CN1837352A

Movatterモバイル変換

Info

Publication number: CN1837352A
Application number: CN 200610078003
Authority: CN
Inventors: 吴庆余; 徐瀚
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2006-04-29
Filing date: 2006-04-29
Publication date: 2006-09-27

Abstract

The invention discloses a small ball algae cultivation method with high density and high quality, which comprises the following steps: adding the culture medium with glucose and a little of CSL addictive in the gas culture bottle; proceeding rapid small ball algae culture in the heterotrophy condition. The invention simplifies the producing craft and reduces the cost, which is benefit to cultivate large-scale high-density small ball algae. When the substrate glucose density is 100 g per L, the obtaining biomass of small ball algae is 34.97 g per L.

Description

The method of culturing heterotrophic chlorella with high density

Technical field

The invention belongs to biological technical field, relate to the method for a kind of culturing heterotrophic chlorella with high density of fast culture chlorella under the heterotrophism condition.

Background technology

Chlorella is Chlorophyta Chlorella (Chlorella) unicell green alga, and ecological the distribution extensively is easy to cultivate fast growth, using value height.Chlorella contains rich in protein, polysaccharide, lipid, chlorophyll, VITAMIN, trace element and some biologically active metabolite products, is widely used in protective foods, feed, foodstuff additive, fine chemical product and pharmaceutical preparation raw material.Chlorella cells can also utilize organic carbon source to carry out growth and breeding under the heterotrophism condition except utilizing under the autotrophy condition luminous energy and carbonic acid gas grow normally, and the speed of growth is following faster than illumination condition, is similar to the metabolism growth of bacterium.At present the most cheap, optimum and the organic carbon source that is widely used is a glucose.Carry out heterotrophism with organism as the sole carbon source of chlorella and cultivate, have the illumination of need not, cell proliferation is fast, the concentration height, and production system is easy to realize automatic control, can utilize advantages such as existing fermentation equipment.

Up to now, the large scale culturing overwhelming majority of the heterophytic chlorella of having reported is carbon source with glucose, but the utilization ratio of glucose is all lower, and cultivation surplus sugar in latter stage is more, needs to collect use again or be wasted.Not of the same race according to heterophytic chlorella, the highest glucose concn of its tolerance is not quite similar.For original chlorella Chlorella protothecoides, the research of Shi etc. (1999) thinks that the glucose concn of the most suitable production xenthophylls is 40g/l, but Liu Shiming etc. (2000) think that the glucose concn that helps chlorella growth most is 10g/l, and the conclusion of Yan Hai etc. (2005) then is that glucose concn is higher than 13.8g/l just to chlorella growth generation detrimentally affect.The terms of settlement of their unanimity is exactly to adopt stream to add or the mode of feed supplement batch fermentation is cultivated, and with the control glucose concn, but the technology difficulty when this mode has obviously strengthened the scale operation chlorella has improved production cost simultaneously.

The method of the culturing heterotrophic chlorella with high density that we propose is then off the beaten track, by finding a kind of special additive, makes chlorella be greatly improved to the utilization ratio of glucose, has strengthened the tolerance of chlorella to high concentration glucose simultaneously.Adopt this additive, consumption is few, and price is low, saves the step that reclaims glucose, without flow feeding, has reduced production difficulty and production cost, helps the suitability for industrialized production heterophytic chlorella.

Summary of the invention

The objective of the invention is to strengthen the tolerance of chlorella, be implemented in the high-density culture of simplifying heterophytic chlorella under the processing condition high concentration glucose for solving the heterophytic chlorella problem lower to the utilization ratio of glucose.It is characterized in that described cultivation heterophytic chlorella has following dual mode:

Mode a: adding pH value is 5.0～7.0 substratum and adds the addition of C SL that is calculated as 0.5 of substratum～3 ‰ by volume in 1000ml aerated culture bottle, make chlorella can make full use of glucose, and alleviate the injury of high concentration glucose frustule;Inoculum size 5～15% (v/v), 25～30 ℃ of culture temperature, air flow 0.3～1.0vvm adds silicone antifoam agent 0.5/10000th; Finish when the highest to cultivate to the heterophytic chlorella cell density.

Described heterotrophism nutrient solution consists ofglucose 10～100g/l, KH substantially₂PO₄0.3～1.0g/l, K₂HPO₄3H₂O 0.2～1.0g/l, MgSO₄7H₂O 0.1～0.5g/l, FeSO₄7H₂O 1～6mg/l, glycine 0.05～0.3g/l,V_B15～15 μ g/l, A₅Liquid microelement 1ml/l, surplus is a water; A wherein₅The composition of liquid microelement is H₃BO₃2.86g/l, Na₂MoO₄.2H₂O 0.039g/l, ZnSO₄.7H₂O0.222g/l, MnCl₂.4H₂O 1.81g/l, CuSO₄.5H₂O 0.074g/l.

Mode b: in 5～11000L fermentor tank, add above-mentioned substratum and addition of C SL, coefficient 60%,inoculum size 5～10% (v/v); 25～30 ℃ of culture temperature, mixing speed 200～450rpm, air flow 1.0～2.0vvm, tank pressure 0.04Mpa adds silicone antifoam agent 0.5/10000th; After reaching maximum biomass, stop to cultivate.

Described addition of C SL is Tsing-Hua University's Biotechnology Experiment chamber self-control, is a kind of mixture of complicated component, is used for fermentative production; Its main component comprises the potassiumphosphate of 0.5～4% the protein that accounts for addition of C SL gross weight, 1～3% glucose, 2～5% saltpetre 0.4～2%, 0～1% total free aminoacids, 0.01～1% plant hormone.

The invention has the beneficial effects as follows that this technology adds a little addition of C SL in substratum, solved heterophytic chlorella to the inadequate problem of glucose utilization, make heterophytic chlorella utilize the efficient of glucose to be greatly improved, and make chlorella strengthen greatly to the tolerance of high concentration glucose, help to alleviate the injury of high concentration glucose, thereby help the extensive high-concentration culturing of heterophytic chlorella frustule.This method can make the utilization ratio of glucose bring up to 70.9～99.6%, and the glucose concn scope of suitable heterophytic chlorella growth reaches 10～100g/l, and the frustule biomass reaches 34.97g/l.This technology is suitable for the heterophytic chlorella of various scales cultivates, and is particularly advantageous in and improves the technology that large scale fermentation is cultivated, and reduces production costs.

Description of drawings

Fig. 1 is under substratum glucose concn 10g/l condition, adds the comparison of heterophytic chlorella growth curve under CSL and the not interpolation situation in the aerated culture bottle.

Fig. 2 is under substratum glucose concn 30g/l condition, adds the comparison of heterophytic chlorella growth curve under CSL and the not interpolation situation in the aerated culture bottle.

Fig. 3 is under substratum glucose concn 50g/l condition, adds the comparison of heterophytic chlorella growth curve under CSL and the not interpolation situation in the aerated culture bottle.

Fig. 4 is under substratum glucose concn 100g/l condition, adds the comparison of heterophytic chlorella growth curve under CSL and the not interpolation situation in the aerated culture bottle.

Embodiment

The present invention is a kind of method of culturing heterotrophic chlorella with high density, and the key of this technology is to add a kind of heterophytic chlorella that can significantly improve to the utilization ratio of glucose with to the additive of high concentration glucose tolerance.

Concrete steps are as follows:

The heterotrophism nutrient solution consists ofglucose 10～100g/l, KH substantially₂PO₄0.3～1.0g/l, K₂HPO₄3H₂O 0.2～1.0g/l, MgSO₄7H₂O 0.1～0.5g/l, FeSO₄7H₂O 1～6mg/l, glycine 0.05～0.3g/l,V_B15～15 μ g/l, A₅Liquid microelement 1ml/l, surplus is a water.A wherein₅The composition of liquid microelement is H₃BO₃2.86g/l, Na₂MoO₄.2H₂O 0.039g/l, ZnSO₄.7H₂O0.222g/l, MnCl₂.4H₂O 1.81g/l, CuSO₄.5H₂O 0.074g/l.Regulating pH is 6.3 ± 0.2.

Ventilation shake-flask culture condition is as follows: after substratum and cultivation vessel are carried out autoclaving, heterophytic chlorella is carried out sterile culture,inoculum size 5～15% (v/v) in cultivating vessel.28～30 ℃ of control culture temperature, air flow 0.3～1.0vvm.Under the general culture condition, heterophytic chlorella was grown after 6 days and is almost stopped, growth dynamically shows as typical S sigmoid growth curve: promptly 0-24h is the lag phase, 24-120h is a fast growing period, 120-144h enters stationary phase, the heterophytic chlorella cell density arrives the highest, and cell begins apoptosis behind the 144h, finishes to cultivate.

Centrifugal to nutrient solution or fermented liquid collection, rotating speed 8000rpm, 8 ℃ of temperature, centrifugal 10min.Remove supernatant liquor, after obtaining algae mud washing recentrifuge and collecting ,-20 ℃ of freeze overnight, lyophilize obtains dry algae powder then.Weigh, be converted into cell density.

In conjunction with specific embodiments this novel chlorella culture technique is further described with reference to the accompanying drawings below.

Embodiment 1

To under substratum glucose concn 10g/l condition, add CSL whether to the influence of the growth conditions of heterotrophism frustule, the result shows as shown in Figure 1.

The glucose concn of 10g/l is by the suitable condition of the heterophytic chlorella of wide coverage growth.Cultivation through 144h is not adding under the condition of CSL, and algae cell density is 3.74g/l; After adding 1 ‰ CSL, cell density is 293% of control group up to 10.95g/l.The nutrient solution residual sugar that does not add CSL is up to 6.49g/l, and the nutrient solution residual sugar that adds CSL only has 0.12g/l, and the utilization ratio of glucose brings up to 98.8% from 35.1%.The adding that shows CSL promotes the growth of heterophytic chlorella greatly, and glucose is fully used.

Embodiment 2

To under substratum glucose concn 30g/l condition, add CSL whether to the influence of the growth conditions of heterotrophism frustule, the result shows as shown in Figure 2.

Cultivation through 144h is not adding under the condition of CSL, and algae cell density is 0.22g/l, and contrast inoculation back frustule concentration 0.17g/l can think that the high concentration glucose of 30g/l has suppressed the growth of algae substantially.After adding 1.5 ‰ CSL, cell density reaches 18.01g/l, residual sugar 0.13g/l, the utilization ratio 99.6% of glucose.The adding that shows CSL has alleviated the injury of high concentration glucose to chlorella, the growth that has stimulated chlorella.

Embodiment 3

To under substratum glucose concn 50g/l condition, add CSL whether to the influence of the growth conditions of heterotrophism frustule, the result shows as shown in Figure 3.

Cultivation through 192h is not adding under the condition of CSL, and the growth of algae is suppressed.After adding 2 ‰ CSL, cell density reaches 22.97g/l, residual sugar 8.40g/l, the utilization ratio 83.2% of glucose.Show the raising along with glucose concn, harvested cell density increases, but frustule descends to the utilization ratio of glucose, and growth cycle prolongs, and the sugar efficient that is converted into biomass reduces simultaneously.

Embodiment 4

To under substratum glucose concn 100g/l condition, add CSL whether to the influence of the growth conditions of heterotrophism frustule, the result shows as shown in Figure 4.

Cultivation through 192h is not adding under the condition of CSL, and the growth of algae is suppressed.After adding 3 ‰ CSL, cell density reaches 34.97g/l, residual sugar 29.09g/l, the utilization ratio 70.81% of glucose.Show the further raising along with glucose concn, harvested cell density further increases, but frustule further descends to the utilization ratio of glucose.

According to above embodiment as can be seen, the raising of success of the present invention heterophytic chlorella to the utilization ratio of glucose with to the tolerance of high concentration glucose, realized the high-density culture of heterophytic chlorella, simplified raise craft condition, cultivated heterophytic chlorella for large scale fermentation and lay good basis.

Claims

1. the method for a culturing heterotrophic chlorella with high density is characterized in that: add addition of C SL in substratum, make chlorella can make full use of glucose, and alleviate the injury of high concentration glucose to frustule; Described cultivation heterophytic chlorella has following dual mode:

Mode a: adding pH value is 5.0～7.0 substratum and adds the addition of C SL that is calculated as 0.5 of substratum～3 ‰ by volume in 1000ml aerated culture bottle, inoculum size 5～15% (v/v), 25～30 ℃ of culture temperature, air flow 0.3～1.0vvm adds silicone antifoam agent 0.5/10000th; Finish when the highest to cultivate to the heterophytic chlorella cell density;

Mode b: in 5～11000L fermentor tank, add above-mentioned substratum and addition of C SL, coefficient 60%, inoculum size 5～10% (v/v); 25～30 ℃ of culture temperature, mixing speed 200～450rpm, air flow 1.0～2.0vvm, tank pressure 0.04Mpa adds silicone antifoam agent 0.5/10000th; After reaching maximum biomass, stop to cultivate.

2. according to the method for the described culturing heterotrophic chlorella with high density of claim 1, it is characterized in that: described heterotrophism substratum consist of glucose 10～100g/l, KH₂PO₄0.3～1.0g/l, K₂HPO43H₂O 0.2～1.0g/l, MgSO₄7H₂O 0.1～0.5g/l, FeSO₄7H₂O 1～6mg/l, glycine 0.05～0.3g/l, V_B15～15 μ g/l, A₅Liquid microelement 1ml/l, surplus is a water; A wherein₅The composition of liquid microelement is H₃BO₃2.86g/l, Na₂MoO₄.2H₂O 0.039g/l, ZnSO₄.7H₂O 0.222g/l, MnCl₂.4H₂O 1.81g/l, CuSO₄.5H₂O 0.074g/l.

3. according to the method for the described culturing heterotrophic chlorella with high density of claim 1, it is characterized in that: described addition of C SL is for " Tsing-Hua University's Biotechnology Experiment chamber self-control is a kind of mixture of complicated component, is used for fermentative production; Its main component comprises the potassiumphosphate of 0.5～4% the protein that accounts for addition of C SL gross weight, 1～3% glucose, 2～5% saltpetre 0.4～2%, 0～1% total free aminoacids, 0.01～1% plant hormone.