Table of Contents (cont)

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National Renewable Energy Laboratory

II.B.1.f.

Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae ............................................................. 83

II.B.1.g.

Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae ............................................................. 85

II.B.1.h.

Transformation and Somatic Cell Genetics for the Improvement of Energy Production in Microalgae ....... 89

Background ................................................................................................................................................................. 89

Isolatation and characterization of natural hosts for the algal viruses. ........................................................................ 91

Screening of the SERI algal collection for infection with the Chlorella viruses. ........................................................ 92

Use of the lytic enzyme(s) produced by the virus for degradation of the host cell wall for the production of

microalgal protoplasts. ................................................................................................................................................ 92

Characterization of the chloroplast and mitochondrial genomes of microalgal strains. .............................................. 93

II.B.2.

Physiology, Biochemistry, and Molecular Biology of Lipid Production: NREL In-House

Researchers ........................................................................................................................................................ 97

II.B.2.a.

Introduction .................................................................................................................................................. 97

II.B.2.b.

Lipid Accumulation Induced by Nitrogen Limitation................................................................................... 98

II.B.2.c.

Studies on Photosynthetic Efficiency in Oleaginous Algae .......................................................................... 98

II.B.2.d.

Lipid Accumulation in Silicon-Deficient Diatoms...................................................................................... 100

II.B.2.e.

Isolation and Characterization of Acetyl-CoA Carboxylase from C. cryptica ............................................ 104

II.B.2.f.

Cloning of the Acetyl-CoA Carboxylase Gene from C. cryptica ................................................................ 107

II.B.2.g.

Biochemistry of Lipid Synthesis in Nannochloropsis ................................................................................. 110

II.B.2.h.

Biochemistry and Molecular Biology of Chrysolaminarin Synthesis ......................................................... 110

II.B.3

Manipulation of Lipid Production in Microalgae via Genetic Engineering....................................... 115

II.B.3.a.

Introduction ................................................................................................................................................ 115

II.B.3.a.

Mutagenesis and Selection.......................................................................................................................... 116

II.B.3.c.

Development of a Genetic Transformation System for Microalgae ............................................................ 118

Introduction:.............................................................................................................................................................. 118

Analysis of Microalgal DNA Composition: .............................................................................................................. 119

Transient Expression of Luciferase in Chlorella ellipsoidea: .................................................................................... 122

Development of Homologous Selectable Markers for Monoraphidium and Cyclotella: ........................................... 126

Transformation of Chlamydomonas reinhardtii Using Silicon Carbide Whiskers: ................................................... 129

Development of a Genetic Transformation System for the Diatoms Cyclotella and Navicula: ................................. 132

II.B.3.d.

Attempts to Manipulate Microalgal Lipid Composition via Genetic Engineering ...................................... 139

II.B.3.e.

The Effect of Different Promoters on Expression of Luciferase in Cyclotella ............................................ 141

II.B.4.

Microalgal Strain Improvement - Conclusions and Recommendations ...................................................... 144

III.

OUTDOOR STUDIES AND SYSTEMS ANALYSIS............................................................................ 146

III.A. PROJECTS FUNDED BY ERDA/DOE 1976-1979 ....................................................................................... 146

III.A.1.

Introduction .................................................................................................................................... 146

III.A.2.

Species Control in Large-Scale Algal Biomass Production ........................................................... 148

III.A.3

An Integrated System for the Conversion of Solar Energy with Sewage-Grown Microalgae ........ 153

III.A.4.

Large-Scale Freshwater Microalgal Biomass Production for Fuel and Fertilizer ........................ 157

III.A.5.

Other Microalgae Projects during the ERDA/DOE Period ........................................................... 162

III.B. THE ASP MICROALGAL MASS CULTURE .................................................................................................... 163

III.B.1. Introduction ........................................................................................................................................ 163

III.B.2.

The ARPS Project in Hawaii, 1980-1987 ....................................................................................... 166

III.B.2.a.

Hawaii ARPS Project Initiation, 1980-1981............................................................................................... 166

III.B.2 b.

Second Year of the Hawaii ARPS Project, 1981-1982 ............................................................................... 166

III.B.2.c.

Third Year of the Hawaii ARPS Project, 1982-1983.................................................................................. 170

III.B.2.d.

Fourth Year of the Hawaii ARPS Project, 1983-1984 ........................................................................... 171

III.B.2.e.

Fifth Year of the ARPS Project, 1984-1985 ............................................................................................... 173

III.B.2.f.

Sixth Year of the Hawaii ARPS Project, 1985-1986 .................................................................................. 173

III.B.2.g.

Seventh Year of the Hawaii ARPS Project, 1986-1987 ......................................................................... 174

III.B.2.h.

Hawaii ARPS Project, Conclusions ....................................................................................................... 174

A Look Back at the Aquatic Species Program--Technical Review