Triglyceride accumulation and the cell cycle in chlorella by Robert Michael Thomas A thesis ...

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Triglyceride accumulation and the cell cycle in chlorella
byRobert Michael Thomas
A thesis submitted in partial fulfillment pfthe degree requirements for the degree of Master of Science
in Microbiology
Montana State University
© Copyright byRobert Michael Thomas (1990)
Abstract:
Triglyceride (TG) storage lipid accumulates in microalgae during exposure to stresses which inhibit
algal cell cycles. Such inhibition is typically induced bynutrient limitations such as depletion of
nitrogen forgreen algae and silicate fordiatoms. No direct mechanism has been described forlow
nutrient-induced lipid accumulation. It is proposed that TG accumulation in microalgae could be based
ona synthesis/utilization pattern which is constitutive within the cell division cycle, as opposed to a
specifically-induced synthesis ofTG. Triglyceride accumulation occurs when a lag is induced in the
cell cycle such that TG synthesis exceeds utilization. Historically, total lipids have been quantified and
the cycling of TG independent ofother lipid fractions has often been overlooked.
Recognizing the limitations ofearlier researchers, trends in TG levels were examined using the neutral
lipid (NL) specific fluorochrome, Nile Red, throughout complete cell cycles of synchronized cultures
of Chlorella CHLORl under conditions ofalkaline stress, nitrogen deprivation, and with
monofluoroacetate (MFA) an inhibitor ofthe tricarboxylic acid (TCA) cycle. Alkaline pH stress, nitrate
deprivation and TCA inhibition affects lipid accumulation byinhibiting the cell division cycle prior to
TG utilization. Low levels ofcalcium were not foundto have any noticeable effects onTG synthesis.
An accumulation of2.5to 5.5times the initial NL level per cell resulted after 25hours of culturing
under alkaline conditions. Nitrogen deprivation yielded increases of 1.4and 2.5times the initial NL
level after 36hours and 14days, respectively. Inless than 30hours, inhibition ofthe tricarboxylic acid
(TCA) with MFA resulted in NL levels of1.4to 3.8times the initial NL level per cell.
Radiolabeled acetate is assimilated at a constant rate into the neutral lipid fraction. Cultures with
growth inhibited byMFA assimilated more radiolabel into neutral lipids and less radiolabel into the
glyco- and polar lipid fractions than in control cultures.
TRIGLYCERIDE ACCUMULATION AND THE CELL CYCLE IN CHT.QRET.TA
by
Robert Michael Thomas
A thesis submitted in partial fulfillment
pf the degree requirements for the degree
of
Master of Science
in
Microbiology
MONTANA |5TATE UNIVERSITY
Bozekan, Montana
July 1990
/iZ3^
APPROVAL
of a thesis submitted by
Robert Michael Thomas
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
Date Chairperson,Graduate Committee
Approved for the Major Department
Approved for the College of Graduate Studies
Date Graduate Dean
iii
STATEMENT OF PERMISSION TO OSE
In presenting this thesis in partial fulfillment of
the requirements for a master's degree at Montana State
University, I agree that the Library shall make it
available to borrowers under rules of the Library. Brief
quotations from this thesis are allowable without special
permission, provided that accurate acknowledgement of
source is made.
Permission for extensive quotation from or
reproduction of this thesis may be granted by my major
professor, or in his absence, by the Director of Libraries
when, in the opinion of either, the proposed use of the
material is for scholarly purposes. Any copying or use of
the material in this thesis for financial gain shall not
be allowed without my written permission.
Signature
iv
ACKNOWLEDGEMENTS
I extend my special thanks to Dr. Keith Cooksey for his
support , guidance, and encouragement on this project. I
am grateful to all of the members of the Department of
Microbiology for their willingness to share equipment and
knowledge. I would particularly like to thank the other
members of my graduate committee. Dr. Jim Cutler and Dr.
Gordon McFeters for their advice and assistance with this
project. I owe a special debt of gratitude to Dr. Jim
Guckert for his inspiring enthusiasm and knowledge. I
would also like to thank Barbara Cooksey for her
methodological suggestions and friendship throughout this
project. I would also like to express my thanks to the
following people and groups:
All of the members of the MSU Biochemistry group,
especially Dr. Larry Jackson for sharing equipment.
The MSU Chemistry Department for use of plotting
facilities. Dr. Pat Callis for use of the
Spectrofluorometer and Dr. Scott Williams for sharing his
expert knowledge of spectrofluorometry. Dr. Barry Pyle
for discussions of viability studies and plotting
assistance. Dr. Dave Ward for his interest and
stimulating discussions about my project. Deb Berglund
for flow cytometric analyses and suggestions. The Solar
Energy Research Institute for funding this work and the
Sigma Xi Grants in Aid of Research Foundation for funds to
purchase a shaker.
V
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS . . . . . .......................... iv
LIST OF TABLES . ....................................Viii
LIST OF FIGURES....................................... ix
ABSTRACT.............................................. xi
INTRODUCTION ...................................... I
Project Goal ................ m
Literature Review and Rationale t
r
Aquatic Species Program Overview i
n
Lipids as Storage Molecules .............. . . . . 10
Stress Effects and the Cell Cycle ..................11
Biochemistry of Fatty Acid Synthesis . . . . . . . 15
Pyruvate dehydrogenase ...................... 16
Regulation of Fatty Acid Synthesis .......... 18
Pyruvate Dehydrogenase ................ 18
Acetyl-CoA Carboxylase ................ 18
Fatty Acid Synthetase................... 20
Desaturation of Fatty Acids ............ 21
Precursor Fatty Acids ................ . . . 22
Two Pathway Model....................... 23
Utilization of Triglycerides ................ 25
Beta Oxidation or Membrane Lipids? . . . 25
The Lipid Trigger....................... 25
Sequential Synthesis of Lipid .............. 26
Some Hypotheses for Control of Lipid
Synthesis................................... 28
Nitrate Deprivation .................... 28
Biosynthetic TCA Cycle ................ 28
TCA Inhibition......................... 30
Other Stress Conditions........... 31
Calcium Effects...................... . . . 31
Alkaline Stress ............................ 31
Synchronous Cultures ............................ 33
Induction of Synchrony in Various Systems . .36
Growth Parameters of Synchronous Cultures . .37
Viability ........................................39
Relative DNA Quantification ...................... 40
MATERIALS AND METHODS ................................ 42
Growth of Algal Cells ............................ 42
Incubator ................................ 45
vi
TABLE OF CONTENTS— Continued
Page
Analysis of Media Components .................... 47
Nitrate . ....................................47
Calcium..................................... 47
pH 48
Culture Synchrony ................................ 48
Viability ....................................... 49
Relative DNA Quantification ...................... .50
Alkaline Stress.............. 52
Nitrogen and Calcium Deprivation ................ 52
Lipid Analysis Using Nile Red ......................52
Microscopy and Photomicrography .............. 54
Radiolabeling Experiments ............ 54
Light/Dark Effects on Acetate Assimilation . . . . 56
Influence of MFA on Lipid Accumulation........... 56
Determination of Labeled Lipids . . . . . ......... 58
RESULTS .62
Synchrony ....................................... 62
Assessment of Chlorella Viability ............. 64
Plate Counts as a Viability Indicator . . . . 64
Fluorescein Diacetate as a Viability
Indicator.......... 64
Fluorometric Observation of DNA Levels
with Hoechst Bisbenzimide Dye 33258 65
Cell Cycle Patterns of Triglyceride and
DNA Levels Under Optimal Growth Conditions . . 70
Visualization of Nile Red-Stained
Triglyceride Droplets ...................... 73
Alkaline Stress Effects
on Neutral Lipid Accumulation ............ .76
Relative DNA Quantification by FlowC ytometry . . . 81
Nitrate and Calcium Deficiency .................. 86
TCA Inhibition with Monofluoroacetate ............ 89
TCA Inhibition with Monofluoroacetate Coupled
to 14C Acetate Uptake......................... 94
14C Acetate Assimilation ijito Lipid
Fractions of Prelabeled Cultures and
the Associated Effects of TCA Inhibition
by M F A .................. . . .'............98
DISCUSSION 102
Synchrony ...................................... 103
Viability ...................................... 104
DNA Quantification.............................. 105
Triglyceride Synthesis-Utilization ........... 108
vii
TABLE OF CONTESTS— Continued
Pagefv
Alkaline Stress . . . . . . . . . . . . . 109
Nutrient Limitation . . . .............. HO
Nitrogen . . ............... . . . . . . HO
Calcium . . . . . . Hl
MOnofluoroacetate . . . . . . . . . . . . . . . . Ill
Acetate Assimilation .................... 114
Practical Applications of Findings ........ . , 115
CONCLUSIONS 118
BIBLIOGRAPHY .................. 120
/
viii
LIST OF TABLES
Table Page
1. Composition of Bold's Basal Medium ........ 42
2. Assessment of Viability by Plate Count . . . . 64
5. Nitrate and Calcium Concentrations . . . . . .88
4. Concentrations of Acetate Incorporated
into Lipid Fractions ........ . . . . . 95
5. Cell Cycle Parameters and Observation
Methods .............................. 102
6. Effects of Various Environmental Factors
oh TG Accumulation . . . . . . . lie
ix
LIST OF FIGURES
Figure Page
1. Lipid Composition of Microalgae ............ 6
2. Proposed Neutral Lipid Accumulating
and Non-Accumulating Conditions ........ 13
3. Pathways of Acyl Biosynthesis
in the Chloroplast.............. . . . 15
4. Control of Lipid Synthesis in Some
Microalgae.......... 19
5. Scatterplots of Relative Cell Number for
Asynchronous and Synchronous Chlorella . 63
6. Histograms of Fluorescein Diacetate
Activity ................................66
7. Bar Graph of Percent Viable C e l l s ..........67
8. Relative Fluorescent Excitations of Unbound
and Bound Hoechst 33258. ............. 68
9. Fluorescent Emissions of Unbound
and Bound Hoechst 33258 ................ 69
10. Time Course of Staining Chlorella
with Hoechst 33258 71
11. Cell Cycle of Chlorella at Neutral pH . . . . 72
12. Fluorescent Micrographs of Nile Red-Stained
Chlorella at 0.5 and 7.5 h o u r s ......... 74
13. Fluorescent Micrographs of Nile Red-Stained
Chlorella at 12 hours and 31 hours . . . 75
14. Alkaline Stress Effects on Cell Cycle
and TG Levels.................. . . . . 77
15. Calibration Curve for Spectrophotometric
Determination of Nitrate Concentrations . 79
16. Cell Cycle Parameters for Alkaline Stress
and Control at Elevated Light .......... 80