CN109797106A - A kind of novel two stages autotrophy-is photosynthetic and supports the method for improving chlorella lipid - Google Patents
A kind of novel two stages autotrophy-is photosynthetic and supports the method for improving chlorella lipid Download PDFInfo
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- CN109797106A CN109797106A CN201910147423.9A CN201910147423A CN109797106A CN 109797106 A CN109797106 A CN 109797106A CN 201910147423 A CN201910147423 A CN 201910147423A CN 109797106 A CN109797106 A CN 109797106A
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Abstract
The present invention discloses a kind of method that novel two stages autotrophy-is photosynthetic and supports raising chlorella lipid, and this method effectively can purify brewing wastewater simultaneously and significantly improve the yield of chlorella lipid.This method includes two stages, and the first stage is the microalgae photoautotrophy stage: chlorella UTEX-265 carries out culture 8-10 days in brewing wastewater BWW#2 (handling by anaeroic digestor), and after completing chlorella collection, purified waste water is discharged.This stage TN content drops to 7mg/L from 75mg/L, and TP content drops to 2mg/L from 20mg/L, reaches emission request.Second stage is the photosynthetic and feeding stage: mixotrophic cultivation is carried out in the BWW#2 that the micro algae growth collected in the first stage during culture in 5-7 days adds organic carbon source outside, achieve the purpose that purify waste water and improve microalgae grease productivity, wherein chlorella lipid maximum production is 65.94mg/L day.The cost advantages that this method has purification brewing wastewater, inhibits germ contamination, collects without Wastewater Pretreatment, saving microalgae.
Description
Technical field
The invention belongs to wastewater treatments and bio-fuel production field, and it is photosynthetic simultaneous to be related to a kind of novel two stages autotrophy-
Support the method for improving chlorella lipid.
Background technique
Microorganism is not only able to produce bio-fuel and can capture carbon dioxide from atmosphere.In microculture
Nutrient and water are the maximum places of main expense in system.In fact, chemical fertilizer resulted in as nutrient source it is very high
It spends and therefore the consumption of energy is obtained nutrient from waste water using microorganism and generation fuel is that one kind passes through very much
The method of Ji type.
Currently, the quantity of whole world beer brewing is about 1339 × 106Hetoliters (hL), wherein every hL generates 5-
The waste water BWW of 6hL contains enough total nitrogens (TN) and total phosphorus (TP) for microdisk electrode[1].Traditionally, brewing wastewater (BWW) exists
It is handled in anaeroic digestor to reduce the high concentration organic carbon in waste water, therefore, brewing wastewater (BWW) is in microdisk electrode mistake
Not only there are the potentiality as inorganic nutrients source in journey, but also have enough organic carbons as substitution carbon source.
There are various microdisk electrode modes, be directed to photoautotrophy, heterotrophism and and the different training methods such as supports.With these sides
Formula, which cultivates chlorella, has positive influence to biomass and grease production power.It is simultaneous to support in its higher biomass and grease production power
Aspect is better than light autotrophy and heterotrophism mode.Waste water single stage and feeding two main problem relevant with Heterotrophic culture are organic bottoms
The high cost of object and growth containing harmful bacteria caused by organic carbon source.In a true waste water system, have a large amount of
Endogenetic bacteria and a large amount of waste water need to handle, Wastewater Pretreatment step by become influence microalgae biodiesel produce another at
This factor.
Nutrient abundance be microalgae biomass accumulation necessary factor, but under Nutrient Stress state microalgae lipid energy
Enough a large amount of accumulations, so obtaining very high biomass simultaneously under single stage training method and lipid production is impossible.
Summary of the invention
The purpose of the present invention is overcoming the problems, such as that above-mentioned background exists, a kind of novel two stages training mode is proposed, it can be with
Carry out the processing to brewing wastewater, at the same can abundant algae production rouge rate, be a kind of multi-functional mode, cultivated in two stages
In mode, microalgae bacterial strain in the BWW (being expressed as BWW#2) first after anaerobic digestion with photoautotrophy pattern growth, with effective
Using inorganic nutritive element, achieve the purpose that Biomass accumulation and preferably control bacterium increase；In micro algae growth late-exponential rank
Section, microalgae adds outside BWW#2 to be grown in the mixed culture medium of glucose or glycerine or brewing wastewater (being expressed as BWW#1).This
Kind microalgae culture system can efficiently use the inorganic and organic nutrient substance in waste water, and inhibit germ contamination, be very suitable to big
The plantation of scale.In photoautotrophy growth phase, micro algae biomass is generated, and in second stage, under nutrient limiting conditions, i.e.,
Under conditions of nutriment consumption, organic carbon such as glucose, glycerol or BWW#1 are put into, the process of lipid accumulation is reached.
The present invention is achieved by the following technical solutions:
Two stages photoautotrophy-is photosynthetic and supports mode, process are as follows:
1) by chlorella Chlorella vulgaris (UTEX-265) to be inoculated with volume as 10%-30% (V inoculum/V
Culture medium) it is inoculated in TAP culture medium, it is placed under illumination and cultivates 5-10 days；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1And on the oscillator of revolving speed 150rpm
Oscillation, absorbance of the chlorella algae suspension in matrix culture medium are adjusted to 1.0；
3) the photoautotrophy stage is the first stage of mode: to be inoculated with volume as 10%-30% (V inoculum/V culture medium)
Chlorella is inoculated in chlorella UTEX- after cultivating 8-10 days, 8-10 days in the brewing wastewater BWW#2 after Anaerobic Digestion
Waste water is discharged after 265 sedimentation 10-24hrs, collects bottom chlorella UTEX-265；
Brewing wastewater (BWW) is collected from city's winery, the TN=30-45mg/L, TP=12- of brewing wastewater (BWW#1)
16mg/L, TOC=1400-1500mg/L, COD=2000-3000mg/L；BOD=1300 ± 2.6mg/L, TSS=300-
320mg/L, PH=5.5-6.0；
The TN=50-75mg/L of brewing wastewater BWW#2, TP=15-20mg/L, TOC=80-100mg/L, COD=100-
150mg/L；BOD=150 ± 2.3mg/L, TSS=100-130mg/L, PH=6.5-7.5；
3) the photosynthetic and feeding stage is the second stage of mode: in closed environment (such as hydrostatic column, the culture of 2L
The relative closures environment such as case, large-scale photosynthetic reactor, in order to reduce and control germ contamination) the investment culture first stage
The chlorella UTEX-265 of collection, culture medium are the mixed liquor of BWW#2 and additional carbon；
4) discharge of wastewater of after-purification in 5-7 days, collecting chlorella can be used.
5) grease is monitored.
Compared with prior art, processing of the present invention to brewing wastewater, while the production rouge rate of algae can be sufficiently improved, it is
A kind of multi-functional mode, in two stages training mode, microalgae is in the BWW (being expressed as BWW#2) first after anaerobic digestion
With photoautotrophy pattern growth, to efficiently use inorganic nutritive element, reaches Biomass accumulation and preferably control what bacterium increased
Purpose.In the micro algae growth late-exponential stage, microalgae is exposed in the BWW#2 of additional carbon.This microalgae culture system can have
Effect inhibits germ contamination using the inorganic and organic nutrient substance in waste water, is very suitable to plant on a large scale.It is photosynthetic from
Growth phase is supported, micro algae biomass is generated, and in second stage, it under nutrient limiting conditions, that is, is more than nutriment consumption
Under the conditions of, organic carbon such as glucose or BWW#1 are put into, the process of lipid accumulation is reached.It is specific as follows:
It 1) can be without the organic and inorganic nutritive element in waste water be pre-processed, made full use of to waste water and is utilized small
Self sedimentation of ball algae UTEX-265 is collected microalgae, greatly reduces culture microalgae and handles the cost of waste water.
2) mode effectively controls the growth of bacterium, is suitble to large-scale plantation.
3) relative to traditional single phase training mode, the present invention substantially increases the lipid production of chlorella UTEX-265
Power.
Detailed description of the invention
Fig. 1 (a), which is represented, cultivates chlorella with the mixture (single-stage mixotrophic cultivation) of different mixing proportion BWW#2 and BWW#1
The lipid yield of chlorella when UTEX-265；
Fig. 1 (b) represents (single as outer plus organic carbon source in the glucose of sterilizing or unpasteurized BWW#2 or addition 3g/L
Grade and support) culture chlorella UTEX-265 when chlorella dry cell weight and lipid content:
Wherein: S=sterilizing；NS=does not sterilize；G=glucose；Wherein, the black and white arrow of each filling distinguishes table
Show left and right y-axis.
Fig. 1 (c) represents mixture (single-stage mixotrophic cultivation) the culture chlorella of different mixing proportion BWW#2 and BWW#1
Removal effect of the chlorella to the TOC of waste water when UTEX-265.
Fig. 2 (a) is represented and is cultivated chlorella UTEX-265 in single-stage photoautotrophy training mode, and the growth of chlorella is bent
Line, BWW#2 water quality PH, TN and TP content variation；
Fig. 2 (b) representative uses BWW#1 as carbon source, under two stages photoautotrophy-mixotrophic cultivation mode of BWW#2
Growth curve and nutrients consumption curve during C.vulagris (UTEX-265)；
Fig. 2 (c) is represented simultaneous using the two stages photoautotrophy-of different organic carbon sources (BWW#1, glucose or glycerine)
It supports under mode culture, the dry cell weight and lipid of chlorella (UTEX-265) and endogenous chlorella (separation and Extraction from BWW) contain
Amount；
Wherein solid circles and square symbols respectively represent the FAME content of chlorella (UTEX-265) and endogenous chlorella.
In x-axis:
1=single-stage photoautotrophy culture；
2=two stages photoautotrophy-mixotrophic cultivation (25%BWW#1)；
3=two stages photoautotrophy-mixotrophic cultivation (3g/L glucose)；
4=two stages photoautotrophy-mixotrophic cultivation (3g/L glycerine).
Fig. 3 represent under two stages photoautotrophy-and feeding mode using different organic carbon source (BWW#1 or glucose or
Glycerol) culture when chlorella (UTEX-265) lipid composition.
In x-axis, 1=single-stage photoautotrophy culture；
2=two stages photoautotrophy-mixotrophic cultivation (25%BWW#1)；
3=two stages photoautotrophy-mixotrophic cultivation (3g/L glucose)；
4=two stages photoautotrophy-mixotrophic cultivation (3g/L glycerine).
Specific embodiment
Below by specific embodiments and the drawings, the present invention is further illustrated.The embodiment of the present invention is in order to more
So that those skilled in the art is more fully understood the present invention well, any limitation is not made to the present invention.
Specific embodiment
Embodiment 1
The present embodiment microalgae cell is grown in 500mL conical flask；
Steps are as follows for the method for the present invention (single-stage and feeding mode):
It 1) is 10% (V inoculum/V culture to be inoculated with volume by chlorella Chlorella vulgaris (UTEX-265)
Base) it is inoculated in TAP culture medium, it is placed under illumination and cultivates 8 days；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation, absorbance of the chlorella algae suspension in matrix culture medium are adjusted to 1.0；
2) 250mL different mixing proportion (100% BWW, 75%BWW, 50%BWW is respectively connected in 500mL conical flask
And 25%BWW) BWW and BWW#2 mixed liquor, be that chlorella is inoculated with by 10% (V inoculum/V culture medium) to be inoculated with volume
It is cultivated 15 days in conical flask；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation；
Brewing wastewater (BWW) is collected from city's winery, the TN=30-45mg/L, TP=12- of brewing wastewater (BWW#1)
16mg/L, TOC=1400-1500mg/L, COD=2000-3000mg/L；BOD=1300 ± 2.6mg/L, TSS=300-
320mg/L, PH=5.5-6.0；
The TN=50-75mg/L of brewing wastewater BWW#2, TP=15-20mg/L, TOC=80-100mg/L, COD=100-
150mg/L；BOD=150 ± 2.3mg/L, TSS=100-130mg/L, PH=6.5-7.5；
4) removal effect that chlorella carries out grease measurement and TOC is collected after cultivation cycle.
Embodiment 2
The present embodiment microalgae cell is grown in the 500mL conical flask of the BWW#2 containing 250mL；
Steps are as follows for the method for the present invention (single-stage photoautotrophy mode):
It 1) is 10% (V inoculum/V culture to be inoculated with volume by chlorella Chlorella vulgaris (UTEX-265)
Base) it is inoculated in TAP culture medium, it is placed under illumination and cultivates 5 days；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation, absorbance of the chlorella algae suspension in matrix culture medium are adjusted to 1.0；
2) BWW#2 of 250mL is accessed in 500mL conical flask, it will for 10% (V inoculum/V culture medium) to be inoculated with volume
Chlorella is inoculated in conical flask and cultivates 14 days, is monitored daily to the water quality of BWW#2；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation；
Brewing wastewater (BWW) is collected from city's winery, the TN=50-75mg/L of brewing wastewater BWW#2, TP=15-20mg/
L, TOC=80-100mg/L, COD=100-150mg/L；BOD=150 ± 2.3mg/L, TSS=100-130mg/L, PH=
6.5-7.5；
3) chlorella is collected after cultivation cycle carry out grease measurement.
Embodiment 3
The present embodiment microalgae cell is grown in 500mL conical flask；
Steps are as follows for the method for the present invention (two stages photoautotrophy-is photosynthetic and supports mode):
It 1) is 10% (V inoculum/V culture to be inoculated with volume by chlorella Chlorella vulgaris (UTEX-265)
Base) it is inoculated in TAP culture medium, it is placed under illumination and cultivates 5 days；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation, absorbance of the chlorella algae suspension in matrix culture medium are adjusted to 1.0；
2) the photoautotrophy stage is the first stage of mode: accessing the BWW#2 of 250mL, in 500mL conical flask with inoculation
Chlorella is inoculated in conical flask for 10% (V inoculum/V culture medium) and cultivates 8 days by volume, and chlorella UTEX-265 carries out light
It closes autotrophy and reaches Biomass accumulation.Cluster and automatic sedimentation occur for chlorella UTEX-265 after 7 days, and settling will purification after 10hrs
Waste water discharge afterwards, bottom chlorella UTEX-265 will be collected, and give over to second stage use；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation；
Brewing wastewater (BWW) is collected from city's winery, the TN=30-45mg/L, TP=12- of brewing wastewater (BWW#1)
16mg/L, TOC=1400-1500mg/L, COD=2000-3000mg/L；BOD=1300 ± 2.6mg/L, TSS=300-
320mg/L, PH=5.5-6.0；
The TN=50-75mg/L of brewing wastewater BWW#2, TP=15-20mg/L, TOC=80-100mg/L, COD=100-
150mg/L；BOD=150 ± 2.3mg/L, TSS=100-130mg/L, PH=6.5-7.5；
2) photosynthetic and feeding stage is the second stage of mode: accessed in 500mL conical flask the 25%BWW of 250mL with
Mixed liquor, 100%BWW#2 and the glucose mixed liquor of 75%BWW#2 and 100%BWW#2 glycerine mixed liquor (glucose and
Glycerol concentration is 3g/L), the chlorella UTEX-265 investment for completing first stage growth is placed in light incubator and cultivates 5
It, during which chlorella largely consumes organic carbon, and by Nutrient Stress (TN, TP content are insufficient) and lipid largely accumulates.
3) discharge of wastewater of after-purification in 5 days collects chlorella and carries out grease measurement, and maximum lipid production is photosynthetic from single phase
The 31.1mg/L of autotrophy culture is promoted to 65.94mg/L.
Embodiment 4
The present embodiment microalgae cell is grown in photosynthetic reactor；
Steps are as follows for the method for the present invention (two stages photoautotrophy-is photosynthetic and supports mode):
It 1) is 30% (V inoculum/V culture to be inoculated with volume by chlorella Chlorella vulgaris (UTEX-265)
Base) it is inoculated in TAP culture medium, it is placed under illumination and cultivates 10 days；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation, absorbance of the chlorella algae suspension in matrix culture medium are adjusted to 1.0；
3) the photoautotrophy stage is the first stage of mode: accessing BWW#2 in photosynthetic reactor, is to be inoculated with volume
Chlorella is inoculated in conical flask by 10% (V inoculum/V culture medium) to be cultivated 10 days, and chlorella UTEX-265 progress is photosynthetic certainly
It supports and reaches Biomass accumulation.Cluster and automatic sedimentation occur for chlorella UTEX-265 after 10 days, and sedimentation for 24 hours will be purified after rs
Waste water discharge, bottom chlorella UTEX-265 will be collected, and give over to second stage use；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1 and in the oscillator of revolving speed 150rpm
Upper oscillation；
Brewing wastewater (BWW) is collected from city's winery, the TN=30-45mg/L, TP=12- of brewing wastewater (BWW#1)
16mg/L, TOC=1400-1500mg/L, COD=2000-3000mg/L；BOD=1300 ± 2.6mg/L, TSS=300-
320mg/L, PH=5.5-6.0；
The TN=50-75mg/L of brewing wastewater BWW#2, TP=15-20mg/L, TOC=80-100mg/L, COD=100-
150mg/L；BOD=150 ± 2.3mg/L, TSS=100-130mg/L, PH=6.5-7.5；
4) photosynthetic and feeding stage is the second stage of mode: accessed in 500mL conical flask the 25%BWW of 250mL with
Mixed liquor, 100%BWW#2 and the glucose mixed liquor of 75%BWW#2 and 100%BWW#2 glycerine mixed liquor (glucose and
Glycerol concentration is 3g/L), it will complete chlorella UTEX-265 investment culture 7 days of first stage growth, during which chlorella is big
Amount consumption organic carbon, and by Nutrient Stress (TN, TP content insufficient) and lipid largely accumulates.
5) discharge of wastewater of after-purification in 7 days collects chlorella and carries out grease measurement.
Lipid yield is maximum when can be seen that mixed proportion is the BWW#2 of 75% BWW#2 and 25% by Fig. 1 (a), by scheming
It is very high to the removal efficiency of TOC when chlorella grows in BWW#2 the and BWW mixed liquor of above two ratio known to 1 (c)；
The lipid production rate of chlorella is more considerable under non-sterilizing state when knowing 3g/L glucose additional to BWW#2 by Fig. 1 (b), Fig. 1
Show to add the organic carbon sources such as 3g/L glucose and BWW#1, chlorella UTEX-265 biomass under non-sterilized environment outside in BWW#2
Will not be greatly affected with lipid yield, can save the cost, therefore this mode can be adopted in two stage second stage
With.
The TN content of BWW#2 is dropped from 75mg/L after photoautotrophy cultivation stage, culture 8 days it can be seen from Fig. 2 (a)
To 7mg/L, TP content drops to 2mg/L from 20mg/L, reaches emission request.In the photosynthetic and feeding stage it can be seen from Fig. 2 (b),
Chlorella UTEX-265 growth rate becomes faster after additional carbon, and water quality situation is also improved.
Table 1 represents that chlorella (UTEX-265) is single under TAP culture medium and BWW or two stages photoautotrophy-is photosynthetic different
Biomass and lipid content under the mode of supporting
This method has dual synergistic effect to lipid accumulation, one is the Nutrient Stress at the end of due to the first stage,
Another kind is the supply of second stage organic carbon.Table 1 carries out chlorella UTEX-265 to a variety of methods of two stages training mode
Lipid production be measured, discovery two stages training mode have facilitation, bead to the biomass and lipid of UTEX-265
Algae UTEX-265 highest lipid production is lipid production (the 31.5mg/L day) in TAP culture medium up to 65.94mg/L day
2 times.
Fig. 3 can be seen that under the photosynthetic and feeding mode of two stages photoautotrophy-, and the use value such as C16 and C18 are higher
Fatty acid (may be used as many aspects such as cosmetics, bio-fuel, health care product) accounts for 80% or more, there is very high utility value.
The ratio of oleic acid C18:1 (having good bio-fuel property, can produce object diesel oil) is very high in lipid components, accounts for about
Between 36%-45%, and the lipid production of this mode chlorella UTEX-265 is preferable, and has very high use value.
It should be understood that embodiment and example discussed herein simply to illustrate that, to those skilled in the art
For, it can be improved or converted, and all these modifications and variations all should belong to the protection of appended claims of the present invention
Range.
Claims (1)
1. a kind of novel two stages autotrophy-is photosynthetic and supports the method for improving chlorella lipid, characterized in that improve microalgae oil-producing
With purify the economic hybrid system of waste water simultaneously, and germ contamination not can be effectively controlled to Wastewater Pretreatment but；
Specific step is as follows:
1) by chlorella Chlorella vulgaris (UTEX-265) to be inoculated with volume as 10%-30% (V inoculum/V culture
Base) it is inoculated in TAP culture medium, it is placed under illumination and cultivates 5-10 days；
Condition of culture are as follows: 25 DEG C of temperature, white fluorescent illuminates 100 μm of ol.m2.s-1And it shakes on the oscillator of revolving speed 150rpm
It swings, absorbance of the chlorella algae suspension in matrix culture medium is adjusted to 1.0；
2) the photoautotrophy stage is the first stage of mode: will be small for 10%-30% (V inoculum/V culture medium) to be inoculated with volume
Ball algae is inoculated in cultivated 8-10 days, 8-10 days in the brewing wastewater BWW#2 after Anaerobic Digestion after chlorella UTEX-265
Waste water is discharged after sedimentation 10-24hrs, collects bottom chlorella UTEX-265；
Brewing wastewater (BWW) is collected from city's winery, TN=30-45mg/L, the TP=12-16mg/L of brewing wastewater (BWW#1),
TOC=1400-1500mg/L, COD=2000-3000mg/L；BOD=1300 ± 2.6mg/L, TSS=300-320mg/L, PH
=5.5-6.0；
The TN=50-75mg/L of brewing wastewater BWW#2, TP=15-20mg/L, TOC=80-100mg/L, COD=100-
150mg/L；BOD=150 ± 2.3mg/L, TSS=100-130mg/L, PH=6.5-7.5；
3) photosynthetic and feeding stage is the second stage of mode: in closed environment (such as the hydrostatic column of 2L, incubator,
The relative closures environment such as large-scale photosynthetic reactor, in order to reduce and control germ contamination) investment culture first stage receipts
The chlorella UTEX-265 of collection, culture medium are the mixed liquor of BWW#2 and additional carbon；
4) discharge of wastewater of after-purification in 5-7 days, collecting chlorella can be used.
5) grease is monitored.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113136339A (en) * | 2020-01-19 | 2021-07-20 | 中国石油化工股份有限公司 | Method for mixotrophic-autotrophic continuous culture of photosynthetic microorganisms, culture system and application thereof |
| CN115678785A (en) * | 2021-11-18 | 2023-02-03 | 珠海光藻生命科学有限公司 | Food-grade chlorella culture medium and culture method |
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