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 Chang
Date: June 26, 2014

2
Outline
• The background
• Research overview
• Results and discussion
• Conclusion
Energy/Environmental Biotechnology &
Biochemical Engineering Laboratory

The Background
of this study
Microalga
Lutein
Microalgae as promising feedstock for lutein production
3

The Background ─ Microalga
4
• Transform the sunlight into chemical
energy via photosynthesis
• Various essential nutrients, such as
DHA, EPA, protein, and pigments
GH-B4

The Background ─ Lutein
5
Photosynthetic pigments
Chlorophylls Carotenoids
Xanthophylls
(CxHyOz)
• Photosynthesis pigments
• Classified into xanthophylls, because its structure
consists of two hydroxyl functional groups

The Background ─ Lutein
6
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
7
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 overview
of this study
The Schematic Diagram of Research
8

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 discussion
of optimal condition for cultivation
Microalgal Strain Selecting
Suitable Medium Chosen
Effect of Sodium Acetate Concentration, Effect of Sodium Nitrate Concentration, RSM
Two-level Fractional Factorial Experimental Design, Steepest Ascent Method, RSM
Effect of light intensity
10

- 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
11

- The procedure of experiments
12
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

- Summary of experimental data
13
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
cultivation
Find out
its optimal conditions
*calculated on the period of maximal lutein productivity

Suitable Medium Choice
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:
14

15
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

- Conclusion of medium choice
16
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
17
Response
Surface Methodology
Effect of
Substrate Concentration
Confirmation of
RSM model

- Effect of substrate concentration
18
Response
Surface Methodology
Effect of
Confirmation of
RSM model
Sodium acetate concentration (g/L)
0 2 4 6 8 10 12
Luteinproductivity(mg/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
2.8
3.0
3.2
3.4
3.6
3.8

- 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
6
7
8
9
1.6
1.8
2.0
2.2
Sodium acetate (g/L)
Sodium
nitrate (g/L)
- The procedure of optimum of sodium acetate and sodium nitrate
20
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

- The procedure of optimum of sodium acetate and sodium nitrate
21
Response
Surface Methodology
Effect of
Confirmation of
RSM model
Biomassproduction(g/L)
0.0
0.5
1.0
1.5
2.0
2.5
Sodiumnitrate(g/L)
0.0
0.5
1.0
1.5
2.0
2.5
pH
4
6
8
10
12
14
Sodiumacetate(g/L)
0
1
2
3
4
5
Time (d)
0 1 2 3
Luteincontent(mg/g)
0
1
2
3
4
5
0
1
2
3
4
Biomassproductivity(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

- Conclusion of optimum of sodium acetate and sodium nitrate
22
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
23
In briefly, that is
Experimental preparation
Two level fractional
factorial design
Steepest ascent method
Response surface
methodology

24
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

25
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
2.8
3.0
3.2
3.4
3.6
3.8
4.0
26
X22
X21
factorial design
Response surface
methodology

- Experimental design matrix of RSM of CaCl2·2H2O and NaCl
R
U
n
Comment Code value Real value RV
X41 X42 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

28
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
0
50
100
150
200
250
300
350
400
Ca 2+
concentration (mg/L) Sodium
chloride (mg/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

29
factorial design
Response surface
methodology
Confirmation of
RSM model
0.0
0.5
1.0
1.5
2.0
2.5
Sodiumnitrate(g/L)
0.0
0.5
1.0
1.5
2.0
2.5
pH
4
6
8
10
12
14
Sodiumacetate(g/L)
0
1
2
3
4
5
Time (d)
0 1 2 3
Luteincontent(mg/g)
0
1
2
3
4
5
0
1
2
3
4
Biomassproductivity(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

- Conclusion of optimum of trace metal
30
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
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
31

- Investigating the influence on growth and available for outdoor cultivation
32
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
Sodiumacetate
(g/L)
0
1
2
3
4
5
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Biomassproductivity
(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.

33
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 discussion
of application of engineering strategies
Semi-batch system
Semi-batch integrated with two-stage system
34

Semi-batch System
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%
35

Semi-batch system
- The illustrated diagram of semi-batch operation
36
Batch cultivation
start to conduct
the semi-batch
operation
continue to culture microalga
till it can be harvested
take out specific
ratio of medium
supplement
fresh medium
X(g/L)
t(d)
(about 2 day)

Semi-batch system
- Summary of semi-batch experimental data
37
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

- Introductory remark
38
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.

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%
39

- The illustrated diagram of semi-batch operation
40
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)

41
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

Conclusion
of this study
Overview of this research
Comparison with the other previous studies
42

Conclusion
- Overview of this research
43

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 Chemical
Engineering
conference attended
4 of internal conf.
2 of international conf.
main author or coauthor
3 of accepted publication
1 of modification
some skills has learned
in the past period
I

Optimization of lutein production with mixotrophic cultivation of an indigenous microalga

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