CN105755091B - A method of it co-cultures using general bacterium raw in rice and chlorella and improves chlorella lipid-producing - Google Patents
A method of it co-cultures using general bacterium raw in rice and chlorella and improves chlorella lipid-producing Download PDFInfo
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Abstract
The method for improving chlorella lipid-producing is co-cultured using general bacterium raw in rice and chlorella the invention discloses a kind of, comprising: 1) general bacterium raw in rice and chlorella are subjected to bacterium algae co-cultivation under light illumination, after culture, obtain bacterium algae co-culture media；2) cell separation is carried out to bacterium algae co-culture media, obtain chlorella cells precipitating and includes the supernatant bacterium solution of raw general bacterium in rice, chlorella cells precipitating is obtained into chlorella cells dry product after processing；3) chlorella cells dry product is crushed, obtains algae powder, total grease is extracted using organic solvent extracting solution later, obtains total grease.The present invention is co-cultured using general bacterium raw in rice and chlorella, to promote the growth of chlorella, improves bead algae biomass, while also improving fat content and lipid-producing.The method of the present invention is easy to operate, and the remarkable benefit in terms of the application and development of chlorella has a extensive future.
Description
Technical field
The present invention relates to microorganisms and field of biotechnology, and in particular to a kind of total using general bacterium raw in rice and chlorella
The method of culture and improvement chlorella lipid-producing.
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 can be applied to the every field such as food development, healthcare products, beauty and drug development.Chlorella is rich in short chain
Fatty acid is that (creep waits the screening and its grease analysis Hydrobiology of high oil-producing chlorella to good biodiesel raw material
Report, 2012,36 (3): 426-432.), and linoleic acid in chlorella institute Lipid-producing and gamma-Linolenic acid etc. have and adjust blood
Rouge, drop blood it is glutinous and other effects [Zhang Qi waits chlorella nutritional activities progress food research and development, 2015,36 (13):
139-142.].Raising bead algae biomass and the synthesis for promoting frustule nutriment are that the technology of development and application chlorella is closed
Key.Existing research shows there is extensive interaction phenomenon, the certain micro-organisms pair such as from parasitism to symbiosis between microalgae and microorganism
The growth metabolism of frustule influences significant (Ueda H, et al..Bacterial communities constructed in
artificial consortia of bacteria and Chlorella vulgaris.Microbes Environment,
2010,25(1):36-40.)。
Endophyte of plant is can to colonize in health plant, and establish a kind of micro- of harmonious symbiosis with host plant
Biology.Rice plant interior raw microorganism containing there are many, is important endogenetic bacteria resource, from the general bacterium of interior life of rice plant
The biomass, chlorophyll and phosphorus content of host's rice can be significantly improved, and influences transfer (FengY, the et of photosynthate
al.Rice endophyte Pantoea agglomeransYS19 promotes hostplant growth and
affects allocations of hostphotosynthates.Journal ofApplied Microbiology,
2006,100 (5): 938-945. Liu Jia, wait in raw pantoea agglomerans HAUM1 to host's rice colonize and the Hubei growth-promoting functions
Agricultural sciences, 2011,50 (23): 4820-4824.).Since chlorella has chloroplaset as plant cell, it is able to carry out
Photosynthesis, biochemical metabolism feature and plant have similitude, theoretically, in the plant to plant with Metabolism regulation effect
Raw bacterium can also influence growth and the metabolic activity of chlorella, but be had no both at home and abroad so far using the plant including rice endophyte
Object endophyte promotes the technical research and application of chlorella growth and biochemical component dynamic accumulation.
The Chinese invention patent application of 103045481 A of application publication number CN (application No. is 201310004747.X) is public
A kind of method for promoting chlorella growth and improving its chlorophyll and mycoprotein content is opened, which is in chlorella
The inner ether sugar from biomass pyrolytic is added in culture solution or the cellulose pyrolytic behavior through detoxification treatment, inner ether sugar additive amount are
5mM-50mM, cellulose pyrolytic behavior additive amount 0.25%-2% by weight.In cellulose biomass high temperature pyrolysis product
Ether sugar or pyrolytic behavior are as promotor, the results showed that inner ether sugar or pyrolytic behavior can be obviously promoted the growth of chlorella, and promotion rate is most
Up to 194%, and it is remarkably improved chlorophyll and mycoprotein content, so that the utility value of cellulose biomass can be improved.
Summary of the invention
The present invention provides a kind of sides that raising chlorella lipid-producing is co-cultured using general bacterium raw in rice and chlorella
Method, this method can be improved bead algae biomass, fat content and lipid-producing.
A method of it co-cultures using general bacterium raw in rice and chlorella and improves chlorella lipid-producing, comprising:
1) general bacterium raw in rice and chlorella are subjected to bacterium algae co-cultivation under light illumination, after culture, it is total to obtain bacterium algae
Culture solution；
2) cell separation is carried out to bacterium algae co-culture media, obtains chlorella cells precipitating and include to give birth to the upper of general bacterium in rice
Chlorella cells precipitating is obtained chlorella cells dry product by clear bacterium solution after processing；
3) chlorella cells dry product is crushed, obtains algae powder, total grease is extracted using organic solvent extracting solution later, is obtained
Total grease.
The present invention is co-cultured using general bacterium raw in rice and chlorella, to promote the growth of chlorella, improves bead
Algae biomass, while also improving fat content and lipid-producing.
It is used as the preferred technical solution of the present invention below:
In step 1), the condition of the 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 and mix 2~6 times
Until cultivation cycle terminates, cultivation cycle is 9~15 days.Further preferably, the condition that the bacterium algae co-cultures are as follows: culture item
Part is 28 DEG C, light intensity 2000Lux, and the photoperiod is 12hr daytime/12hr night, predominantly stationary culture, and it is mixed to be shaken every day culture bottle
Even 4 times until cultivation cycle terminate, cultivation cycle be 12 days.
The bacterium algae ratio of raw general bacterium and chlorella is 0.01~10000:1 in the rice, in a certain range, in rice
The ratio of raw general bacterium is higher, can more promote the growth of chlorella, more can be improved fat content and lipid-producing.It is further excellent
It selects, the bacterium algae ratio of raw general bacterium and chlorella is 1~1000:1 in the rice.
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 cell separation includes: that bacterium algae co-culture media is first handled under ultrasonic conditions, Zhi Houli
The heart obtains chlorella cells precipitating and includes the supernatant bacterium solution of raw general bacterium in rice.
Further preferably, the Ultrasonic Conditions are as follows: under 140W~180W, 30kHz~50kHz Ultrasonic Conditions
Manage 15s~45s.The centrifugation is in 1000~1400r/min low-speed centrifugal, 3~10min.Still more preferably, described
Ultrasonic Conditions are as follows: handle 30s under 160W, 40kHz Ultrasonic Conditions.The centrifugation is in 1200r/min low-speed centrifugal
5min.30s is handled under 160W, 40kHz Ultrasonic Conditions, separates raw general bacterium cell and chlorella cells in rice,
1200r/min low-speed centrifugal 5min, so that biggish chlorella cells is precipitated and on raw general bacterium still suspends in lesser rice
In clear, raw general bacterium cell in the rice in the chlorella cells and supernatant in precipitating is collected respectively.
The processing includes: washing, centrifugation and drying, chlorella cells is precipitated after distilled water resuspension washing is added,
It is collected after 6000~10000r/min is centrifuged 5~20min, is dried under 30 DEG C~50 DEG C cryogenic conditions later, obtain bead
Algae frustule dry product.Further preferably, chlorella cells are precipitated after distilled water resuspension washing is added, is centrifuged through 8000r/min
It collects after 10min, is dried under 40 DEG C of cryogenic conditions later, obtain chlorella algae cell dry product.
In step 3), the organic solvent extracting solution is chloroform-methanol extracting solution, the chloroform-methanol extracting solution
It is made of the chloroform and methanol of 1~3:1 of volume ratio.Further preferably, the chloroform-methanol extracting solution is by volume ratio 2:1's
Chloroform and methanol composition.
The quality of the algae powder and the volume of organic solvent extracting solution are 1g:45~75m1.Further preferably, described
The quality of algae powder and the volume of organic solvent extracting solution are 1g:60m1.
Compared with prior art, the present invention has the advantage that
The present invention produces raw general bacterium is applied to chlorella in rice culture, creates a kind of appliable plant endophyte and mentions
The bacterium algae Coculture techniques of high bead algae biomass and lipid-producing provide new way for the industrialized production of chlorella.I
The common chlorella type of state has chlorella pyrenoidosa, chlorella ellipsoidea, chlorella vulgaris etc..The present invention is with chlorella pyrenoidosa
For, develop the bacterium algae Coculture techniques that raw pantoea agglomerans in a kind of rice promotes bead algae biomass and oil and fat accumulation, but this
Invention is not only limited in chlorella pyrenoidosa.
Increase the year-on-year control group of harvest dry cell weight of chlorella pyrenoidosa using bacterium algae Coculture techniques of the invention
42.80%~150.52%, it compares the fat content of frustule on year-on-year basis and increases 85.4%~133.66%, make frustule
Average daily lipid-producing improves 1.6~4.8 times than control, and extracts the algae-residue protein content obtained after grease and exist
38% or more, it can be used for preparing feed, bait and amino acid preparation, drug extraction etc..The technology of the present invention is easy to operate,
Remarkable benefit in terms of the application and development of chlorella, 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
Percentage in lower embodiment is in case of no particular description weight percent.
Embodiment one:
1) raw general bacterium strain is purchased from Chinese agriculture Microbiological Culture Collection administrative center (ACCC), coding in the rice used
Be 10454, strain activates according to a conventional method choose single colonie after, be inoculated into the triangular flask of the LB liquid medium containing 10mL, carry out
Amplification cultivation, the bacterium solution of logarithmic growth phase will be in after amplification cultivation 16h by bacterium solution: LB liquid medium volume ratio=1:10 connects
Carried out after kind it is secondary 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 seed cell co-cultured as bacterium algae, according to growth curve, bacterium growth is in logarithmic phase, using conventional panel counting method
The living bacteria count (CFU, Colony-Forming Units) in culture solution is measured, bacterial concentration is about 2 × 10 at this time8CFU/
mL.Bacterium cell quantity needed for being co-cultured according to bacterium algae, takes appropriate seed bacterium solution to be centrifuged 10min in room temperature (25 DEG C) 8000r/min,
Precipitating washed twice with 3 times of volume sterile waters, then be centrifuged 10min collect somatic cells, be added with seed bacterium solution in equal volume
BG11 fluid nutrient medium suspends spare.
2) Chinese Academy of Sciences's wildlife Germplasm Bank fresh water algae library (FACHB) is purchased from using chlorella algae, specially
Chlorella pyrenoidosa, number FACHB-1222 take chlorella pyrenoidosa original algae solution, by 10-2, 10-3, 10-4It is dilute to carry out gradient
It releases, dilution 100 μ L of algae solution is taken to be coated on BG11 solid medium (to add 2% agar in BG11 fluid nutrient medium), culture dish
It sets and cultivates in the light incubator, after 7 days, bottle-green single algae is grown in BG11 culture medium and is fallen, with sterile pipette tips picking list algae
It falls in the triangular flask of the fluid nutrient medium of BG11 containing 10mL, carries out amplification cultivation.Logarithmic growth will be in after amplification cultivation 7 days
The frustule of phase 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 altogether 3 times,
Culture 7 days every time.Condition of culture are as follows: 28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, sooner or later each daily to shake
Once.After 3rd amplification cultivation, the chlorella cells concentration in cell culture fluid is calculated using conventional blood cell plate counting method
It is 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-cultivation group chlorella vulgaris daughter cell
Initial inoculation concentration is 2 × 105A cell/mL, raw general strain daughter cell initial inoculation concentration is 2 × 10 in rice5CFU/mL,
Such bacterium algae cell inoculation ratio is 1:1, and bacterium algae co-cultivation is carried out after mixing.It is careful that pure algae culture control group is only inoculated with chlorella vulgaris
Born of the same parents, initial inoculation concentration are 2 × 105A cell/mL.Two groups of cultivating systems are 500ml × 3 bottle, and condition of culture is 28 DEG C,
Light intensity 2000Lux, photoperiod are 12hr daytime/12hr night, predominantly stationary culture, are shaken every day culture bottle and mix 4 times until training
End cycle is supported, cultivation cycle is 12 days.
4) after cultivation cycle, bacterium algae co-cultivation group takes bacterium algae co-culture media at 160W, 40kHz Ultrasonic Conditions
30s is managed, separates raw general bacterium cell and chlorella cells in rice, 1200r/min low-speed centrifugal 5min makes biggish small
Ball frustule precipitates and raw general bacterium still suspends in supernatant in lesser rice, collect respectively chlorella cells in precipitating and
Raw general bacterium cell in rice in supernatant.
5) the supernatant bacterium solution that bacterium algae co-cultivation group obtains step 4) centrifugation, raw general bacterium is effective in rice in bacterium solution after measured
Viable count is 2 × 105This supernatant bacterium solution 8000r/min centrifugation 10min is obtained raw general bacterium somatic cells in rice and sunk by CFU/mL
It forms sediment, and is seeded to the thallus seed cell that fresh LB culture is co-cultured to logarithmic growth phase as bacterium algae again, with reality
The repeated recycling utilize of existing bacterium cell.
6) after distilled water resuspension washing is added in the chlorella cells precipitating that bacterium algae co-cultivation group obtains step 4), warp
8000r/min is centrifuged 10min and collects chlorella cells precipitating；Step 3) is obtained chlorella cells culture by pure algae culture control group
Liquid directly collects frustule precipitating through 8000r/min centrifugation 10min；Frustule is dried under 40 DEG C of cryogenic conditions is precipitated to constant weight
Chlorella cells dry product is obtained, weighing and calculating frustule dry weight yield (mg/L) is carried out to frustule dry product, the results are shown in Table 1.
7) the frustule dry product for obtaining step 6) accurately weighs algae powder 0.05g through mechanical lapping smudge cells powdering
(m1), it is transferred to the dry centrifuge tube of 10mL, by bacterium algae powder (g): the leaching of 3mL chloroform-methanol is added in chloroform-methanol extracting solution=1:60
Extract (chloroform: methanol volume ratio=2:1) (25 DEG C) oscillations of room temperature in dry closed container extract 20min, in 8000r/min
Centrifugation 10min collects supernatant grease extracting solution and algae-residue precipitating respectively, precipitates to algae-residue and repeats to extract 2 times, merges centrifugation supernatant
Total grease extracting solution is obtained, total grease extracting solution is placed in Rotary Evaporators under the conditions of 40 DEG C and is concentrated in vacuo 2h or more until removal
Organic solvent obtains total oil and fat product, in an oven 70 DEG C of dry 2h to constant weight, weighs oil quality (m2), calculates fat content
And yield, experiment set 3 repetitions, results are averaged, is shown in Table 1.
Frustule fat content (%)=(m2/m1) × 100%；
Algae lipid-producing (mg/Ld)=frustule dry weight (g) × frustule fat content (%)/[frustule culture body
Product (L) × incubation time (d)]；
8) algae-residue product is obtained after the algae-residue that step 7) obtains being removed volatile organic solvent in vacuum concentration instrument, is claimed
Re-computation algae-residue yield measures algae-residue protein content through Micro-kjoldahl method, the results are shown in Table 1.
Bacterium algae co-cultivation group is compared with pure algae cultivates control group lipid-producing and index of correlation when 1 bacterium algae of table is than=1:1
Note: data are three groups of statistical averages in table.
Bacterium algae co-cultivation improves efficiency=and [(bacterium algae co-cultivation group-pure algae culture control group)/pure algae culture control group] ×
100%.
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
It spreads cultivation, is continuously spread cultivation three times again, condition of culture is 37 DEG C, dark culturing in 200rpm constant-temperature table.Third time spreads cultivation 10h extremely
OD600=1 seed cell co-cultured as bacterium algae, according to growth curve, bacterium growth is in logarithmic phase, using conventional panel meter
Living bacteria count (CFU, Colony-Forming Units) in number methods measurement culture solutions, at this time bacterial concentration be about 2 ×
108CFU/mL.Bacterium cell quantity needed for being co-cultured according to bacterium algae, takes appropriate seed bacterium solution to be centrifuged in room temperature 8000r/min
10min, precipitating are washed twice with 3 times of volume sterile waters, then are centrifuged 10min and are collected somatic cells, are added and the bodies such as seed bacterium solution
Long-pending BG11 fluid nutrient medium suspends spare.
2) Chinese Academy of Sciences's wildlife Germplasm Bank fresh water algae library (FACHB) is purchased from using chlorella algae, specially
Chlorella pyrenoidosa, number FACHB-1222 take chlorella pyrenoidosa original algae solution, by 10-2, 10-3, 10-4It is dilute to carry out gradient
It releases, dilution 100 μ L of algae solution is taken to be coated on BG11 solid medium (to add 2% agar in BG11 fluid nutrient medium), culture dish
It sets and cultivates in the light incubator, after 7 days, bottle-green single algae is grown in BG11 culture medium and is fallen, with sterile pipette tips picking list algae
It falls in the triangular flask of the fluid nutrient medium of BG11 containing 10mL, carries out amplification cultivation.Logarithmic growth will be in after amplification cultivation 7 days
The frustule of phase 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 altogether 3 times,
Culture 7 days every time.Condition of culture are as follows: 28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, sooner or later each daily to shake
Once.After 3rd amplification cultivation, the chlorella cells concentration in cell culture fluid is calculated using conventional blood cell plate counting method
It is 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-cultivation group chlorella vulgaris daughter cell
Initial inoculation concentration is 2 × 106A cell/mL, raw general strain daughter cell initial inoculation concentration is 2 × 10 in rice8CFU/mL,
Such bacterium algae cell inoculation ratio is 100:1, and bacterium algae co-cultivation is carried out after mixing.Pure algae culture control group is only inoculated with chlorella seed
Cell, initial inoculation concentration are 2 × 106A cell/mL.Two groups of cultivating systems are 500ml × 3 bottle, and condition of culture is 28
DEG C, light intensity 2000Lux, the photoperiod be 12hr daytime/12hr night, predominantly stationary culture, be shaken every day culture bottle mix 4 times until
Cultivation cycle terminates, and cultivation cycle is 20 days.
4) after cultivation cycle, bacterium algae co-cultivation group takes bacterium algae co-culture media at 160W, 40kHz Ultrasonic Conditions
30s is managed, separates raw general bacterium cell and chlorella cells in rice, 1200r/min low-speed centrifugal 5min makes biggish small
Ball frustule precipitates and raw general bacterium still suspends in supernatant in lesser rice, collect respectively chlorella cells in precipitating and
Raw general bacterium cell in rice in supernatant.
5) the supernatant bacterium solution that bacterium algae co-cultivation group obtains step 4) centrifugation, raw general bacterium is effective in rice in bacterium solution after measured
Viable count is 2 × 106This supernatant bacterium solution 8000r/min centrifugation 10min is obtained raw general bacterium somatic cells in rice and sunk by CFU/mL
It forms sediment, and is seeded to the thallus seed cell that fresh LB culture is co-cultured to logarithmic growth phase as bacterium algae again, with reality
The repeated recycling utilize of existing bacterium cell.
6) after distilled water resuspension washing is added in the chlorella cells precipitating that bacterium algae co-cultivation group obtains step 4), warp
8000r/min is centrifuged 10min and collects chlorella cells precipitating；Step 3) is obtained chlorella cells culture by pure algae culture control group
Liquid directly collects frustule precipitating through 8000r/min centrifugation 10min；Frustule is dried under 40 DEG C of cryogenic conditions is precipitated to constant weight
Chlorella cells dry product is obtained, weighing and calculating frustule dry weight yield (mg/L) is carried out to frustule dry product, the results are shown in Table 2.
7) the frustule dry product for obtaining step 6) accurately weighs algae powder 0.05g through mechanical lapping smudge cells powdering
(m1), it is transferred to the dry centrifuge tube of 10mL, by bacterium algae powder (g): 3mL chloroform-first is added in chloroform-methanol extracting solution (m1)=1:60
Alcohol leaching liquor (chloroform: methanol volume ratio=2:1) shaken at room temperature in dry closed container extracts 20min, in 8000r/min from
Heart 10min collects supernatant grease extracting solution and algae-residue precipitating respectively, precipitates to algae-residue and repeats to extract 2 times, merges centrifugation supernatant and obtains
Total grease extracting solution is placed in Rotary Evaporators under the conditions of 40 DEG C and is concentrated in vacuo 2h or more until removal has by total grease extracting solution
Solvent obtains total oil and fat product, in an oven 70 DEG C of dry 2h to constant weight, weigh oil quality (m2), calculate fat content and
Yield, experiment set 3 repetitions, and results are averaged:
Frustule fat content (%)=(m2/m1) × 100%；
Algae lipid-producing (mg/Ld)=frustule dry weight (g) × frustule fat content (%)/[frustule culture body
Product (L) × incubation time (d)]；
Concrete outcome is shown in Table 2.
8) algae-residue product is obtained after the algae-residue that step 7) obtains being removed volatile organic solvent in vacuum concentration instrument, is claimed
Re-computation algae-residue yield measures algae-residue protein content through Micro-kjoldahl method, the results are shown in Table 2.
The ratio of bacterium algae co-cultivation group and pure algae culture control group lipid-producing and index of correlation when 2 bacterium algae of table is than=100:1
Compared with
Note: data are three groups of statistical averages in table.
Bacterium algae co-cultivation improves efficiency=and [(bacterium algae co-cultivation group-pure algae culture control group)/pure algae culture control group] ×
100%
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
It spreads cultivation, is continuously spread cultivation three times again, condition of culture is 37 DEG C, dark culturing in 200rpm constant-temperature table.Third time spreads cultivation 10h extremely
OD600=1 seed cell co-cultured as bacterium algae, according to growth curve, bacterium growth is in logarithmic phase, using conventional panel meter
Living bacteria count (CFU, Colony-Forming Units) in number methods measurement culture solutions, at this time bacterial concentration be about 2 ×
108CFU/mL.Bacterium cell quantity needed for being co-cultured according to bacterium algae, takes appropriate seed bacterium solution to be centrifuged in room temperature 8000r/min
10min, precipitating are washed twice with 3 times of volume sterile waters, then are centrifuged 10min and are collected somatic cells, are added and the bodies such as seed bacterium solution
Long-pending BG11 fluid nutrient medium suspends spare.
2) Chinese Academy of Sciences's wildlife Germplasm Bank fresh water algae library (FACHB) is purchased from using chlorella algae, specially
Chlorella pyrenoidosa, number FACHB-1222 take chlorella pyrenoidosa original algae solution, by 10-2, 10-3, 10-4It is dilute to carry out gradient
It releases, dilution 100 μ L of algae solution is taken to be coated on BG11 solid medium (to add 2% agar in BG11 fluid nutrient medium), culture dish
It sets and cultivates in the light incubator, after 7 days, bottle-green single algae is grown in BG11 culture medium and is fallen, with sterile pipette tips picking list algae
It falls in the triangular flask of the fluid nutrient medium of BG11 containing 10mL, carries out amplification cultivation.Logarithmic growth will be in after amplification cultivation 7 days
The frustule of phase 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 altogether 3 times,
Culture 7 days every time.Condition of culture are as follows: 28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, sooner or later each daily to shake
Once.After 3rd amplification cultivation, the chlorella cells concentration in cell culture fluid is calculated using conventional blood cell plate counting method
It is 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-cultivation group chlorella vulgaris daughter cell
Initial inoculation concentration is 2 × 105A cell/mL, raw general strain daughter cell initial inoculation concentration is 2 × 10 in rice8CFU/mL,
Such bacterium algae cell inoculation ratio is 1000:1, and bacterium algae co-cultivation is carried out after mixing.Pure algae culture control group is only inoculated with chlorella vulgaris
Daughter cell, initial inoculation concentration are 2 × 105A cell/mL.Two groups of cultivating systems are 500ml × 3 bottle, and condition of culture is
28 DEG C, light intensity 2000Lux, the photoperiod is 12hr daytime/12hr night, predominantly stationary culture, is shaken every day culture bottle and mixes 4 times directly
Terminate to cultivation cycle, cultivation cycle is 8 days.
4) after cultivation cycle, bacterium algae co-cultivation group takes bacterium algae co-culture media at 160W, 40kHz Ultrasonic Conditions
30s is managed, separates raw general bacterium cell and chlorella cells in rice, 1200r/min low-speed centrifugal 5min makes biggish small
Ball frustule precipitates and raw general bacterium still suspends in supernatant in lesser rice, collect respectively chlorella cells in precipitating and
Raw general bacterium cell in rice in supernatant.
5) the supernatant bacterium solution that bacterium algae co-cultivation group obtains step 4) centrifugation, raw general bacterium is effective in rice in bacterium solution after measured
Viable count is 5 × 106This supernatant bacterium solution 8000r/min centrifugation 10min is obtained raw general bacterium somatic cells in rice and sunk by CFU/mL
It forms sediment, and is seeded to the thallus seed cell that fresh LB culture is co-cultured to logarithmic growth phase as bacterium algae again, with reality
The repeated recycling utilize of existing bacterium cell.
6) after distilled water resuspension washing is added in the chlorella cells precipitating that bacterium algae co-cultivation group obtains step 4), warp
8000r/min is centrifuged 10min and collects chlorella cells precipitating；Step 3) is obtained chlorella cells culture by pure algae culture control group
Liquid directly collects frustule precipitating through 8000r/min centrifugation 10min；Frustule is dried under 40 DEG C of cryogenic conditions is precipitated to constant weight
Chlorella cells dry product is obtained, carrying out weighing and calculating frustule dry weight yield (mg/L) to frustule dry product the results are shown in Table 3.
7) the frustule dry product for obtaining step 6) accurately weighs algae powder 0.05g through mechanical lapping smudge cells powdering
(m1), it is transferred to the dry centrifuge tube of 10mL, by bacterium algae powder (g): 3mL chloroform: first is added in chloroform-methanol extracting solution (m1)=1:60
Alcohol (2:1) extracting solution mixes, and 25 DEG C of oscillations of room temperature extract 20min, collects supernatant in 8000r/min centrifugation 10min.It repeats
It extracts twice, the supernatant for extracting acquisition three times is incorporated into 10mL centrifuge tube, be placed in vacuum concentration instrument vacuum under the conditions of 40 DEG C
1-2h is concentrated and removes organic solvent, 70 DEG C of dry 2h to constant weight in an oven are weighed oil quality (m2), calculate fat content with
Yield.Experiment sets 3 repetitions, and results are averaged, the results are shown in Table 3:
Frustule fat content (%)=(m2/m1) × 100%；
Lipid-producing (mg/Ld)=frustule dry weight (g) × frustule fat content (%)/[frustule volume of culture
(L) × incubation time (d)]；
8) algae-residue product is obtained after the algae-residue that step 7) obtains being removed volatile organic solvent in vacuum concentration instrument, is claimed
Re-computation algae-residue yield measures algae-residue protein content through Micro-kjoldahl method, the results are shown in Table 3.
The ratio of bacterium algae co-cultivation group and pure algae culture control group lipid-producing and index of correlation when 3 bacterium algae of table is than=1000:1
Compared with
Note: data are three groups of statistical averages in table.
Bacterium algae co-cultivation improves efficiency=and [(bacterium algae co-cultivation group-pure algae culture control group)/pure algae culture control group] ×
100%
Increase the year-on-year control group of harvest dry cell weight of chlorella pyrenoidosa using bacterium algae Coculture techniques of the invention
42.80%~150.52%, it compares the fat content of frustule on year-on-year basis and increases 85.4%~133.66%, make frustule
Average daily lipid-producing improves 1.6~4.8 times than control, and extracts the algae-residue protein content obtained after grease and exist
38% or more, it can be used for preparing feed, bait and amino acid preparation, drug extraction etc..The technology of the present invention is easy to operate,
Remarkable benefit in terms of the application and development of chlorella, has a extensive future.
Claims (8)
1. a kind of co-culture the method for improving chlorella lipid-producing using general bacterium raw in rice and chlorella, which is characterized in that
Include:
1) general bacterium raw in rice and chlorella are subjected to bacterium algae co-cultivation under light illumination, after culture, obtain bacterium algae co-cultivation
Liquid；The bacterium algae ratio of raw general bacterium and chlorella is 1~1000:1 in the rice；
2) cell separation is carried out to bacterium algae co-culture media, obtain chlorella cells precipitating and includes the supernatant bacterium of raw general bacterium in rice
Chlorella cells precipitating is obtained chlorella cells dry product by liquid after processing；
3) chlorella cells dry product is crushed, obtains algae powder, total grease is extracted using organic solvent extracting solution later, obtains total oil
Rouge.
2. according to claim 1 co-culture the side for improving chlorella lipid-producing using general bacterium raw in rice and chlorella
Method, which is characterized in that in step 1), the condition of the 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 9~15 days.
3. according to claim 1 co-culture the side for improving chlorella lipid-producing using general bacterium raw in rice and chlorella
Method, which is characterized in that in step 2), the cell separation includes: that bacterium algae co-culture media is first handled under ultrasonic conditions, it
After be centrifuged, obtain chlorella cells precipitating and include in rice the supernatant bacterium solution of raw general bacterium.
4. according to claim 3 co-culture the side for improving chlorella lipid-producing using general bacterium raw in rice and chlorella
Method, which is characterized in that in step 2), the Ultrasonic Conditions are as follows: in 140W~180W, 30kHz~50kHz Ultrasonic Conditions
Lower processing 15s~45s.
5. according to claim 3 co-culture the side for improving chlorella lipid-producing using general bacterium raw in rice and chlorella
Method, which is characterized in that in step 2), the centrifugation is in 1000~1400r/min low-speed centrifugal, 3~10min.
6. according to claim 1 co-culture the side for improving chlorella lipid-producing using general bacterium raw in rice and chlorella
Method, which is characterized in that in step 2), the processing includes: washing, centrifugation and drying, and chlorella cells are precipitated to be added and are steamed
After washing is resuspended in distilled water, collected after 6000~10000r/min is centrifuged 5~20min, later in 30 DEG C~50 DEG C cryogenic conditions
Lower drying obtains chlorella algae cell dry product.
7. according to claim 1 co-culture the side for improving chlorella lipid-producing using general bacterium raw in rice and chlorella
Method, which is characterized in that in step 3), the organic solvent extracting solution is chloroform-methanol extracting solution, the chloroform-methanol
Extracting solution is made of the chloroform and methanol of 1~3:1 of volume ratio.
8. according to claim 1 co-culture the side for improving chlorella lipid-producing using general bacterium raw in rice and chlorella
Method, which is characterized in that in step 3), the quality of the algae powder and the volume of organic solvent extracting solution are 1g:45~75m1.
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