge or its cells produce biological siliceous material specifically.
Background technology
Sponge is the multicellular animals of minimum grade, has the ability of utilizing synthetic self bone of lower concentration orthosilicic acid in the water body.Compare ordinary method, this biosynthetic process is to carry out under physical environment, extremely low concentration of substrate.The essentially consist unit of sponge bone is a spicule, and its chemical ingredients is the silicon-dioxide ((SiO that has crystal water
2)
2 ~ 5H
2O), consistent with the chemical constitution of commercial opticglass, simultaneously, studies show that this biological siliceous material has excellent biological compatibility and chemical stability, its material property such as breaking tenacity etc. are better than the artificial glass material of comparable size, therefore have great application prospect in fields such as biology, chemical industry, optics, semi-conductors.But because the main component of natural sponge spicule is a silicon-dioxide, its chemical stability has also influenced the further application to the spongy biological siliceous material.Silane coupling agent is the chemical that a class is widely used in chemical field, can be used in the physico-chemical property of improving or reconciling the macromolecular material of silicon rubber.Therefore, introducing silane coupling agent in the spicule building-up process will be a good try.
Simultaneously, sponge still is important medicine source biology, reactor culturing of sponge or sponge cell cultures will be not solve the most probable approach in biomass source, but the siliceous material in the sponge has accounted for more than 50% of sponge dry weight biomass, be used as processing waste material at present mostly and handle, caused the great wasting of resources.The reasonable utilization of this part spicule will produce huge economic benefit and environmental benefit.
Summary of the invention
The method that the purpose of this invention is to provide a kind of reactor culture sponge or its cells produce biological siliceous material, make up the culture system that adapts to sponge motion growth phase, form by silicon source in the regulation and control water body, introduce silane coupling agent, carry out the controlled sponge source biological siliceous material production of chemical ingredients.
The method principle: what play an important role in sponge siliceous material production process is the albumen that a class is called as silicon polysaccharase (silicatein), this proteinoid has the ability that the catalysis orthosilicic acid aggregates into silica polymer, and experiment in vitro shows that this proteinoid can also catalysis phenyltrimethoxysila,e polymeric ability simultaneously.This makes finally introduces organic group and carries out modification and become possibility by changing in the culture system silicon source category in the sponge siliceous material.
Spongy tissue constantly produces new spicule material in the growth movement process, and abandons old part.Under the guiding of current and growing carrier, spongy tissue can constantly move.Therefore, reasonably make up the sponge growth reactor, can under the prerequisite of not damaging spongy tissue, directly obtain sponge spicule.Be subjected to the restriction of spongy tissue cultivation level up till now, it was decided to be for 6 weeks with the growth of spongy tissue.
In the sponge cell aggregation culturing process, also can produce spicule, satisfying its growth needs, and be removed in the cell dissociation process at original sponge spicule, therefore, the newborn spicule of generation will be the spicule after the modification fully.
For achieving the above object, the technical solution used in the present invention is:
The present invention is based on method spongy tissue deblocking reaction device culture system or sponge cell culture system, that produce sponge source biological siliceous material with certain chemically modified or modification; Utilize sponge self biological property to carry out the production of biological siliceous material, the silicon source category serves as that the basis produces the modification biological siliceous material in the sponge growth system to change;
Be specially, adopt natural sea-water, drop into conventional sponge bait micro-algae cultivation sponge or its cell in reactor, in reactor, be provided with from the horizontal by the adherance of 45-135 degree aufwuchsplate, sponge or its cell can be grown along aufwuchsplate, be added with silane coupling agent at natural sea-water, the volumetric molar concentration of silane coupling agent in natural sea-water is 10-100 μ M, utilize the phenomenon of abandoning the original bone of part in the spongy tissue growth movement process to carry out the spicule results, or in stripped sponge cell culture system, produce spicule, obtain biological siliceous material.
Further, can carry out purifying to obtaining biological siliceous material, with bone resistates or spongy tissue wherein carry out pickling, remove organicly, obtain having the sponge source biological siliceous material that particular chemical is modified; Described acid is that concentrated nitric acid or volume ratio are 0.1-4: 1 the vitriol oil and the mixing acid of concentrated nitric acid, the treatment temp of pickling are 25-80 ℃, and the treatment time is more than 0.5 hour or 0.5 hour.
Described silane coupling agent is siloxanes and derivative (as tetramethoxy-silicane, tetraethoxysilane etc.) thereof based on orthosilicic acid; Siloxanes and derivative (as propyl trimethoxy silicane, 3-urea groups propyl trimethoxy silicane etc.) thereof with a silicon-carbon bonds; Or other has the silane derivative with the orthosilicic acid polymerization reaction take place.
Produce sponge source biological siliceous material by reactor culture sponge, carry out the biological siliceous material chemically modified, specific operation process is,
1) the wild sponge that will gather utilizes the natural sea-water clean surface through 0.22 μ m membrane filtration degerming, with pollutant removals such as top layer silt, epiphytic algaes;
2) sponge after will cleaning is cut into the fritter that area is about 1-3cm * 1-3cm with scalpel;
3) with step 2) in the sponge fritter that cuts shift into through 0.22 μ m membrane filtration degerming and add in the natural sea-water of 200-400mg/L gentamicin, left standstill 5-10 minute;
4) adding wherein is added with silane coupling agent through the natural sea-water of 0.22 μ m membrane filtration degerming in reactor, and is stand-by;
5) sponge fritter treated in the step 3) is fixed on the adherance in the reactor, in reactor, cultivates 15 ~ 23 degrees centigrade of culture temperature;
6) add little algae (as: diatom, flat algae, chrysophyceae) of final concentration 10-30 ten thousand cells/mL as bait.
In spongy tissue piece culturing process, by glass needle, the spongy tissue piece is fixed on the cultivation base, cultivate in the reactor at the liquid lift-type and cultivate; In culture system, add a certain amount of silane coupling agent; In culturing process, regularly original bone of abandoning in the sponge growth movement process is collected; After 6 weeks, spongy tissue is collected whole spongy tissues.In the sponge cell cultivation process, under the type culture mode, in culture system, add silane coupling agent, cultivate; After 30 days, collect the cell aggregation that is obtained.Collected bone (or cell aggregation) is utilized the nitration mixture (vitriol oil: concentrated nitric acid=3: 1 (volume ratio)) handle, remove organic; It is also air-dry to utilize deionized water that institute's acquisition spicule is cleaned.Get sample segment at last and utilize the introducing of infrared affirmation organic group.The present invention has made up a kind of spongy biological reactor of novelty, and has utilized biological instinct to carry out the production of biological siliceous material, environmental friendliness, and mild condition is a technology a kind of initiative, novel.
Reference literature (Primmorphs from archaeocytes-dominant cell populationof the sponge Hymeniacidon perleve:Improved cell proliferation andspiculogenesis (Biotechnology and Bioengineering, v84-5,583-590)) described sponge cell isolated culture method, set up sponge cell isolated culture system, carry out the biological siliceous material production of sponge source, specific operation process
1) the no calcium magnesium seawater that the spongy tissue utilization of weight in wet base 1 gram is contained volumetric molar concentration 2-10mM ethylenediamine tetraacetic acid (EDTA) disperses, and utilizes density gradient centrifugation method to collect the archeocyte enriched layer in the cell that is obtained, and being adjusted to final concentration is 5 * 10
6Cells/mL inoculates; Substratum is for adding the ironic citrate of 10-30 μ M through the filtering natural sea-water of 0.22 μ m; 18 ~ 23 degrees centigrade of culture temperature;
2) add silane coupling agent in substratum and cultivate, the replacing substratum was 1 time in per two days to three days 100%;
3) stopped in the 20 to 30 day cultivating, collect and cultivate the cell aggregation that obtains;
4) handle results sponge source biological siliceous material according to claim 4 steps 4～6.
Described reactor can be liquid lift-type inserted block and cultivates reactor:
It comprises a container, and the container inner bottom part is provided with hollow sheeting, and hollow sheeting is provided with aperture, and hollow sheeting links to each other with water pump by pipeline, and circulation enters into hollow sheeting inside through water pump to cultivate seawater, upwards flows out from the water outlet duct of hollow sheeting surface arrangement;
Be placed with the vertical slender glass pin that has glass base on the hollow sheeting, in order to fixing spongy tissue piece, the fixed arrangement of spongy tissue inserted block by glass needle is on hollow sheeting;
Spongy tissue is fixed on the inserted block, and can be under flow action upwards growth of glass needle on the inserted block; Be provided with the admission passage that links to each other with air pump in the container, by air pump to the system tonifying Qi;
The bottom-up growth of spongy tissue is peeled off the residue bone of spongy tissue below and collect from adherance weekly.
The present invention compared with prior art has following advantage:
1. the invention provides a kind of brand-new liquid lift-type inserted block and cultivate reactor, can make spongy tissue in culturing process, prolong the specific direction motion, be convenient to the collection of product based on sponge motion growth characteristics.
2. described in the invention is a brand-new technical process, with chemical synthesis process in the past essential distinction is arranged, and is to utilize the organism self-characteristic to carry out the production of biological siliceous material in conjunction with the growth reactor of uniqueness.Cultivating reactor (or sponge cell culture reactor) by making up spongy tissue liquid lift-type inserted block, is the production that substrate carries out sponge spicule with the orthosilicic acid in silane coupling agent and the natural sea-water; Be specially, structure meets the spongy tissue piece liquid lift-type inserted block of spongy tissue motion growth rhythm and cultivates reactor, silane coupling agent is incorporated in the natural sea-water of culture system, chemical constitution to sponge spicule is regulated and control, make sponge or sponge cell become " factory " of production particular organisms siliceous material, also deeply using for sponge trichite synthetic provides possibility.
3. environmental friendliness.The present invention carries out in being similar to physical environment fully.Produce for the sponge that is retrieved as purpose with biomass, can also effectively utilize its production " waste " spicule.
4. concentration of substrate is low.Operational path of the present invention is to add an amount of silane coupling agent on the natural sea-water basis, and related concentration of substrate is no more than 60 μ M, deducts therefore that sponge utilizes and self degraded, does not have residually substantially, can directly discharge.
5. can effectively combine with other technical process.Sponge itself can also be used in the organic granular of aquaculture water and remove and the purification aspect, and as operating unit, the inventive method can combine with total system as one of them unit operation, improves overall efficiency.
In a word, the present invention is the production technique of a kind of novelty, environmental protection and effective biological siliceous material.
Description of drawings
Fig. 1 is a culture system synoptic diagram of the present invention; Figure A is a side-view, and wherein: 1 for cultivating used container, and 2 is the spongy tissue on the adherance, and 3 for entering the current of culture systems, and 4 for having the adherance of glass needle, and 5 for having the hollow sheeting of posticum; Figure B is a vertical view, and wherein: 6 is the posticum on the hollow sheeting;
Fig. 2 is biomass changing trend diagram in the liquid lift-type inserted block reactor of the present invention;
Fig. 3 is the spongy tissue photo in the liquid lift-type inserted block reactor;
Fig. 4 is the infrared detection result of spicule;
Fig. 5 is the newborn spicule (shown in the arrow) in the cell culture system.
Embodiment
Below by specific embodiment method of the present invention and result are described, the culture systems of using among the present invention is to be 1.8L liquid lift-type inserted block reactor, the silane coupling agent that uses is a standard commercial reagent, here be that representative is described with 3-urea groups propyl trimethoxy silicane, seawater is through the filtering natural sea-water of 0.22 μ M, sponge is for being grown in the geographic film sponge in great numbers in tideland, Dalian, and the infrared detection of chemical constitution utilizes Bruker Tensor 27FT-IR to carry out in the spicule.
With the film sponge in great numbers of fresh collection, clean through the natural sea-water of 0.22 μ M membrane filtration with 200mL, remove surface attachment algae and silt etc., in the laboratory, supported three days temporarily; With scalpel sponge is cut into 0.5 * 0.5cm
2Fritter, totally ten, about 1 gram shifts 200mL through 0.22 μ m membrane filtration degerming and add in the natural sea-water of 400mg/L gentamicin, leaves standstill 10 minutes; In liquid lift-type inserted block reactor, add the natural sea-water of 1.8L through 0.22 μ m membrane filtration degerming; The sponge block of handling is inserted on the glass base of liquid lift-type inserted block reactor, in cultivating reactor, cultivates; The chrysophyceae that adds final concentration weekly and be 200,000 cell/mL is as food; Weekly sponge is together taken out together with inserted block, remove surface contaminants and wipe away dried surperficial seawater, weigh.The increase that spongy tissue has shown biomass in first three week, but along with constantly original bone being abandoned in the histokinesis, the spongy biological amount falls back to initial value when 6 weeks, stop test.Show this reaction system suit in, Short-term Culture, and biological siliceous Metabolic activity is strong.The results are shown in Figure 2, Fig. 3.
Get the laboratory and support sponge temporarily, the underwent operative cutter is cut into 0.5 * 0.5cm
2Fritter, totally ten, 1.3g, transfer is advanced 200mL process filtration sterilization and is added in the natural sea-water of 400mg/L gentamicin, leaves standstill 10 minutes; In liquid lift-type inserted block reactor, add the natural sea-water of 1.8L through 0.22 μ m membrane filtration degerming; The sponge block of handling is inserted on the glass base of liquid lift-type inserted block reactor, in cultivating reactor, cultivates; The chrysophyceae that adds final concentration weekly and be 200,000 cell/mL adds the silane coupling agent 3-urea groups propyl trimethoxy silicane of 60 μ m simultaneously as food; The bone of when weighing weekly sponge being abandoned is collected, and dries; After the 6th week finished, spongy tissue is collected, about 1.2g weighs; With the whole spongy tissues and the bone resistates vitriol oil: the mixing acid 20mL of concentrated nitric acid=3: 1 (volume ratio) handled one hour at 80 ℃; Utilize the resulting mixing solutions of acid funnel suction filtration, filter cake is a spicule; Utilize the washed with de-ionized water filter cake to neutral, air-dry, weigh, obtain final sponge source biological siliceous material.Wherein derive from the heavily about 0.17g of spicule of the bone of being abandoned in the cultivation.With film sponge weight psychrometric ratio in great numbers 1: 10, spicule accounted for dry weight 50% and calculates, and these spicules are equivalent to the spicule amount that 3.4g weight in wet base film spongy tissue in great numbers is provided.That is to say, utilize this mode, though to the biomass that obtained in the 6th week close with initial biomass,, the spicule amount that is obtained is far above directly directly handling these sponges, and its difference is exactly that this special " biological factory " manufacturing is come out.By the spicule that is obtained is carried out infrared detection, with the spicule of natural sponge in contrast, find that the spicule that utilizes this mode to obtain has successfully been introduced organic group, sees Fig. 4.Among the figure sign place be 3-urea groups propyl trimethoxy silicane C-C skeleton vibrations characteristic peak (on), and in normal sponge spicule, be not have tangible this characteristic peak (descending).In the detection contrast used natural spicule consumption be 10 times to 3-urea groups propyl trimethoxy silicane interpolation group.Illustrate, utilize this method not only can carry out the production of the biological siliceous material in sponge source, can also in the biological siliceous material of sponge source, introduce the chemical modification group effectively.
After sponge is gathered, after supporting temporarily in 24 hours, utilize 7ppmCuSO4 and 400mg/L GentamycinSulfate (Justaware Pharmaceutical Co.Ltd) to handle three hours.Reference literature (Primmorphs from archaeocytes-dominant cell population of thesponge Hymeniacidon perleve:Improved cell proliferation andspiculogenesis (Biotechnology and Bioengineering, v84-5, method 583-590)) is carried out Ficoll cell density gradient separations, gets 12 ~ 15% confluent monolayer cells and carries out the ADCP cultivation.Cell cultures is carried out in 9cm culture dish (Corning), inoculum density 5 * 10
6Cell/mL.Substratum is through the filtering natural sea-water of 0.22 μ m, adds 60 μ M silane coupling agent 3-urea groups propyl trimethoxy silicanes and 30 μ M ironic citrates.18 ℃ of static cultivations of lucifuge were changed liquid in per two days 100%.Cultivate the back and in cell aggregation, can find the existence of newborn spicule, see Fig. 5.Arrow is depicted as newborn spicule.
Claims (7)
1. the method for reactor culture sponge or its cells produce biological siliceous material, it is characterized in that: reactor adopts natural sea-water, drop into conventional sponge bait micro-algae and cultivate sponge or its cell, in reactor, be provided with from the horizontal by the adherance of 45-135 degree aufwuchsplate, sponge or its cell can be grown along aufwuchsplate, be added with silane coupling agent at natural sea-water, the volumetric molar concentration of silane coupling agent in natural sea-water is 10-100 μ M, utilize the phenomenon of abandoning the original bone of part in the spongy tissue growth movement process to carry out the spicule results, or in stripped sponge cell culture system, produce spicule, obtain biological siliceous material.
2. according to the described method of claim 1, it is characterized in that: carry out purifying to obtaining biological siliceous material, wherein bone resistates or spongy tissue carried out pickling, remove organicly, obtain sponge source biological siliceous material; Described acid is that concentrated nitric acid or volume ratio are 0.1-4: 1 the vitriol oil and the mixing acid of concentrated nitric acid, the treatment temp of pickling are 25-80 ℃, and the treatment time is more than 0.5 hour or 0.5 hour.
3. according to the described method of claim 1, it is characterized in that: described silane coupling agent is siloxanes and derivative thereof based on orthosilicic acid; Siloxanes and derivative thereof with a silicon-carbon bonds; Or other has the silane derivative with the orthosilicic acid polymerization reaction take place.
4. according to the described method of claim 3, it is characterized in that: described orthosilicic acid is tetramethoxy-silicane or tetraethoxysilane for the siloxanes and the derivative thereof on basis; Siloxanes and derivative thereof with a silicon-carbon bonds are propyl trimethoxy silicane or 3-urea groups propyl trimethoxy silicane.
5. according to the described method of claim 1, it is characterized in that: produce sponge source biological siliceous material by reactor culture sponge, carry out the biological siliceous material chemically modified, specific operation process is,
1) the wild sponge that will gather utilizes the natural sea-water clean surface through 0.22 μ m membrane filtration degerming, and the pollution of surface thing is removed;
2) sponge after will cleaning is cut into the fritter that area is about 0.5-3cmX0.5-3cm with scalpel;
3) with step 2) in the sponge fritter that cuts shift into through 0.22 μ m membrane filtration degerming and add in the natural sea-water of 200-400mg/L gentamicin, left standstill 5-10 minute;
4) adding wherein is added with silane coupling agent through the natural sea-water of 0.22 μ m membrane filtration degerming in reactor, and is stand-by;
5) sponge fritter treated in the step 3) is fixed on the adherance in the reactor, in reactor, cultivates 15～23 degrees centigrade of culture temperature;
6) add little algae of final concentration 10-30 ten thousand cells/mL as bait.
6. according to the described method of claim 1,, set up the sponge cell culture system that exsomatizes, carry out the biological siliceous material production of sponge source, it is characterized in that with reference to conventional sponge cell isolated culture method: specific operation process,
1) the no calcium magnesium seawater that the spongy tissue utilization of weight in wet base 1 gram is contained volumetric molar concentration 2-10mM ethylenediamine tetraacetic acid (EDTA) disperses, and utilizes density gradient centrifugation method to collect the archeocyte enriched layer in the cell that is obtained, and being adjusted to final concentration is 5X10
6Cell/mL inoculates; Substratum is for adding the ironic citrate of 10-30 μ M through the filtering natural sea-water of 0.22 μ m; 18～23 degrees centigrade of culture temperature;
2) add silane coupling agent in substratum and cultivate, the replacing substratum was 1 time in per two days to three days 100%;
3) stopped in the 20 to 30 day cultivating, collect and cultivate the cell aggregation that obtains;
4) handle results sponge source biological siliceous material according to claim 5 step 4～6.
7. according to the device of the described method of claim 1, it is characterized in that: described reactor is that liquid lift-type inserted block is cultivated reactor,
Comprise a container, the container inner bottom part is provided with hollow sheeting, and hollow sheeting is provided with aperture, and hollow sheeting links to each other with water pump by pipeline, and circulation enters into hollow sheeting inside through water pump to cultivate seawater, upwards flows out from the water outlet duct of hollow sheeting surface arrangement;
Be placed with the vertical slender glass pin that has glass base on the hollow sheeting, in order to fixing spongy tissue piece, the fixed arrangement of spongy tissue inserted block by glass needle is on hollow sheeting;
Spongy tissue is fixed on the inserted block, and can be under flow action upwards growth of glass needle on the inserted block; Be provided with the admission passage that links to each other with air pump in the container, by air pump to the system tonifying Qi.
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| CN111012916A (en) * | 2018-10-09 | 2020-04-17 | 厦门大学 | Surface-modified sponge spicule and preparation method and application thereof |
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| CN102071166B (en) * | 2009-11-20 | 2012-12-26 | 中国科学院大连化学物理研究所 | Method for tissue artificial culture of hymeniacidon perleve by using program for temperature regulation and control |
| CN105858669B (en) * | 2016-04-27 | 2017-10-27 | 厦门大学 | The preparation method of high-purity sponge spicule |
| CN108163861B (en) * | 2018-02-09 | 2019-08-30 | 四川理工学院 | The purification process and purification system of a kind of Silica Sponge Spicule and application |
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| CN1775864A (en) * | 2004-11-17 | 2006-05-24 | 信越化学工业株式会社 | Silicon rubber composition for sponge |
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| Alan Duckworth ETAL.Sponge aquaculture forthe production of biologically active metabolites : theinfluence of farming protocols and environment.Aquaculture221.2003,221311-329. * |
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| CN111012916A (en) * | 2018-10-09 | 2020-04-17 | 厦门大学 | Surface-modified sponge spicule and preparation method and application thereof |
| CN111012916B (en) * | 2018-10-09 | 2021-10-01 | 厦门大学 | Surface-modified sponge spicule and preparation method and application thereof |
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