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Metabolic Engineering and Molecular

Biotechnology of Microalgae for Fuel

Production

​

​

Tse-Min Lee

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Microalgae as feedstock

  1. They grow extremely fast and hence produce high biomass yield quickly.
  2. Microalgae-based fuels do not compete with the food

supply and hence present no food security concerns.

  1. Biofuels generated from microalgae are renewable and can be carbon-reducing [generation of 100 tons of algal biomass is equivalent to removing roughly 183 tons of carbon dioxide from the atmosphere (Chisti, 2008)].
  2. Microalgal farming does not require arable land and can utilize industrial flue gas as a carbon source.
  3. Selected oleaginous microalgae do not require fresh water and can grow in seawater, brackish water, or waste water.
  4. Biodiesel fuels derived from microalgae can be integrated

into the current transportation infrastructure.

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s

ro

m

ic

a

d

alga about 10micrometres in diameter that swims with t

hydro shap "eyes

ignificant advances in unco components required for p

olecules in microalgae

  • The availability of genom

green alga Chlamydomon

  • Chlamydomonas reinhar

vering molecular duction of fuel

​

sequences in the model

s reinhardtii.

tii is a single-cell green

wo flagella. It has a c

ll wall made of

xyproline-rich glycopr

oteins, a large cup-

ed chloroplast, a large

pyrenoid, and an

pot" that senses light.

Kingdom:

Phylum:

Class:

Order:

Family:

Genus:

Species:

C. reinhardtii

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Genetics of C. reinhardtii

  • The Chlre3 draft of the Chlamydomonas nuclear genome sequence prepared by Joint Genome Institute of the U.S. Dept of Energy comprises 1557 scaffolds totaling 120 Mb.
  • Roughly half of the genome is contained in 24 scaffolds all at least 1.6 Mb in length. The current assembly of the nuclear genome is available online.
  • The ~15.8 Kb mitochondrial genome (database accession: NC_ 001638) is available online at the NCBI database.
  • The complete >200 Kb chloroplast genome is available online.

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Culture of C. reinahrdtii

  • TAP or HSM medium
  • Other medium
  • On the plate and liquid flask

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BIODIESEL

  • Oleaginous green microalgae species that have the capacity to accumulate oil in the form of triacylglycerols, or TAGs (Chisti, 2007; Sheehan et al., 1998), have been isolated and possess great potential as a feedstock for biodiesel fuels (Converti et al., 2009; Liu et al., 2008; Xu et al., 2006).

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Triacylglycerol (TAG)

neutral lipid – stained by Nile Red

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Nitrogen starvation (-N)

fast accumulation

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營養缺乏誘導TAG-Chlorella sp.

‘Nile Red’ yellow fluorescence

Light microscopic observation

小球藻 (Chlorella sorokiniana T-89) 缺氮下之中性脂肪細胞細胞數量及含 量(粘珮嫆,李澤民,周德珍,方孟德)。

● 誘導期 - 尼羅紅(Nile Red)黃螢光鏡檢

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產油藻類生產生質柴油

引用自Chisti (2007) Biotechnology Advances 25: 294-306

●產油藻類之粗油脂含量高

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●產油藻類之生產油脂效率高

⮚比較不同植物及藻類生產油脂所需面積。

⮚微細藻類是最有效率的生產油脂植物，遠高於棕櫚樹。

引用自Chisti (2007) Biotechnology Advances 25: 294-306

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BIODIESEL from algae – not yet

  • However, microalgae-based biodiesel is far from being commercially feasible, because it is not economically practical at present.
  • Why?
  • High cost

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FEEDSTOCK

  • Algal Biology
  • Algal Cultivation
  • Harvesting and Dewatering

​

CONVERSION

  • Extraction and Fractionation
  • Fuel Conversion
  • Co-products

INFRASTRUCTURE

  • Distribution and Utilization
  • Resources and Siting

PURSUING STRATEGIC R&D: TECHNO- ECONOMIC MODELING AND ANALYSIS

微藻生質柴油生產程序及成本

  • A. Cultivation
  • B. Harvesting/Collection

•

•

•

•

•

•

•

    • Hydrolysis
    • Drying and handling

D. Processing

  1. Storage/transportation
  2. Sale
  1. Finance
  2. Laws and social factors

(台科大李篤中教授)

• 10-20%

• 10-25%

• 10-20%

• 20-30%

• 10-20%

• 5-10%

• 10-20%

• 20-40%

• 5-10%

戶外栽培成本估算

A+B+C+D佔微藻能源成本 >50%

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微細藻類生產生質柴油之量產程序

第1階段 - 藻種篩選及實驗室內小量栽培 第2階段 –於光反應器內之中量栽培

第3階段 - 川流式養殖場(raceway culture pond)之大量栽培

第4階段 –生質柴油之後製

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Algal biology

  • Genetic st
  • Physiology
  • Lipid body

udies

studies

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From a biological point of view, one of the obvious solutions is to increase oil

content

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Mechanism for TAG accumulation

  • C partition

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l

枷『k Hi

Marine Biology Research Division,

Scripps Institution 。f Ocean。graphy, University of Calif。rn祠 San Dieg。

Growth Conditions A何ect Carbon Partitioning In Some Algae

co,

co,

Plh

直圈

／迦

／＼

Lipid

Carbohydrate Synth開is

Syr咐 晶is

Nutrient Replete

﹛Abundant Grow旬 ﹜

Nutrient Deficient

(P。。r Gr< wthl

ldentificati。n of pathway-levelregulati。n may be especially usefulin metab。lie

engineering to increase lipid yields under n。n-limiting gr。wth conditions.

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BIOHYDROGEN

However, the observed emission of hydrogen was transient and the amount was very minima.

In green microalgae, production of hydrogen is catalyzed by [FeFe]-hydrogenases.

  • The ability of microalgae to produce hydrogen was first reported by Gaffron and Rubin in 1942 (Gaffron and Rubin, 1942).

•

​

•

​

  • [FeFe]-hydrogenases catalyze the reversible reduction of protons toH2 (Equation 3.1).
  • [FeFe]-hydrogenases have been isolated and identified from microalgae such as C. reinhardtii (Forestier et al., 2001; Happe and Kaminski, 2002), Scenedesmus obliquus (Florin et al., 2001), and Chlorella fusca (Winkler et al., 2002).

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Anoxia induction and

photosynthesis dependence

  • Two highly similar [FeFe]-hydrogenases are present in the genome of C. reinhardtii and are named HydA1 and HydA2. Both [FeFe]-hydrogenases are activated by anaerobic conditions induced by purging with neutral gas or by sulfur deprivation of the cultures (Forestier et al., 2003).
  • Gene expression and mRNA stability as well as the enzymatic activities of HydA1 and HydA2 are extremely sensitive to oxygen. For this reason, hydrogen production rapidly stops as soon as cells begin oxygenic photosynthesis.
  • Therefore, establishing anoxic culture conditions is crucial for

induction of hydrogen in algal cultures.

  • In addition to creating anaerobic conditions, electrons required to reduce H ions are derived from photosystem II (PSII)-dependent activity (Antal et al., 2003; Antal et al., 2009) and PSII- independent plastoquinone (PQ) reduction pathways, as shown in Figure 3.2 (Chochois et al., 2009; Hemschemeier et al., 2008).

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Pathway for H2 production

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BIOHYDROGEN

  • The ability of microalgae to produce hydrogen was first reported by Gaffron and Rubin in 1942 (Gaffron and Rubin, 1942).
  • However, the observed emission of hydrogen was transient and the amount was very minima

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3.4 OTHER STRATEGIES

  • 3.4.1 Optimization of Light Conversion Efficiency

(LHCB)

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SDS-PAGE electrophoresis

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Optimization of light conversion

efficiency (LCE)

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苟會

看捧 毛 看車

警告

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Optimization of light conversion

efficiency (LCE)

  • The sunlight intensities are much higher than those

required to saturate photosynthesis.

  • To avoid overexcitation of the photosystem, plants and green microorganisms deal with excess light by dissipating heat and emitting fluorescence.
  • As a consequence, the realistic LCE converts solar energy to biomass is much lower than the theoretical calculation (Dismukes et al., 2008; Melis, 2009; Wijffels and Barbosa, 2010).

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NPQ and photoinhibition

  • Around 80% of the absorbed photons could be wasted due to dissipation of excitation by nonphotochemical quenching and photoinhibition of photosynthesis (Melis, 1999; Melis et al., 1999).

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Adjustment of photosynthesis to light intensity

alterations

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Increase in Lhc number (antenna

size)

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Arrangement of PSII, PSI, Cytb6f, and ATPase

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Increase in PSII number

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Improvement of light absorption

  • To improve solar illumination distribution of the microalgal culture, mutants with reduced light-harvesting chlorophyll antenna sizes that would allow for efficient utilization of light energy, and therefore would increase productivity, have been proposed.
  • The rationale of this approach is to minimize light absorption by cells on the surface and to permit greater sunlight penetrance into the deeper layers of the culture.

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truncated light-harvesting chlorophyll antenna size (tla) mutants (Lee et al., 2002; Polle et al.,

2000; Polle et al., 2003)

Reduction of photosystem chlorophyll antenna size in tla mutants has been demonstrated to improve solar energy

conversion efficiency and productivity.

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RNAi approach

  • Reduction of the light harvest complex I(LHCI) and LHCII antenna complex system by knocking down light harvest complex B major proteins results in

improved photon capture efficiency, enhanced growth rate, and reduced photoinhibition (Mussgnug et al., 2007).

Mautusi Mitra and Anastasios Melis (2008) Optical properties of microalgae for enhanced biofuels production. Optics Express, Vol. 16, Issue 26, pp. 21807-21820.

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吾先

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