Optimization of lutein production with mixotrophic cultivation of an indigenous microalga

Optimization of lutein production with mixotrophic

cultivation of an indigenous microalga

Reporter: Chen-Chun Liu (劉振群)Advisor: Jo-Shu ChangDate: June 26, 2014

2

Outline

• The background

• Research overview

• Results and discussion

• Conclusion

Energy/Environmental Biotechnology &

Biochemical Engineering Laboratory

The Backgroundof this study

MicroalgaLutein

Microalgae as promising feedstock for lutein production


Biochemical Engineering Laboratory3

The Background ─ Microalga



• Transform the sunlight into chemical

energy via photosynthesis

• Various essential nutrients, such as

DHA, EPA, protein, and pigments

GH-B4

The Background ─ Lutein



Photosynthetic pigments

Chlorophylls Carotenoids

Xanthophylls

(CxHyOz)

• Photosynthesis pigments

• Classified into xanthophylls, because its structure

consists of two hydroxyl functional groups

The Background ─ Lutein



Diseases Preventing mechanisms by lutein

Xerophthalmia Quenching active oxygen species

Age macular degeneration Protect cells from blue light-induced

damage and scavenge free radicals

Colorectal cancer N.A.

Light-induced erythema Filtering of blue light and scavenging

reactive intermediates

Cardiovascular diseases Protect against the development of early

atherosclerosis

Reference: Brown, 2008; Slattery et al., 2000; Mares-Perlman et al., 2002

The Background─ Microalgae as promising feedstock for lutein production



1) No limitation of seasonal harvesting

2) Higher growth rate than marigolds

3) Sufficient lutein content inside biomass

4) High lutein productivity

5) Existence of lutein in free form

6) No need for an extra separating step in

comparison with other plants

Source Lutein content

(μg/100g)

Egg yolk 384-1320

Broccoli 710-3300

Carrot 170-650

Lettuce 1000-47806

Orange 64

Papaya 38

Spinach 5930–7900

Corn 2190

Tomato 10-200

Marigold (Tagetes erecta L.) 30000

Microalgae 300000-700000

10 time ↑

Present source

Promising source

Reference: Abdel-Aal et al., 2013; Del Campo

et al., 2007; Fraser & Bramley, 2004;

Hammershøj et al., 2010

Research overviewof this study

The Schematic Diagram of Research



The Schematic Diagram of Research

9

Semi-batch integrated

with two-stage

Microalga Strain

Medium Optimizing Engineering StrategiesLight Intensity

Semi-batch

RSM of C/N

RSM of Trace metal

Medium Choice

Optimizing of cultivation condition for lutein production The use of engineering strategies to enhance

the performance on lutein production

1) Two-level factional factorial design

2) Steepest ascent

3) Central composite design

4) Confirmed experiment

1) Central composite design

2) Confirmed experiment

RSM:

Response surface

methodology

Results and discussionof optimal condition for cultivation

Microalgal Strain Selecting

Suitable Medium ChosenEffect of Sodium Acetate Concentration, Effect of Sodium Nitrate Concentration, RSM

Two-level Fractional Factorial Experimental Design, Steepest Ascent Method, RSM Effect of light intensity



Microalgal Strain Selecting- Condition of experiments

Microalgal strain:

Chlorella sp.

Scenedesmus abundans GH-D11

Scenedesmus obliquus AS-6-1

Chlorella sorokiniana HCH-2

Operated system:

Mixotrophic cultivation, 1L batch

Media:

BG-11 medium

Organic carbon source:

3 g/L sodium acetate

Nitrogen source:

1 g/L sodium nitrate

Light intensity:

150 mmol/m2/s

(TL5, Fluorescent lamp)

Inoculum size:

0.04 g/L

Aeration:

0.2 vvm with 2.5% CO2



Microalgal Strain Selecting- The procedure of experiments



Microalga isolation Acetate-tolerant strains

Chlorella sp.

Chlorella

sorokiniana

Scenedesmus

abundans

Scenedesmus

obliquus

Mixotrophic cultivation

Objective:

1) The strain was higher tolerance of acetate,

2) and had potential to produce lutein

Microalgal Strain Selecting- Summary of experimental data



Strain Cultivation

time*

(d)

Maximal specific

growth rate

(1/d)

Biomass

Productivity*

(g/L/d)

Lutein

Content*

(mg/g)

Maximal Lutein

productivity*

(mg/L/d)

Chlorella sp. 1.0 2.263 1.10 2.55 2.79

HCH-2 1.2 1.320 0.66 3.65 2.42

GH-D11 2.5 1.042 0.53 3.83 2.01

AS-6-1 2.0 1.071 0.53 1.80 0.95

The highest performance

Batch

cultivationFind out

its optimal conditions

*calculated on the period of maximal lutein productivity

Suitable Medium Choice- Condition of experiments

Microalgal strain:

Chlorella sp.

Operated system:


Media:Basal medium

modified Bold Basal medium (MBBM)

modified Bristol's medium (MBM)

Blue Green medium (BG-11)


3 g/L sodium acetate

Nitrogen source:

1 g/L sodium nitrate

Light intensity:

150 mmol/m2/s


Inoculum size:

0.04 g/L

Aeration:




Suitable Medium Choice- Summary of experimental data



Medium Maximal specific

growth rate

(1/d)

Biomass

productivity*

(g/L/d)

Lutein

content*

(mg/g)

Maximal lutein

productivity*

(mg/L/d)

COST

BM 2.133 1.23 1.78 2.19 High

MBM 2.497 1.35 2.30 3.09 Low

MBBM 2.377 1.29 2.29 2.96 Medium

BG-11 2.628 1.32 2.57 3.39 Low


Suitable Medium Choice- Conclusion of medium choice



Ion optimum

C/N optimum

Medium choice

BG-11 medium

1) was suitable for growth and has better lutein performance of

production.

2) the chemical cost of BG-11 was the lowest among these medium

Carbon and Nitrogen Optimizing- The procedure of the optimum of acetate and nitrate concentration



Response

Surface Methodology

Effect of

Substrate Concentration

Confirmation of

RSM model

Carbon and Nitrogen Optimizing- Effect of substrate concentration



Response

Surface Methodology

Effect of


Confirmation of

RSM model

Sodium acetate concentration (g/L)

0 2 4 6 8 10 12

Lu

tein

pro

du

ctiv

ity

(m

g/L

/d)

1.0

1.5

2.0

2.5

3.0

3.5

Sodium nitrate concentration (g/L)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Lu

tein

pro

du

ctiv

ity

(m

g/L

/d)

2.8

3.0

3.2

3.4

3.6

3.8

Carbon and Nitrogen Optimizing- Experimental design matrix of RSM of acetate and nitrate

R

u

n

Comment Code value Real value RV

X31 X32 𝝃31

(g/L)

𝝃32

(g/L)

Y3

(mg/L/d)

1 FF -1 -1 3 1.5 3.60

2 FF -1 1 3 2.5 3.49

3 FF 1 -1 9 1.5 2.71

4 FF 1 1 9 2.5 3.07

5 Axial -1 0 3 2.0 3.60

6 Axial 1 0 9 2.0 2.64

7 Axial 0 -1 6 1.5 3.89

8 Axial 0 1 6 2.5 3.08

9 Center-Ax 0 0 6 2.0 3.66

10 Center-Ax 0 0 6 2.0 3.78

11 Center-Ax 0 0 6 2.0 4.06

12 Center-Ax 0 0 6 2.0 4.06

13 Center-Ax 0 0 6 2.0 4.21

X31, 𝜉31, quantity of acetate; X32, 𝜉32, quantity of nitrate; Y3, maximal lutein productivity

19

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

3

4

5

67

89

1.6

1.8

2.0

2.2

Lu

tein

pro

du

ctiv

ity (

mg

/L/d

)

Sodium acetate (g/L)

Sodium nitrate (g/L)

Carbon and Nitrogen Optimizing- The procedure of optimum of sodium acetate and sodium nitrate



Response

Surface Methodology

Effect of


Confirmation of

RSM model

Optimum value:

Sodium acetate concentration: 4.88 g/L

Sodium nitrate concentration: 1.83 g/L

Maximal lutein productivity: 3.96 mg/L/d

Carbon and Nitrogen Optimizing- The procedure of optimum of sodium acetate and sodium nitrate



Response

Surface Methodology

Effect of


Confirmation of

RSM model

Bio

mass

prod

ucti

on

(g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

Sod

ium

nit

rate

(g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

pH

4

6

8

10

12

14

Sod

ium

aceta

te (

g/L

)

0

1

2

3

4

5

Time (d)

0 1 2 3

Lu

tein

con

ten

t (m

g/g

)

0

1

2

3

4

5

Lu

tein

prod

ucti

vit

y (

mg/L

/d)

0

1

2

3

4

Bio

mass

prod

ucti

vit

y (

g/L

/d)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Maximal lutein productivity:

3.97±0.19 mg/L/d

Carbon and Nitrogen Optimizing- Conclusion of optimum of sodium acetate and sodium nitrate



Ion optimum

C/N optimum

Medium choice

4.88 g/L of sodium acetate and 1.83 g/L of sodium nitrate

1) were the optimal composition of carbon and nitrate source for

Chlorella sp. to produce lutein.

2) The growth curve and the time course of lutein content changing

were investigated

Trace Metal Optimizing- The procedure of the optimum of trace metal



In briefly, that is

Experimental preparation

Two level fractional

factorial design

Steepest ascent method

Response surface

methodology




1) Calcium chloride dehydrate

2) Copper sulfate pentahydrate

3) Ferric ammonium citrate

4) Magnesium sulfate heptahydrate

5) Sodium chloride

6) Zinc sulfate heptahydrate



factorial design


Response surface

methodology




1) Calcium chloride dehydrate*

2) Copper sulfate pentahydrate

3) Ferric ammonium citrate

4) Sodium chloride*

* whose p-value were less than 0.05



factorial design


Response surface

methodology

Y = 𝛽0 +

i=1

k

𝛽𝑖χi

Y: the predicted response

β0: the intercept

βi: linear coefficients

Step

O-Δ O O+Δ O+2Δ O+3Δ

Lu

tein

pro

du

ctiv

ity (

mg

/L/d

)

2.8

3.0

3.2

3.4

3.6

3.8

4.0




X22

X21



factorial design


Response surface

methodology

Trace Metal Optimizing- Experimental design matrix of RSM of CaCl2·2H2O and NaCl

R

U

n

Comment Code value Real value RV

X41X42 𝝃41

(mg/L)

𝝃42(mg/L)

Y4

(mg/L/d)

1 FF 1 1 55 61 3.44

2 FF 1 -1 55 356 3.18

3 FF -1 1 45 61 3.03

4 FF -1 -1 45 356 2.94

5 Axial 1.414 0 57 208 3.46

6 Axial 0 1.414 50 0 3.16

7 Axial 0 -1.414 50 417 3.11

8 Axial -1.414 0 43 208 3.57

9 Center-Ax 0 0 50 208 4.14

10 Center-Ax 0 0 50 208 4.00

11 Center-Ax 0 0 50 208 3.80

12 Center-Ax 0 0 50 208 3.75

13 Center-Ax 0 0 50 208 3.71

X41, 𝜉41, quantity of CaCl2·2H2O ; X42, 𝜉42, quantity of NaCl; Y4, maximal lutein productivity



2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

34

36

38

40

42

050

100150

200250

300350

400

Lu

tein

pro

du

ctiv

ity (

mg/L

/d)

Ca 2+ concentration (mg/L) Sodium chloride (m

g/L)

Optimum value:

CaCl2∙2H2O concentration: 51 mg/L

NaCl concentration: 218 mg/L

Predicted lutein productivity: 3.88 mg/L/d



factorial design


Response surface

methodology






factorial design


Response surface

methodology

Confirmation of

RSM model

Bio

ma

ss p

ro

du

cti

on

(g

/L)

0.0

0.5

1.0

1.5

2.0

2.5

So

diu

m n

itra

te (

g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

pH

4

6

8

10

12

14

So

diu

m a

ceta

te (

g/L

)

0

1

2

3

4

5

Time (d)

0 1 2 3

Lu

tein

co

nte

nt

(mg

/g)

0

1

2

3

4

5

Lu

tein

pro

du

cti

vit

y (

mg

/L/d

)

0

1

2

3

4B

iom

ass

pro

du

cti

vit

y (

g/L

/d)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Maximum lutein productivity:

4.10±0.04 mg/L/d

Trace Metal Optimizing- Conclusion of optimum of trace metal



Ion optimum

C/N optimum

Medium choice

51 mg/L of CaCl2∙2H2O and 218 mg/L of NaCl

1) were the optimal composition of ion, especially for

calcium chloride and sodium chloride for Chlorella sp.

to produce lutein.

2) It was the end step of optimizing medium

Effect of Light intensity- Condition of experiments

Microalgal strain:

Chlorella sp.

Operated system:


Medium:

BG-11 medium


4.88 g/L sodium acetate

Nitrogen source:

1.83 g/L sodium nitrate

Light intensity:

150 - 600 mmol/m2/s


Inoculum size:

0.04 g/L

Aeration:


Ion concentration:

CaCl2∙2H2O: 51 mg/L

NaCl: 218 mg/L



Effect of light intensity- Investigating the influence on growth and available for outdoor cultivation



Bio

mass

pro

du

ctio

n (

g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

150 umol/m2/s

300 umol/m2/s

450 umol/m2/s

600 umol/m2/s

Sod

ium

ace

tate

(g/L

)

0

1

2

3

4

5

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Bio

mass

pro

du

ctiv

ity

(g/L

/d)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

(a)

(b)

(c)

There were no statistically significant

results for biomass productivity

And, the acetate still could be

exhausted regardless of the light

intensity.

Effect of light intensity- Summary of experimental data



Light intensity

(μmol/m2/s)

Biomass productivity*

(g/L/d)

Lutein content*

(mg/g)

Lutein productivity

(mg/L/d)

150 (Origin) 1.06±0.01 3.87±0.09 4.10±0.04

300 1.02±0.01 3.78±0.06 3.86±0.06

450 1.03±0.02 3.66±0.11 3.78±0.11

600 1.01±0.04 3.69±0.05 3.72±0.14


Results and discussionof application of engineering strategies

Semi-batch system

Semi-batch integrated with two-stage system



Semi-batch System- Condition of experiments

Microalgal strain:

Chlorella sp.

Operated system:

Mixotrophic cultivation, 1L

Medium:

BG-11 medium



Nitrogen source:


Light intensity:

150 mmol/m2/s


Inoculum size:

0.04 g/L

Aeration:


Ion concentration:

CaCl2∙2H2O: 38.55 mg/L

NaCl: 218 mg/L

Replacement ratio:

20% - 80%



Semi-batch system- The illustrated diagram of semi-batch operation



Batch cultivation

start to conduct

the semi-batch

operation

continue to culture microalga

till it can be harvested

take out specific

ratio of mediumsupplement

fresh medium

X(g/L)

t(d)

(about 2 day)

Semi-batch system- Summary of semi-batch experimental data



Replacement

ratio

(%)

Cultivation

time*

(hr)

Biomass

productivity*

(g/L/d)

Lutein

content*

(mg/g)

Lutein

productivity*

(mg/L/d)

Batch 42 1.06±0.01 3.87±0.09 4.10±0.04

20% 9.50±0.45 1.00±0.06 3.93±0.02 3.95±0.24

40% 15.40±2.37 1.18±0.15 3.78±0.17 4.43±0.46

60% 22.60±2.52 1.32±0.08 3.70±0.22 4.86±0.20

80% 25.00±1.67 1.55±0.12 3.58±0.21 5.51±0.21

Forty percent time saved

• The cultivation time could be reduced indeed; even for 80%

replacement ratio, and it still reduced about forty percent time.

• The biomass productivity and lutein productivity increased

under the replacement ratio enhanced.

• The lutein content in biomass was seemingly no difference


Semi-batch integrated with two-stage system- Introductory remark



Despite of the good results with conventional semi-batch,

it still had some disadvantages:

a) repeated adaptation of different environment (with or without acetate),

b) extra timeframe for accumulation of lutein,

c) the insufficient stability of cultivation system,

d) and the poor utilization of mixed gas.

Semi-batch integrated with two-stage system- Condition of experiments

Microalgal strain:

Chlorella sp.

Operated system:

Mixotrophic cultivation, 1L

Medium:

BG-11 medium



Nitrogen source:


Light intensity:

150 mmol/m2/s


Inoculum size:

0.04 g/L

Aeration:


Ion concentration:

Calcium (II): 38.55 mg/L

Sodium chloride: 218 mg/L

Replacement ratio:

60, and 80%



Semi-batch integrated with two-stage system- The illustrated diagram of semi-batch operation



Semi-batch

operation

Lutein

accumulation

Buffer

tank

1. cell growth

2. mixotrophic

3. acetate existing

1. lutein

accumulation

2. autotrophic

3. acetate free

X(g/L)

t(d)

Content (mg/g)

t(d)

Semi-batch integrated with two-stage system- Summary of experimental data



Replacement

ratio

(%)

Cultivation

time*

(hr)

Biomass

productivity*

(g/L/d)

Lutein

content*

(mg/g)

Lutein

productivity*

(mg/L/d)

Batch 42 1.06±0.01 3.87±0.09 4.10±0.04

Semi-batch 60% 22.60±2.52 1.32±0.08 3.70±0.22 4.86±0.20

Semi-batch 80% 25.00±1.67 1.55±0.12 3.58±0.21 5.51±0.21

Integrated system

60%13.2±1.9 1.44±0.10 3.93±0.09 5.66±0.30

Integrated system

80%16 1.98±0.04 3.85±0.13 7.62±0.21

Integrated system: semi-batch integrated with two-stage*calculated on the period of maximal lutein productivity

Conclusionof this study

Overview of this research

Comparison with the other previous studies



Conclusion- Overview of this research



Conclusion- Comparison with the other previous studies

Microalga strain Operation

system

Cultivation

condition

Biomass

productivity

(g/L/day)

Lutein

content

(mg/g)

Lutein

productivity

(mg/L/day)

References

Chlorella protothecoides Batch Heterotrophic 1.90 1.90 3.6 (Wei et al., 2008)

Chlorella zofingiensis Batch Autotrophic 0.88 3.4 3.0 (Del Campo et al.,

2007)

Scenedesmus obliquus FSP-3 Batch Autotrophic 0.92 4.52 4.15 (Ho et al., 2014a)

Chlorella zofingiensis Batch Autotrophic 0.45 7.2 3.2 (Del Campo et al.,

2004)

Coccomyxa onubensis Semi-batch Autotrophic 0.55 6.2 3.41 (Vaquero et al., 2012)

Scenedesmus obliquus FSP-3 Semi-batch Autotrophic 1.23±0.03 4.57±0.26 5.56±0.31 (Chan, 2012)

Desmodesmus sp. F51 Fed-batch Autotrophic 0.65 5.5 3.56 (Xie et al., 2013)

Scenedesmus almeriensis Continuous Autotrophic 0.87 5.5 4.77 (Sánchez et al., 2008a)

Scenedesmus almeriensis Continuous Autotrophic 0.72 5.3 3.8 (Sánchez et al., 2008b)

Muriellopsis sp. Continuous Autotrophic 1.67 4.3 7.2 (Del Campo et al.,

2001)

Coccomyxa acidophila Batch Mixotrophic 0.26 3.50 0.9 (Casal et al., 2011)

Chlorella sp. Batch Mixotrophic 1.03±0.04 3.86±0.22 3.97±0.19 This study

Chlorella sp. Semi-batch Mixotrophic 1.55±0.12 3.58±0.21 5.51±0.21 This study

Chlorella sp. Integrated system Mixotrophic 1.98±0.04 3.85±0.13 7.62±0.21 This study

Integrated system: semi-batch integrated with two-stage

About me

45

at National Cheng Kung University

master student,second year, major in ChemicalEngineering

conference attended4 of internal conf.2 of international conf.

main author or coauthor3 of accepted publication1 of modification

some skills has learned in the past period

I

Health & Medicine

Optimization of lutein production with mixotrophic cultivation of an indigenous microalga