CN105695355B - A method of it is co-cultured using two-wheeled and flocculence prepares bacterium algae cell - Google Patents
A method of it is co-cultured using two-wheeled and flocculence prepares bacterium algae cell Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention discloses a kind of methods co-cultured using two-wheeled and flocculence prepares bacterium algae cell, it include: that general bacterium raw in the rice of first time addition and chlorella 1) are subjected to first round bacterium algae co-cultivation under light illumination, after culture, first round bacterium algae co-culture media is obtained；2) raw general bacterium in the rice for adding second of addition again in first round bacterium algae co-culture media carries out the second wheel bacterium algae under light illumination and co-cultures, after culture, obtains the second wheel bacterium algae co-culture media；3) flocculation harvesting processing is carried out to the second wheel bacterium algae co-culture media, obtains bacterium algae cell.The present invention is by carrying out the growth rate and biomass that bacterium algae co-cultures raising frustule using general bacterium raw in rice and chlorella, improve frustule flocculating property simultaneously, reduce chemical floc use and the murder by poisoning to frustule, improve frustule harvest efficiency, it reduces frustule and harvests cost, so that the industrial production and development and application cost of pyrenoids bead be greatly reduced.
Description
Technical field
The present invention relates to bacterium algae Coculture techniques fields, and in particular to a kind of co-cultured using two-wheeled prepares bacterium with flocculence
The method of frustule.
Background technique
Chlorella is a kind of monoplast green alga, and environmental suitability is strong, is easy to large-scale culture, rich in cell
Nutriment is widely used in health food, feed, food additives, fine design product and as pharmaceutical preparation raw material.It is small
Ball algae is rich in short chain fatty acids, is good biodiesel raw material (creep, Xu Xudong, Fang Xiantao, Hu Hanhua high oil-producing bead
The screening of algae and its grease analyze aquatile journal, 2012,36 (3): 426-432.).Chlorella pyrenoidosa is that China is common
Chlorella kind, content albumen is high, it is necessary to which amino acid is abundant, is a kind of excellent forage protein source (Li Guoping pyrenoids
Amino acid content and feeding value analyze Chinese Wild plant resources .2003,22 (2): 23-25 in chlorella powder).In recent years
The productivity effect of chlorella is significant, and prospect is very wide.Especially emerging helotism technology is expected to break through current chlorella
Low yield existing for exploitation aspect harvests the technical restrictions such as difficult, at high cost, is paid close attention to by industry.
Existing research shows there is the extensive interaction phenomenon such as from parasitism to symbiosis between microalgae and microorganism, certain is slightly raw
Object can promote frustule growth by secretion growth hormone and Signal Regulation substance, improve frustule biomass and biochemical component
Accumulate [Ueda H, Otsuka S, Senoo K.Bacterial communities constructed in artificial
consortia of bacteria and Chlorella vulgaris.Microbes Environment,2010,25(1):
36-40.].But influence of different types of bacterium to chlorella growth with metabolism is different, some promotions, some inhibition, and effect is poor
It is very not big.Fungal component of the strain excellent as chlorella is obtained by screening, on the one hand artificial constructed helotism system is expected to
Frustule growth rate is set to greatly improve (Park Y, et al.Growth promotion of Chlorella
ellipsoidea by co-inoculation with Brevundimonas sp.isolated from the
microalga.Hydrobiologia,2008,598(1):219-228；Kim B H,et al.Role of rhizobium,a
plant growth promoting bacterium,in enhancing algal biomass through
Mutualistic interaction.Biomass and Bioenergy, 2014,69 (3): 95-105.), overcome chlorella
The low technical problem of biological yield.On the other hand, since chlorella cells are (3-12 μm) small in size, density is low, cell surface
It is negatively charged, it is sufficiently stable under dispersity, it is difficult to settle, these features keep chlorella harvesting difficult, increase energy consumption and
Production cost.Chlorella collecting method has flocculence, centrifugal process, filtration method and gas floatation process etc. at present, wherein centrifuge separation is state
The method that inside and outside factory's microalgae recovery generally uses, but this method equipment investment is big, harvests at high cost, current chlorella cells harvest
Cost accounts about the 1/3 of total production cost, limits the industrial applications of chlorella, and it is thin to seek low cost, efficient microalgae
Born of the same parents harvest one of the problem of approach is microalgae industrialization urgent need to resolve (Lee J, Cho D H, Ramanan R, Kim B H, Oh H
M,Kim H S.Microalgae-associated bacteria play a key role in the flocculation
Of Chlorella vulgaris.Bioresource Technology, 2013,131 (2): 195-201. Haiyang etc.: energy
Source microalgae recovery Research progress.2013,32 (9): 2092-2098.).Flocculation sedimentation mainly utilizes chemical floc
Electrification property make frustule aggregation sedimentation, have advantage low in cost, easy to operate, practical.But pure chemistry is wadded a quilt with cotton
It is solidifying that toxic action, shadow are generated to chlorella to the possible undesirable and excessively high chemical floc concentration of chlorella settlement action
The sound subsequent industrial applications of chlorella [Zuo Zhipeng waits the research hubei agricultural science of flocculation sedimentation harvesting chlorella,
2015,54(9):2206-2213.].Biological flocculant is that microorganism itself generates, with the natural of efficient flocculation
It is multifactor effect that polymer substance, which mainly includes several substances, the effects of biological flocculant such as sugar, glycolipid, protein, DNA,
Common results, mainly by bridge linking effect, electrically neutralize, volume sweep effect and some complexity chemical reaction to microalgae progress
Flocculating setting.Compared with traditional flocculant, biological flocculant is easily degraded by microorganisms, flocculating effect nontoxic to environment
Obviously, applied widely, flocculant producing strains source is more, and (Shi Chunyang waits the research and application progress of microbial flocculant
[J] pollution prevention technique, 2013,26 (3): 48-51.).Andrew etc. is reported ballstone algae (Pleurochrysis
Carterae) and a kind of microbe symbiotic culture, the latter will generate a kind of tool when the nutritional components of cultivating system are depleted
There is the extracellular substance of flocculation, so that ballstone algae be made to flocculate, the rate of recovery is up to 90% (Andrew K L, et
Al.Microbial flocculation, a potentially low-cost harvesting technique for
marine microalgae for the production of biodiesel[J].Journal of Applied
Phycology, 2009,21 (5): 559-567.).Microbe symbiotic method is combined with conventional centrifugal or filter method, is able to achieve
Energy microalgae low cost, high efficiency, free of contamination harvesting (Zhang Haiyang etc.: energy microalgae harvesting technique progress.2013,32
(9): 2092-2098.).Therefore, the excellent fungal component in artificial constructed helotism system is possibly through generation bioflocculation
Agent substance improves the flocculating property of chlorella cells, when being used cooperatively with chemical floc applied to frustule harvesting, reduces
The dosage of chemical floc, mitigates the toxic action of chemical floc to a certain extent, and frustule is greatly reduced
Harvest cost.
The key that novel helotism technology is developed for the industrialized production of chlorella is excellent symbiosis bacteria strain
Screening and application.Endophyte of plant is can to colonize in health plant, and the one of harmonious symbiosis is established with host plant
Quasi-microorganism.Rice plant interior raw microorganism containing there are many, is important endogenetic bacteria resource, wherein general Pseudomonas is the normal of rice
See endophyte, have the function of promoting plant growth and increases plant weights [S á nchez-Matamoros R C, et
al.Structure of the O-antigen of the lipopolysaccharide isolated from Pantoea
ananatis AEP17,a rhizobacterium associated with rice.Carbohydrate Research,
2013,369:25-30.], raw general bacterium promotes the main mechanism of the growth of host's rice to include secretion auxin, promote in rice
(RuizaD, et al..Characterization and screening the ofplant probiotic such as fixed nitrogen and Soluble phosphorus
traits of bacteria isolated from rice seeds cultivatedin Argentina.The
Journal of Microbiology, 2011,49 (6): 902-912), since microalgae has both plant cell photosynthesis and micro-
The characteristic of biological cell fast-growth theoretically there is the endophyte of symbiosis function may also play class to microalgae cell plant
As biological action, the existing a small amount of research evidence of this respect shows there is the microorganism of growth-promoting effect can also mention plant
Biomass (Kim B H, et al.Role of rhizobium, a plant growth promoting of high microalgae
bacterium,in enhancing algal biomass through mutualistic interaction.Biomass
And Bioenergy, 2014,69 (3): 95-105.), however have no being total to using rice endophyte and microalgae both at home and abroad so far
The research of raw effect exploitation chlorella production new technology.
Summary of the invention
The present invention provides a kind of methods co-cultured using two-wheeled and flocculence prepares bacterium algae cell, by applying rice
The growth rate and biomass of interior life general bacterium and chlorella progress bacterium algae co-cultivation raising frustule, while improving frustule flocculation
Performance, reduce chemical floc use and the murder by poisoning to frustule, improve frustule harvest efficiency, reduce frustule harvesting at
This, so that the industrial production and development and application cost of pyrenoids bead be greatly reduced.
A method of it is co-cultured using two-wheeled and flocculence prepares bacterium algae cell, comprising:
1) general bacterium raw in the rice of first time addition and chlorella are subjected to first round bacterium algae co-cultivation under light illumination, cultivated
After, obtain first round bacterium algae co-culture media；
2) raw general bacterium in the rice for adding second of addition again in first round bacterium algae co-culture media carries out the under light illumination
Two wheel bacterium algaes co-culture, and after culture, obtain the second wheel bacterium algae co-culture media；
3) flocculation harvesting processing is carried out to the second wheel bacterium algae co-culture media, obtains bacterium algae cell.
It is used as the preferred technical solution of the present invention below:
In step 1), the condition of the first round bacterium algae co-cultivation are as follows: condition of culture is 23 DEG C~33 DEG C, light intensity
1500~2500Lux, photoperiod are daytime/10 10~14hr~14hr night, predominantly stationary culture, are shaken every day culture bottle mixing
2~6 times until cultivation cycle terminate, cultivation cycle be 6~12 days.Further preferably, the first round bacterium algae co-cultures
Condition are as follows: condition of culture is 28 DEG C, light intensity 2000Lux, and the photoperiod is 12hr daytime/12hr night, predominantly stationary culture, daily
It shakes culture bottle to mix 4 times until cultivation cycle terminates, cultivation cycle is 8 days.
The bacterium algae ratio of raw general bacterium and chlorella is 1~10000:1 in the rice that the first time is added, in a certain range
Interior, the ratio of raw general bacterium is higher in rice, can more promote the growth of chlorella, more can be improved chlorella yield.Further
It is preferred that the bacterium algae ratio of raw general bacterium and chlorella is 10~1000:1 in the rice that is added of the first time.
Commercial product can be used in raw general bacterium in the rice, and Chinese agriculture Organism Depositary (ACCC) can be used
That sells is encoded to raw general bacterium strain in 10454 rice.
The chlorella can be chlorella pyrenoidosa, and commercial product can also be used, it is wild that the Chinese Academy of Sciences can be used
The chlorella pyrenoidosa that the biological commercially available number of Germplasm Bank fresh water algae library (FACHB) is FACHB-1222.
In step 2), the condition of the second wheel bacterium algae co-cultivation are as follows: condition of culture is 23 DEG C~33 DEG C, light intensity
1500~2500Lux, photoperiod are daytime/10 10~14hr~14hr night, predominantly stationary culture, are shaken every day culture bottle mixing
2~6 times until cultivation cycle terminate, cultivation cycle be 2~12 days.Further preferably, the second wheel bacterium algae co-cultures
Condition are as follows: condition of culture is 28 DEG C, light intensity 2000Lux, and the photoperiod is 12hr daytime/12hr night, predominantly stationary culture, daily
It shakes culture bottle to mix 4 times until cultivation cycle terminates, cultivation cycle is 4~8 days.
In the rice of described second of addition in raw general bacterium and the rice being added for the first time the ratio of raw general bacterium be 0.25~
4:1.Further preferably, the ratio of raw general bacterium is in the rice that raw general bacterium and first time are added in the rice of described second addition
1:1, can advantageously promote the growth of chlorella, more can be improved chlorella yield.
In step 3), the flocculation harvesting processing includes: that cation wadding is first added in the second wheel bacterium algae co-culture media
Solidifying agent CaCl2, FeCl is added later3Then flocculant harvests.
Cationic flocculant CaCl is added2Afterwards, CaCl2Concentration be 5~20mM, be added FeCl3After flocculant, FeCl3Wadding
The concentration of solidifying agent is 0.1~0.5mM.
Further preferably, cationic flocculant CaCl is sequentially added in the second wheel bacterium algae co-culture media2To final concentration of 5
~20mM, 100-300rpm, which shake at a slow speed, mixes 1~5min, then adds FeCl3Flocculant to final concentration of 0.1~0.5mM,
100-300rpm shake at a slow speed mix 1~5min, be then allowed to stand 5min-1h, make frustule flocculate to be formed flocculation group settle down,
Then it harvests.Harvesting can use low speed centrifugal process or filtration method in short-term.
Most preferably, cationic flocculant CaCl is sequentially added in the second wheel bacterium algae co-culture media2Extremely final concentration of 10mM,
100-300rpm, which shakes at a slow speed, mixes 2min, then adds FeCl3Flocculant shakes at a slow speed to final concentration of 0.26mM, 100-300rpm
Swing mix 2min, be then allowed to stand 5min-1h, make frustule flocculate to be formed flocculation group settle down.
Bacterium algae co-culture media after flocculation treatment, which is directlyed adopt pp carpet filtering, can be obtained cell products, collection
Bacterium algae living cells can be used as the living cells biological base of the sewage treatments such as harmful substance such as azo dyes biodegrade in water, or warp
Cell products are obtained after drying, as the raw material for preparing feed, extraction grease, frustule pigment etc..
The mechanism that the present invention mends bacterium increasing algae technology is: during the co-cultivation of bacterium algae interaction, due to using small
Ball algae suitable culture medium and condition of culture, symbiotic bacteria can promote the growth of microalgae, on the contrary, there may be bacterium lifes for microalgae
Long inhibiting substances, such as chlorellin inhibit the proliferation of bacterium cell, and so with the extension of cultivation cycle, algae is thin in bacterium algae culture solution
Born of the same parents are in gradually growth trend, and bacterium cell quantity is then gradually decayed, can be fewer and fewer, only few in last bacterium algae cultivating system
The bacterium cell survival of amount.Therefore, bacterium increases the Effect of promoting growth of algae with the increase of inoculation bacterium algae ratio in a certain range,
And become apparent in the increasing algae effect for mending bacterium initial stage than later period, this is also to determine inoculation bacterium algae ratio set by the present invention, be total to
Cultivation cycle period length and the technical foundation for mending bacterium inoculation time.Preferably, the present invention is to be inoculated with bacterium algae ratio 10-1000:
1, first run culture 8 days, second took turns and hereafter mends the bacterium co-cultivation time based on 4-8 days, to reach ideal technical effect.This hair
Bright raising frustule flocculating property, the mechanism for reducing frustule harvesting cost are: raw general bacterium in the rice during co-culturing
Some extracellular bioflocculation substances have been secreted in generation, can be interacted with chemical floc and be improved the flocculating property of frustule, altogether
When a certain amount of chemical floc progress cell harvesting being added after culture, mention the flocculating setting efficiency of frustule substantially
Height, and with the increase of bacteria concentration, the bioflocculation substance for generating secretion is more, and the effect for improving frustule flocculating rate is got over
Obviously, the harvest cost of frustule is so reduced.
Compared with prior art, the present invention has the advantage that
The present invention is low for frustule production efficiency in current chlorella industrialized production, harvesting is difficult, higher cost
Key technology bottleneck develops a kind of method for reducing chlorella pyrenoidosa production harvest cost using general bacterium raw in rice, mainly
It is to co-culture the growth rate and biomass that improve frustule by bacterium algae, while improving frustule flocculating property, reduces chemistry
The use of flocculant improves frustule harvest efficiency, so that the production development cost of pyrenoids bead be greatly reduced.Due to rice
The interior general Pseudomonas endophyte of plant of life, safe and non-toxic, raw general bacterium-chlorella pyrenoidosa is thin in the rice obtained using the method for the present invention
Born of the same parents' product can be applied to the preparation of the Related products such as exploitation feed, bait and biodiesel directly as raw material.It can also pass through
Exploitation after bacteria removing, for products such as food, drug, fine chemistry industries.
Bacterium algae co-culture media after flocculation treatment is directlyed adopt pp carpet filtering by the present invention can be obtained cell products,
The bacterium algae living cells of collection can be used as the living cells biology base of the sewage treatments such as harmful substance such as azo dyes biodegrade in water
Material, or cell products are obtained after drying, as the raw material for preparing feed, extraction grease, frustule pigment etc..
The technology of the present invention is easy to operate, low in cost, has and quicklys increase the significant of chlorella pyrenoidosa cell yield
Effect co-cultures in the range of bacterium algae inoculation is than being 10~1000:1 by two-wheeled plus bacterium, ties within cultivation cycle (12~16 days)
Shu Hou, pure algae culture control can increase 23.68~104 times, and bacterium algae to the chlorella pyrenoidosa cell quantity harvested on year-on-year basis
Than bigger, the second wheel cultivation cycle is longer, and technical effect is more obvious.Meanwhile albumen can be made by two-wheeled bacterium algae Coculture techniques
Core chlorella cells flocculating setting efficiency improves 29.76-37.74 times than control, and frustule flocculating setting rate maximum can reach
95% or more, it is very significant to be applied to frustule industrialization harvesting effect.Method of the present invention can be by quickly improving egg
White nucleus chlorella growth rate reaches reduction chlorella pyrenoidosa production harvest cost with frustule flocculation efficiency is greatly improved
Technical effect, the remarkable benefit in terms of the application and development of chlorella pyrenoidosa has a extensive future.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention is carried out below clear
Chu, complete description.The embodiment provided is of the invention only for illustrating, the application range being not intended to be limiting of the invention.With
The percentage of lower appearance, is such as not particularly illustrated, and is mass percent.
Embodiment one
1) Chinese agriculture Organism Depositary (ACCC) is purchased from using general bacterium strain raw in rice, is encoded to 10454, bacterium
It after single colonie is chosen in activation kind according to a conventional method, is inoculated into the triangular flask of the LB liquid medium containing 10mL, carries out amplification cultivation,
It will be in the bacterium solution of logarithmic growth phase after amplification cultivation 16h by bacterium solution: being carried out after LB liquid medium volume ratio=1:10 inoculation
Secondary to spread cultivation, condition of culture is 37 DEG C, dark culturing in 200rpm constant-temperature table.Spread cultivation 10h to OD for the second time600=1 is used as bacterium
The seed cell that algae co-cultures, according to growth curve, bacterium growth is in logarithmic phase, measures culture solution using conventional panel counting method
In living bacteria count (CFU, Colony-Forming Units), bacterial concentration is about 2 × 10 at this time8CFU/mL.According to bacterium
Bacterium cell quantity needed for algae co-cultures takes appropriate seed bacterium solution in 25 DEG C of 8000r/min centrifugation 10min of room temperature, precipitates 3 times of bodies
Product sterile water washes twice, then is centrifuged 10min and collects somatic cells, is added and the isometric BG11 fluid nutrient medium of seed bacterium solution
It suspends spare.
2) Chinese Academy of Sciences's wildlife Germplasm Bank fresh water algae library (FACHB) is purchased from using chlorella pyrenoidosa algae,
Specifically, number is FACHB-1222.Chlorella pyrenoidosa original algae solution is taken, by 10-2, 10-3, 10-4Gradient dilution is carried out, is taken dilute
It releases 100 μ L of algae solution and is coated on BG11 solid medium (to add 2% agar in BG11 fluid nutrient medium), culture dish is placed in illumination
It is cultivated in incubator, after 7 days, bottle-green single algae is grown in BG11 culture medium and is fallen, is fallen on and is contained with sterile pipette tips picking list algae
In the triangular flask of 10mL BG11 fluid nutrient medium, amplification cultivation is carried out.The algae of logarithmic growth phase will be in after amplification cultivation 7 days
Cell is by algae solution: carried out after the inoculation of liquid B G11 culture volume ratio=1:10 it is secondary spread cultivation, so spread cultivation 3 times, train every time altogether
It supports 7 days.Condition of culture are as follows: 28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, and each shake is primary sooner or later daily.The
After 3 amplification cultivations, using conventional blood cell plate counting method calculate cell culture fluid in chlorella cells concentration be 2.6 ×
107A cell/mL, as chlorella vulgaris daughter cell culture solution.
3) bacterium algae co-cultivation group and pure algae culture control group are concurrently set, wherein bacterium algae co-cultures histone core chlorella vulgaris
Daughter cell initial inoculation concentration is 2 × 105A cell/mL, in rice raw general strain daughter cell initial inoculation concentration be 2 ×
106CFU/mL, such bacterium algae cell inoculation ratio are 10:1, and the co-cultivation of first run bacterium algae is carried out after mixing.Pure algae culture control group is only
It is inoculated with chlorella pyrenoidosa seed cell, initial inoculation concentration is 2 × 105A cell/mL.Two groups of cultivating systems are 500ml
× 3 bottles, condition of culture is 28 DEG C, light intensity 2000Lux, and the photoperiod is 12hr daytime/12hr night, predominantly stationary culture, daily
It shakes culture bottle to mix 4 times, cultivation cycle is 8 days.
4) it co-cultures the 9th day, co-cultures raw general bacterium cell concentration 2 × 10 in the rice of initial inoculation by bacterium algae6CFU/mL
Benefit is inoculated in rice raw general strain daughter cell, carries out the second wheel bacterium algae and co-cultures, cultivation cycle is 4 days.
5) after cultivation cycle, using the frustule concentration in blood cell plate counting method measurement culture solution, as a result pure algae training
Supporting frustule concentration in control group is 3.58 × 107A cell/mL, bacterium algae co-cultivation group frustule concentration are 9.50 × 108It is a
Cell/mL is 23.68 times of control on year-on-year basis.
Embodiment two:
1) Chinese agriculture Organism Depositary (ACCC) is purchased from using general bacterium strain raw in rice, is encoded to 10454, bacterium
It after single colonie is chosen in activation kind according to a conventional method, is inoculated into the triangular flask of the LB liquid medium containing 10mL, carries out amplification cultivation,
It will be in the bacterium solution of logarithmic growth phase after amplification cultivation 16h by bacterium solution: being carried out after LB liquid medium volume ratio=1:10 inoculation
Secondary to spread cultivation, condition of culture is 37 DEG C, dark culturing in 200rpm constant-temperature table.Spread cultivation 10h to OD for the second time600=1 is used as bacterium
The seed cell that algae co-cultures, according to growth curve, bacterium growth is in logarithmic phase, measures culture solution using conventional panel counting method
In living bacteria count (CFU, Colony-Forming Units), bacterial concentration is about 2 × 10 at this time8CFU/mL.According to bacterium
Bacterium cell quantity needed for algae co-cultures takes appropriate seed bacterium solution in 25 DEG C of 8000r/min centrifugation 10min of room temperature, precipitates 3 times of bodies
Product sterile water washes twice, then is centrifuged 10min and collects somatic cells, is added and the isometric BG11 fluid nutrient medium of seed bacterium solution
It suspends spare.
2) Chinese Academy of Sciences's wildlife Germplasm Bank fresh water algae library (FACHB) is purchased from using chlorella pyrenoidosa algae,
Specifically, number is FACHB-1222.Chlorella pyrenoidosa original algae solution is taken, by 10-2, 10-3, 10-4Gradient dilution is carried out, is taken dilute
It releases 100 μ L of algae solution and is coated on BG11 solid medium (to add 2% agar in BG11 fluid nutrient medium), culture dish is placed in illumination
It is cultivated in incubator, after 7 days, bottle-green single algae is grown in BG11 culture medium and is fallen, is fallen on and is contained with sterile pipette tips picking list algae
In the triangular flask of 10mL BG11 fluid nutrient medium, amplification cultivation is carried out.The algae of logarithmic growth phase will be in after amplification cultivation 7 days
Cell is by algae solution: carried out after the inoculation of liquid B G11 culture volume ratio=1:10 it is secondary spread cultivation, so spread cultivation 3 times, train every time altogether
It supports 7 days.Condition of culture are as follows: 28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, and each shake is primary sooner or later daily.The
After 3 amplification cultivations, using conventional blood cell plate counting method calculate cell culture fluid in chlorella cells concentration be 2.6 ×
107A cell/mL, as chlorella vulgaris daughter cell culture solution.
3) bacterium algae co-cultivation group and pure algae culture control group are concurrently set, wherein bacterium algae co-cultures histone core chlorella vulgaris
Daughter cell initial inoculation concentration is 2 × 105A cell/mL, in rice raw general strain daughter cell initial inoculation concentration be 2 ×
107CFU/mL, such bacterium algae cell inoculation ratio are 100:1, and the co-cultivation of first run bacterium algae is carried out after mixing.Pure algae culture control group is only
It is inoculated with chlorella pyrenoidosa seed cell, initial inoculation concentration is 2 × 105A cell/mL.Two groups of cultivating systems are 500ml
× 3 bottles, condition of culture is 28 DEG C, light intensity 2000Lux, and the photoperiod is 12hr daytime/12hr night, predominantly stationary culture, daily
It shakes culture bottle to mix 4 times, cultivation cycle is 8 days.
4) it co-cultures the 9th day, co-cultures raw general bacterium cell concentration 2 × 10 in the rice of initial inoculation by bacterium algae7CFU/mL
Benefit is inoculated in rice raw general strain daughter cell, carries out the second wheel bacterium algae and co-cultures, cultivation cycle is 4 days.
5) after two-wheeled bacterium algae co-cultures, after culture solution concussion is mixed, take culture solution 1ml using blood counting chamber method
The chlorella pyrenoidosa cell number in culture solution is measured, as a result: frustule concentration is average in pure algae culture control group culture solution
Value is that frustule concentration is 3.58 × 10 in the pure algae culture control group of result7A cell/mL, algae in bacterium algae co-cultivation group culture solution
Cell concentration average value is 1.72 × 109A cell/mL increases by 47.05 times than control.
Embodiment three:
1) Chinese agriculture Organism Depositary (ACCC) is purchased from using general bacterium strain raw in rice, is encoded to 10454, bacterium
It after single colonie is chosen in activation kind according to a conventional method, is inoculated into the triangular flask of the LB liquid medium containing 10mL, carries out amplification cultivation,
It will be in the bacterium solution of logarithmic growth phase after amplification cultivation 16h by bacterium solution: being carried out after LB liquid medium volume ratio=1:10 inoculation
Secondary to spread cultivation, condition of culture is 37 DEG C, dark culturing in 200rpm constant-temperature table.Continuously spreading cultivation three times, condition of culture is 37 DEG C,
Dark culturing in 200rpm constant-temperature table.Spread cultivation 10h to OD for the third time600=1 seed cell co-cultured as bacterium algae, according to
Growth curve, bacterium growth be in logarithmic phase, using conventional panel counting method measurement culture solution in living bacteria count (CFU,
Colony-Forming Units), bacterial concentration is about 2 × 10 at this time8CFU/mL.Bacterium cell number needed for being co-cultured according to bacterium algae
Amount takes appropriate seed bacterium solution in 25 DEG C of 8000r/min centrifugation 10min of room temperature, and precipitating is washed twice with 3 times of volume sterile waters, then
It is centrifuged 10min and collects somatic cells, it is spare that the BG11 fluid nutrient medium suspension isometric with seed bacterium solution is added.
2) Chinese Academy of Sciences's wildlife Germplasm Bank fresh water algae library (FACHB) is purchased from using chlorella pyrenoidosa algae,
Specifically, number is FACHB-1222.Chlorella pyrenoidosa original algae solution is taken, by 10-2, 10-3, 10-4Gradient dilution is carried out, is taken dilute
It releases 100 μ L of algae solution and is coated on BG11 solid medium (to add 2% agar in BG11 fluid nutrient medium), culture dish is placed in illumination
It is cultivated in incubator, after 7 days, bottle-green single algae is grown in BG11 culture medium and is fallen, is fallen on and is contained with sterile pipette tips picking list algae
In the triangular flask of 10mL BG11 fluid nutrient medium, amplification cultivation is carried out.The algae of logarithmic growth phase will be in after amplification cultivation 7 days
Cell is by algae solution: carried out after the inoculation of liquid B G11 culture volume ratio=1:10 it is secondary spread cultivation, so spread cultivation 3 times, train every time altogether
It supports 7 days.Condition of culture are as follows: 28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, and each shake is primary sooner or later daily.The
After 3 amplification cultivations, using conventional blood cell plate counting method calculate cell culture fluid in chlorella cells concentration be 2.6 ×
107A cell/mL, as chlorella vulgaris daughter cell culture solution.
3) bacterium algae co-cultivation group and pure algae culture control group are concurrently set, wherein bacterium algae co-cultures histone core chlorella vulgaris
Daughter cell initial inoculation concentration is 2 × 105A cell/mL, in rice raw general strain daughter cell initial inoculation concentration be 2 ×
108CFU/mL, such bacterium algae cell inoculation ratio are 1000:1, and the co-cultivation of first run bacterium algae is carried out after mixing.Pure algae cultivates control group
It is only inoculated with chlorella pyrenoidosa seed cell, initial inoculation concentration is 2 × 105A cell/mL.Two groups of cultivating systems are
500ml × 3 bottle, condition of culture are 28 DEG C, light intensity 2000Lux, and the photoperiod is 12hr daytime/12hr night, predominantly stationary culture,
It is shaken every day culture bottle to mix 4 times, cultivation cycle is 8 days.
4) it co-cultures the 9th day, co-cultures raw general bacterium cell concentration 2 × 10 in the rice of initial inoculation by bacterium algae8CFU/mL
Benefit is inoculated in rice raw general strain daughter cell, carries out the second wheel bacterium algae and co-cultures, the second wheel cultivation cycle is set as 4 days two
Group.
5) after two-wheeled bacterium algae co-cultures, after culture solution concussion is mixed, take culture solution 1ml using blood counting chamber method
The chlorella pyrenoidosa cell number in culture solution is measured, as a result: when the second wheel cultivation cycle is 4 days, pure algae culture control
Frustule mean concentration is 3.58 × 10 in group culture solution7A cell/mL, frustule concentration in bacterium algae co-cultivation group culture solution
Average value is 3.40 × 109A cell/mL increases by 93.97 times than control.
Example IV:
1) Chinese agriculture Organism Depositary (ACCC) is purchased from using general bacterium strain raw in rice, is encoded to 10454, bacterium
It after single colonie is chosen in activation kind according to a conventional method, is inoculated into the triangular flask of the LB liquid medium containing 10mL, carries out amplification cultivation,
It will be in the bacterium solution of logarithmic growth phase after amplification cultivation 16h by bacterium solution: being carried out after LB liquid medium volume ratio=1:10 inoculation
Secondary to spread cultivation, condition of culture is 37 DEG C, dark culturing in 200rpm constant-temperature table.Continuously spreading cultivation three times, condition of culture is 37 DEG C,
Dark culturing in 200rpm constant-temperature table.Spread cultivation 10h to OD for the third time600=1 seed cell co-cultured as bacterium algae, according to
Growth curve, bacterium growth be in logarithmic phase, using conventional panel counting method measurement culture solution in living bacteria count (CFU,
Colony-Forming Units), bacterial concentration is about 2 × 10 at this time8CFU/mL.Bacterium cell number needed for being co-cultured according to bacterium algae
Amount takes appropriate seed bacterium solution in 25 DEG C of 8000r/min centrifugation 10min of room temperature, and precipitating is washed twice with 3 times of volume sterile waters, then
It is centrifuged 10min and collects somatic cells, it is spare that the BG11 fluid nutrient medium suspension isometric with seed bacterium solution is added.
2) Chinese Academy of Sciences's wildlife Germplasm Bank fresh water algae library (FACHB) is purchased from using chlorella pyrenoidosa algae,
Specifically, number is FACHB-1222.Chlorella pyrenoidosa original algae solution is taken, by 10-2, 10-3, 10-4Gradient dilution is carried out, is taken dilute
It releases 100 μ L of algae solution and is coated on BG11 solid medium (to add 2% agar in BG11 fluid nutrient medium), culture dish is placed in illumination
It is cultivated in incubator, after 7 days, bottle-green single algae is grown in BG11 culture medium and is fallen, is fallen on and is contained with sterile pipette tips picking list algae
In the triangular flask of 10mL BG11 fluid nutrient medium, amplification cultivation is carried out.The algae of logarithmic growth phase will be in after amplification cultivation 7 days
Cell is by algae solution: carried out after the inoculation of liquid B G11 culture volume ratio=1:10 it is secondary spread cultivation, so spread cultivation 3 times, train every time altogether
It supports 7 days.Condition of culture are as follows: 28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, and each shake is primary sooner or later daily.The
After 3 amplification cultivations, using conventional blood cell plate counting method calculate cell culture fluid in chlorella cells concentration be 2.6 ×
107A cell/mL, as chlorella vulgaris daughter cell culture solution.
3) bacterium algae co-cultivation group and pure algae culture control group are concurrently set, wherein bacterium algae co-cultures histone core chlorella vulgaris
Daughter cell initial inoculation concentration is 2 × 105A cell/mL, in rice raw general strain daughter cell initial inoculation concentration be 2 ×
108CFU/mL, such bacterium algae cell inoculation ratio are 1000:1, and the co-cultivation of first run bacterium algae is carried out after mixing.Pure algae cultivates control group
It is only inoculated with chlorella pyrenoidosa seed cell, initial inoculation concentration is 2 × 105A cell/mL.Two groups of cultivating systems are
500ml × 3 bottle, condition of culture are 28 DEG C, light intensity 2000Lux, and the photoperiod is 12hr daytime/12hr night, predominantly stationary culture,
It is shaken every day culture bottle to mix 4 times, cultivation cycle is 8 days.
4) it co-cultures the 9th day, co-cultures raw general bacterium cell concentration 2 × 10 in the rice of initial inoculation by bacterium algae8CFU/mL
Benefit is inoculated in rice raw general strain daughter cell, carries out the second wheel bacterium algae and co-cultures, the second wheel cultivation cycle is set as 8 days.
5) after two-wheeled bacterium algae co-cultures, after culture solution concussion is mixed, take culture solution 1ml using blood counting chamber method
The chlorella pyrenoidosa cell number in culture solution is measured, as a result: when bacterium algae co-cultivation group when the second wheel cultivation cycle is 8 days
When, it is 5.88 × 10 that pure algae, which cultivates frustule mean concentration in control group culture solution,7A cell/mL, bacterium algae co-culture tissue culture
Frustule mean concentration is 6.17 × 10 in nutrient solution9A cell/mL increases by 104 times than control.
Summarize the experimental result for comparing 4 embodiments of above-described embodiment one to four, it is known that, rice is applied using the present invention
The method that the interior general bacterium of life reduces chlorella pyrenoidosa production harvest cost, in the range of bacterium algae inoculation is than being 10~1000:1,
It is co-cultured by two-wheeled plus bacterium, after cultivation cycle (12~16 days), the chlorella pyrenoidosa cell quantity harvested is year-on-year
Pure algae culture control can increase 23.68~104 times, to the growth for promoting chlorella pyrenoidosa and improve cell harvest yield effect pole
It is significant.Wherein bacterium algae ratio is bigger, and the second wheel cultivation cycle is longer, and technical effect is more obvious.
Embodiment five:
In order to verify bacterium algae co-culture method of the invention to improvement chlorella pyrenoidosa flocculation sedimentation efficiency of crop
Technical effect operates as follows:
1) the pure algae obtained after one, two, three cultivation cycle of above-described embodiment is compareed into culture solution and bacterium algae co-cultures
Liquid harvests respectively, after mixing fullys shake, takes 5~10ml culture solution stoste, suitable according to being added after known frustule concentration calculation
The dilution of BG11 fluid nutrient medium is measured, so that the frustule final concentration of each culture group is balanced to 6 × 10 after dilution6A/ml.
2) above-mentioned steps 1 are taken) dilution balance frustule final concentration of 6 × 106The pure algae of a/ml cultivates control group culture solution
50ml carries out flocculation treatment: algae solution 100rpm is mixed at a slow speed 1min；Then cationic flocculant CaCl is added2To final concentration
1min is mixed at a slow speed for 10mM, 100rpm；Finally add FeCl3To final concentration of 0.26mM.After algae solution stands 5min, such as herein
1ml supernatant is carried out 3 times using frustule concentration (a/ml) in supernatant after blood cell plate counting method measurement flocculation treatment, experiment after reason
In parallel, results are averaged, and the flocculating setting rate of frustule is calculated as follows:
Flocculating setting rate (%)=[frustule concentration/6 × 10 in supernatant after 1- flocculation treatment6] × 100%
3) above-mentioned steps 1 are taken respectively) dilution balance frustule final concentration of 6 × 106The bacterium algae co-cultivation group each group of a/ml
Culture solution 50ml, carry out flocculation treatment by step 2) and calculate the flocculating rate of frustule, concrete outcome is shown in Table 1:
Influence of the different bacterium algaes of table 1 than in rice after raw general bacterium co-cultivation to chlorella pyrenoidosa flocculating setting rate
Note: data are three groups of statistical averages in table.
Bacterium algae co-cultures algae flocculating setting rate and improves multiple=(bacterium algae co-cultivation group flocculating setting rate-pure algae culture control
Group flocculating setting rate)/pure algae cultivates control group flocculating setting rate
The present embodiment the result shows that, using two-wheeled bacterium algae Coculture techniques of the invention can make chlorella pyrenoidosa cell wad a quilt with cotton
Solidifying settling efficiency improves 29.76-37.74 times than control, and when bacterium algae ratio is greater than 100:1 or more, frustule flocculating setting
Rate reaches 90% or more, and it is very significant to be applied to frustule industrialization harvesting effect.
Claims (7)
1. a kind of method co-cultured using two-wheeled and flocculence prepares bacterium algae cell characterized by comprising
1) general bacterium raw in the rice of first time addition and chlorella are subjected to first round bacterium algae co-cultivation under light illumination, culture terminates
Afterwards, first round bacterium algae co-culture media is obtained；The bacterium algae ratio of raw general bacterium and chlorella is 10 in the rice that the first time is added
~1000:1；
In the rice in the rice for being encoded to 10454 of the raw general bacterium using Chinese agriculture Organism Depositary (ACCC) sale
Raw general bacterium strain；
2) raw general bacterium in the rice for adding second of addition again in first round bacterium algae co-culture media, carries out the second wheel under light illumination
Bacterium algae co-cultures, and after culture, obtains the second wheel bacterium algae co-culture media；In the rice of described second of addition raw general bacterium and
The ratio of raw general bacterium is 0.25~4:1 in the rice being added for the first time；
3) flocculation harvesting processing is carried out to the second wheel bacterium algae co-culture media, obtains bacterium algae cell.
2. the method according to claim 1 co-cultured using two-wheeled and flocculence prepares bacterium algae cell, which is characterized in that
In step 1), the condition of the first round bacterium algae co-cultivation are as follows: condition of culture is 23 DEG C~33 DEG C, light intensity 1500~
2500Lux, photoperiod are daytime/10 10~14hr~14hr night, and stationary culture is shaken every day culture bottle and mixes 2~6 times until training
End cycle is supported, cultivation cycle is 6~12 days.
3. the method according to claim 1 co-cultured using two-wheeled and flocculence prepares bacterium algae cell, which is characterized in that
In step 1), the bacterium algae ratio of raw general bacterium and chlorella is 10~1000:1 in the rice that the first time is added.
4. the method according to claim 1 co-cultured using two-wheeled and flocculence prepares bacterium algae cell, which is characterized in that
In step 2), the condition of the second wheel bacterium algae co-cultivation are as follows: condition of culture is 23 DEG C~33 DEG C, light intensity 1500~
2500Lux, photoperiod are daytime/10 10~14hr~14hr night, and stationary culture is shaken every day culture bottle and mixes 2~6 times until training
End cycle is supported, cultivation cycle is 2~12 days.
5. the method according to claim 1 co-cultured using two-wheeled and flocculence prepares bacterium algae cell, which is characterized in that
In step 3), the flocculation harvesting processing includes: that cationic flocculant is first added in the second wheel bacterium algae co-culture media
CaCl2, FeCl is added later3Then flocculant harvests.
6. the method according to claim 5 co-cultured using two-wheeled and flocculence prepares bacterium algae cell, which is characterized in that
In step 3), cationic flocculant CaCl is added2Afterwards, CaCl2Concentration be 5~20mM, be added FeCl3After flocculant, FeCl3
The concentration of flocculant is 0.1~0.5mM.
7. the method according to claim 1 co-cultured using two-wheeled and flocculence prepares bacterium algae cell, which is characterized in that
In step 3), the flocculation harvesting processing includes: to sequentially add cationic flocculant in the second wheel bacterium algae co-culture media
CaCl2It is 5~20mM to concentration, 100-300rpm, which shakes at a slow speed, mixes 1~5min, then adds FeCl3Flocculant to concentration is
0.1~0.5mM, 100-300rpm, which shake at a slow speed, mixes 1~5min, is then allowed to stand 5min-1h, frustule is made to flocculate to form flocculation
Group settles down, and then harvests.
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