Understanding How Critical Growth Parameters Affect Algal Biofilm Growth and Internal Lipid ...

213 Pages·2016·5.38 MB·English

by  Peter Schnurr

Understanding How Critical Growth Parameters Affect Algal
Biofilm Growth and Internal Lipid Concentrations

By

Peter Schnurr

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy,

Department of Chemical Engineering and Applied Chemistry

University of Toronto

© Copyright by Peter Schnurr, 2016
Understanding How Critical Growth Parameters Affect Algal Biofilm Growth and Internal Lipid
Concentrations
Peter Schnurr
Doctor of Philosophy 2016
Department of Chemical Engineering and Applied Chemistry
University of Toronto
ABSTRACT

Due to their relatively fast growth rates and high concentrations of specific biocompounds, algal
biofilms have great potential for the production of valuable bioproducts. This thesis investigates the
critical factors affecting algal biofilm growth rates and their internal lipid concentrations. Results of
this thesis have implications for operation of algal biofilm culturing systems.
The main factors investigated in this thesis were how nutrient supplies (carbon dioxide, nitrogen
and silicon concentrations), light orientation, and photon flux density affects algal biofilm growth
and internal lipid concentrations. This was accomplished by growing algal biofilms on glass and
polycarbonate growth coupons in culturing systems that facilitated control over the main factors
affecting growth. Two separate biofilm culturing systems were developed and utilized to test distinct
research objectives. Biofilms were grown for 14-26 days, harvested from growth coupons several
times per week, lyophilized to determine productivities (g/m2/d), and in some cases, exposed to
chemical treatments to determine lipid content.
Irradiating algal biofilms from either the water side, material side, or both sides, while
maintaining a total photon flux density (PFD) of 100 µmol/m2/s, did not affect overall algal biofilm
biomass productivities. When well-developed algal biofilms were starved of nitrogen and silicon,
there was no accumulation of neutral lipids. Fatty acid methyl ester (FAME) concentrations
increased from 5% to 8% (w/w) when PFDs were increased from 50 to 150 µmol/m2/s, however,
ii
further FAME accumulations were not observed beyond that PFD range. Algal biofilm starvation
did, however, induce biofilm growth cessation and sloughing from the growth substratum. At fixed
PFDs of 100 µmol/m2/s, increasing inorganic carbon concentrations from 0.04% to 2% (v/v) resulted
in significant biomass productivity increases of 0.5 to 2.0 g/m2/d; however, no biomass productivity
increases occurred when CO concentrations were incrementally increased from 2% to 12%. When
2
PFDs and CO concentrations were simultaneously increased (from 50 to 440 µmol/m2/s and 0.04 to
2
7% CO ), there were significant increases in algae biofilm biomass productivities (from ~1 g/m2/d to
2
4.4 g/m2/d) i.e. there was an interaction effect. Maximal algal biofilm total FAME productivities
were ~0.3 g/m2/d, comparable to the best terrestrial crops.

iii
Foreword Acknowledgements

I would like to express my sincere gratitude to the following people and organizations that supported
me over the course of this doctoral thesis:

My parents, Betty and Terry Schnurr, for raising me with strong principles and work ethic, both of
which have been critical factors in getting me to where I am today.

Professor Grant Allen and George Espie, my supervisors, who supplied me with invaluable guidance
and mentorship through the course of this thesis. Their intelligence, wisdom, and commitment have
been critical to my education and development over the course of this thesis.

Professor Elizabeth Edwards and Gideon Wolfaardt, members of my reading committee, who
continually provided support in the form of constructive criticisms, general feedback, and regular
consultation.

Professors Acosta, Diosady, Aitchison and Short, members of the University of Toronto Algae
Group, who provided additional support and consultation through the course of this thesis.

The Natural Science and Engineering Research Council of Canada for their financial support in the
form of an Alexander Graham Bell Canadian Graduate Scholarship and various strategic grants
provided to our lab group.

The Ontario Graduate Scholarship Program for their financial support in the form of an Ontario
Graduate Scholarship.

Hatch Ltd. for their financial support in the form of a Hatch Graduate Scholarship for Sustainable
Energy Research.

Our industrial partners, Pond Biofuels Ltd., Biox Inc., and Mara Renewables Inc., for their guidance,
partnership, and technical support over the course of this thesis.

The lecturers and administrative and technical staff in the Department of Chemical Engineering &
Applied Chemistry at the University of Toronto for their support: Paul Jowlabar, Chris Ambidge,
Mary Butera, Joan Chen, Leticia Gutierrez, Pauline Martini, Phil Milczarek, Gorette Silva, Daniel
Tomchyshyn.

The summer students who worked hard and helped me with experiments throughout the course of
this thesis: Jian (J.F.) Wang, Ana Luisa Cunha Norte, Gregory Peniuk, and Jin Choi.

My family, friends, lab mates, and colleagues for their moral, emotional, and technical support over
the years.

iv
FOREWORD ACKNOWLEDGEMENTS .................................................................................................................................. IV
1 INTRODUCTION .................................................................................................................................................................... 1
1.1 ALGAE BIOFILM GROWTH FOR BIOFUELS AND BIOPRODUCTS ...................................................................................... 1
1.2 HYPOTHESES ........................................................................................................................................................................... 2
1.3 OBJECTIVES ............................................................................................................................................................................. 3
1.4 OVERALL APPROACH ............................................................................................................................................................. 4
1.5 SIGNIFICANCE .......................................................................................................................................................................... 4
1.6 THESIS OUTLINE ..................................................................................................................................................................... 5
2 LITERATURE REVIEW: FACTORS AFFECTING ALGAE BIOFILM GROWTH AND LIPID
PRODUCTION: A REVIEW ......................................................................................................................................................... 7
2.1 INTRODUCTION ....................................................................................................................................................................... 7
2.2 COMPOSITION AND STRUCTURE OF AN ALGAL BIOFILM ................................................................................................. 9
2.2.1 Extracellular Polymeric Substances and Matrices .......................................................................................... 9
2.2.2 Species and Succession of Photosynthetic Biofilms ....................................................................................... 11
2.3 ALGAE BIOFILM BIOMASS AND LIPID PRODUCTION ..................................................................................................... 15
2.3.1 Algae Biofilm Attachment to Growth Materials ............................................................................................. 16
2.3.1.1 The Effect of Material Properties on Algae Biofilm Growth .................................................................................................. 16
2.3.1.2 Biotic Factors on Biofilm Development and Growth ................................................................................................................ 19
2.3.2 Key Growth Parameters and their Effect on Algae Biofilm Biomass Productivities ....................... 21
2.3.2.1 Light Intensity ........................................................................................................................................................................................... 21
2.3.2.2 Carbon Dioxide Concentrations ......................................................................................................................................................... 25
2.3.2.3 Other Growth Factors ............................................................................................................................................................................. 27
2.3.3 Algae Biofilm Biomass lipid Potential ................................................................................................................. 29
2.3.3.1 Lipids and Lipid Concentration Enhancement ............................................................................................................................ 29
2.3.3.2 Algae Biofilm Productivities ................................................................................................................................................................ 33
2.4 CONCLUSIONS ....................................................................................................................................................................... 35
2.5 REFERENCES ........................................................................................................................................................................ 38
3 THE EFFECT OF NUTRIENT STARVATION ON ALGAE BIOFILM GROWTH AND LIPID
ACCUMULATION ........................................................................................................................................................................ 47
3.1 INTRODUCTION .................................................................................................................................................................... 47
3.2 MATERIALS AND METHODS .............................................................................................................................................. 50
3.2.1 Biofilm Culturing System .......................................................................................................................................... 50
3.2.2 Experimental Approach ............................................................................................................................................ 52
3.2.2.1 Biofilm Growth .......................................................................................................................................................................................... 52
3.2.2.2 Suspended Cultures ................................................................................................................................................................................ 54
3.2.3 Sampling and Analysis ............................................................................................................................................... 55
3.3 RESULTS AND DISCUSSION ................................................................................................................................................ 57
3.3.1 Biofilm Growth Kinetics and the Affects of Nutrient Starvation ............................................................. 57
3.3.2 Lipid Concentration of Algae Grown in Nutrient Replete and Deficient Conditions ...................... 62
3.3.3 Algae Biofilm Biomass and Lipid Productivities and Implications for Algal Biofilm Growth
System for Biofuels ...................................................................................................................................................................... 65
3.4 CONCLUSIONS ....................................................................................................................................................................... 67
3.5 REFERENCES ........................................................................................................................................................................ 68
4 THE EFFECT OF LIGHT DIRECTION AND SUSPENDED CELL CONCENTRATIONS ON ALGAE
BIOFILM GROWTH RATES ...................................................................................................................................................... 71
4.1 INTRODUCTION .................................................................................................................................................................... 71
4.2 MATERIALS AND METHODS .............................................................................................................................................. 73
4.2.1 Algae Biofilm Growth System ................................................................................................................................. 73
4.2.2 Experimental Approach ............................................................................................................................................ 75
4.2.3 Sampling and Analysis ............................................................................................................................................... 76
4.2.4 Data Analysis ................................................................................................................................................................. 77
4.3 RESULTS ................................................................................................................................................................................ 78
v
4.3.1 The Effect of Light Direction on Algal Biofilm Growth Kinetics .............................................................. 78
4.3.2 The Effect of Suspended Algal-Cell Concentration on Biofilm Growth kinetics ................................ 82
4.4 DISCUSSION .......................................................................................................................................................................... 84
4.4.1 Implications for Reactor Design and Operation ............................................................................................. 88
4.5 REFERENCES ........................................................................................................................................................................ 90
5 THE EFFECT OF PHOTON FLUX DENSITY ON ALGAL BIOFILM GROWTH AND INTERNAL FATTY
ACID CONCENTRATIONS ......................................................................................................................................................... 93
5.1 INTRODUCTION .................................................................................................................................................................... 93
5.2 MATERIALS AND METHODS .............................................................................................................................................. 96
5.2.1 Biofilm Growth System .............................................................................................................................................. 97
5.2.2 Experimental Approach ............................................................................................................................................ 99
5.2.3 Sampling and Analysis ............................................................................................................................................ 100
5.2.4 Data Analysis .............................................................................................................................................................. 101
5.3 RESULTS AND DISCUSSION .............................................................................................................................................. 102
5.3.1 Algal Biofilm Growth Kinetics and Productivities at Various Light Intensities ............................. 102
5.3.2 Biomass Yield on Light of Algal Biofilms at Various Photon Flux Densities .................................... 107
5.3.3 The Effect of Light Wavelength on Algal Biofilm Growth ....................................................................... 109
5.3.4 The Effect of Photon Flux Densities on Algal Biofilm Fatty Acid Accumulation ............................ 111
5.3.5 Algal Biofilm Fatty Acid Productivities at Various Photon Flux Densities ....................................... 112
5.4 CONCLUSIONS ..................................................................................................................................................................... 113
5.5 SUPPLEMENTARY MATERIAL .......................................................................................................................................... 114
5.5.1 White-Red PDF Scaling Factor ............................................................................................................................ 114
5.6 REFERENCES ...................................................................................................................................................................... 116
6 THE EFFECT OF CO CONCENTRATION AND INTERACTION EFFECTS OF CO CONCENTRATION
2 2
AND PHOTON FLUX DENSITY ON ALGAE BIOFILM BIOMASS PRODUCTIVITY ................................................... 120
6.1 INTRODUCTION .................................................................................................................................................................. 120
6.2 MATERIALS AND METHODS ............................................................................................................................................ 123
6.2.1 Flat-Plate Parallel Horizontal Algae Biofilm Culturing Systems ......................................................... 123
6.2.2 Experimental Approach ......................................................................................................................................... 124
6.2.2.1 Carbon Dioxide Concentration One-variable-at-a-time Studies ....................................................................................... 124
6.2.2.2 Carbon Dioxide Concentration – Photon Flux Density Interaction Studies ................................................................. 125
6.2.3 Sampling and Analysis ............................................................................................................................................ 126
6.2.4 Statistical Analysis .................................................................................................................................................... 127
6.3 RESULTS AND DISCUSSION .............................................................................................................................................. 128
6.3.1 The effect of carbon dioxide concentration on algae biofilm growth kinetics ............................... 128
6.3.2 Interaction effects of PFD and carbon dioxide concentration on algae biofilm growth kinetics
130
6.3.2.1 Physiological Basis for CO2 and PFD Interaction ..................................................................................................................... 135
6.3.2.2 Detrimental Interactions ................................................................................................................................................................... 136
6.3.2.3 Mass and Light Transport Limitations ......................................................................................................................................... 137
6.3.2.4 Interaction Effects in Other Aquatic Photosynthetic Organisms ..................................................................................... 137
6.4 CONCLUSIONS ..................................................................................................................................................................... 138
6.5 SUPPLEMENTARY MATERIALS ........................................................................................................................................ 139
6.6 REFERENCES ...................................................................................................................................................................... 142
7 OVERALL DISCUSSION & CONCLUSIONS .................................................................................................................. 145
7.1 ALGAE BIOFILM CULTURING SYSTEMS ......................................................................................................................... 145
7.2 ALGAL BIOFILM LIPID ACCUMULATION ........................................................................................................................ 147
7.3 ALGAL BIOFILM GROWTH RATES ................................................................................................................................... 149
7.4 CONCLUSIONS ..................................................................................................................................................................... 151
7.5 RECOMMENDATIONS FOR FUTURE WORK ................................................................................................................... 153
8 APPENDICES ..................................................................................................................................................................... 156
vi
8.1 ALGAE BIOFILM GROWTH SYSTEM CHARACTERIZATION .......................................................................................... 156
8.1.1 Theoretical Inorganic Carbon Required ......................................................................................................... 157
8.1.2 Theoretical Inorganic Carbon Available and Mass Balance .................................................................. 157
8.1.3 Theoretical Nitrogen Required ........................................................................................................................... 159
8.1.4 Theoretical Nitrogen Available and Mass Balance .................................................................................... 159
8.1.5 Residence Times in Biofilm Culturing System ............................................................................................... 160
8.2 SHEAR RATE DERIVATION ............................................................................................................................................... 161
8.3 SYNTHETIC GROWTH MEDIUM ....................................................................................................................................... 163
8.4 ALGAL BIOFILM DNA PELLET RAW DATA LOG SHEET ............................................................................................. 164
8.5 RAW DATA FROM THESIS ................................................................................................................................................ 167
1
vii
LIST OF TABLES

Table 1. Comparing algae biofilm biomass productivities from different studies with different light
intensities. ..................................................................................................................................... 25
Table 2. Comparison of lipid concentrations and productivities of species commonly found in
freshwater photosynthetic biofilms. All values are of organisms grown planktonically under
nutrient replete conditions. ........................................................................................................... 33
Table 3. Comparison of biomass and lipid productivities from algal biofilm growth systems. .......... 34
Table 4: Comparison of the conversion efficiencies of various photon flux densities to biofilm
biomass. Biofilm biomass productivity 95% confidence interval values were determined from
the linear regression of the data . Biofilm biomass photon conversion efficiency confidence
intervals were calculated from the 95% confidence intervals from the biomass productivity
multiplied by the constant used to calculate the conversion efficiencies. .................................. 108
Table 5: Scaling factors for white light sources to yield a unit of red PFD (620 -640 nm). Conversion
ratios comparing the light intensity (µmol/m2/s) of white light photosynthetically active
radiation (400-700nm) in light sources typically used for growing algae, to the light intensity of
photons in the red LED lights used in these experiments (620-640nm) within that same white
light source. ................................................................................................................................ 115
Table 6: Experimental design growth conditions for interaction experiments of coded and uncoded
CO concentrations and red light PFD. ...................................................................................... 126
2
Table 7: A summary of the surface response model used to determine the impact of PFD and CO on
2
biomass productivity. ................................................................................................................. 132
Table 8: Comparing the experimentally determined biofilm biomass productivities data to the
productivities predicted by the model at each of the PFD and CO concentrations tested. The
2
biomass productivity model data was determined from the equation of the line, which was
determined from the coefficients shown in Table 7. .................................................................. 133

viii
LIST OF FIGURES

Figure 1. Conceptual algae biofilm photobioreactor. ............................................................................. 1
Figure 2. Strategies for achieving thesis objectives. ............................................................................. 4
Figure 3. Development of a Mixed Community Algal Biofilm: (A) Growth surfaces are first
'conditioned' with bacteria cells that secrete the initial EPS matrix; (B) Various species of algae
cells present in the bulk medium then begin to colonize the EPS matrix; (C) The algae cells
grow and reproduce, forming a symbiotic relationship with the bacteria present in the EPS
matrix; (D) A mature biofilm matrix is densely populated with algae cells, particularly
cyanobacteria and chlorophytes, and retains nutrients in the EPS matrix. .................................. 12
Figure 4. Schematic of Light Profiles Through an Algae Biofilm at Various Intensities: Photon
penetration through algae biofilms increases with increasing photon flux density. Thick algae
biofilms have relatively thin photosynthetically active regions adjacent to their source of light,
but comparatively thick photosynthetically inactive regions opposite the light source (A); As
light intensity increases the photosynthetically active region increases due to increased photon
penetration, and the subsequent reduction of antennae size/number within this region (B) and
(C). ............................................................................................................................................... 23
Figure 5. Schematic of the algae biofilm culturing system used to grow algae biofilms with controls
on key growth parameters. ........................................................................................................... 52
Figure 6a. Algal biofilm growth kinetics grown at a shear rate of 4 s-1, pH of 7 +/- 0.2, temperature
of 25°C +/- 2°C, CO2 concentration of 2%, and a photon flux density of 100 µmol/m2/s.
Triplicate biological replicates were run to determine the effects of nutrient starvation on
growth kinetics. Nutrient starvation of nitrogen and silicon began at the 13th day of growth. A
fourth control experiment was run for 26 days without any nutrient deficiencies. Inoculum was
a pure culture of Scenedsmus obliquus. Technical replicates were taken as triplicates and the
error bars show the standard error of the mean. ........................................................................... 58
Figure 7a. Algae biofilms inoculated with S. obliquus and grown for 14 days under nutrient replete
conditions. .................................................................................................................................... 60
Figure 8. Internal FAME lipid concentrations of algae grown under nutrient replete and deficient
conditions, and grown planktonically and as a biofilm. Technical replicates were taken as
quadruplicates and the error bars show the deviation. Algal biofilms show no increase in
biofilm lipid concentrations after 3-4 and 6-7 days of nutrient starvation. Conversely, lipid
concentrations in planktonic cultures of both Nitzschia palea and Scenedsmus obliquus were
nearly doubled when exposed to 3 days of nutrient deficient conditions. ................................... 62
Figure 9. Comparing biomass and lipid productivities of N. palea and S. obliquus grown at shear
rates of 7 s-1, pH of 7 +/- 0.2, temperature of 25°C +/- 2°C, CO2 concentration of 2%, and a
photon flux density of 100 µmol/m2/s. Lipid productivities were calculated from the linear
regression of the biomass productivities and the mass fraction of lipids in the biomass before
starvation commenced. The error bars represent the 95% confidence interval of the linear
regression of the biomass productivities. ..................................................................................... 65
Figure 10. Schematic of the algae biofilm culturing system used to grow algae biofilms with controls
on key growth parameters. ........................................................................................................... 75
Figure 11. Biofilms were irradiated from the water side, the materials side, or both the water and
materials side. The PFD at the biofilm surface was 100 µmol/m2/s – 50 µmol/m2/s from each
side when light was provided from both sides. A single experiment was conducted with a PFD
of µmol/m2/s from the water-side only. ........................................................................................ 76
Figure 12. Sampling flow chart for light direction and initial recruitment/regrowth experiments. ..... 77
Figure 13 a, b. Algal biofilm growth kinetics when light was provided from the water side, material
side, or both the water and material side of the biofilm. Throughout the experimental run shear
ix
rates were maintained at 4 s-1, pH at 7 +/- 0.2, temperature at 25°C +/- 2°C, CO2 concentration
at 2%, and total photon flux densities at 100 µmol/m2/s. Triplicate biological replicates were
run when light was provided from both sides, while duplicate biological replicates were run
when light was provided from only the water side or only the material side. A single control
experiment control experiment was run at half the total PFD (50 µmol/m2/s). Triplicate
experimental replicates were taken on sampling days. Inoculum was a pure culture of
Scenedsmus obliquus. ................................................................................................................... 79
Figure 14. Algal biofilm biomass production rates following 26 days of growth with different light
directions, with half light intensity, and with enhanced suspended cell concentrations. Linear
regression of the combined data for biological replicates were used to produce these
productivities. No difference in algal biofilm growth kinetics was observed at the 95%
confidence interval, however there was a difference when light was halved from 100 to 50
µmol/m2/s total photon flux densities. The 95% confidence intervals are shown as the error
bars. .............................................................................................................................................. 80
Figure 15 a, b. Photon flux density of light transmitted through a biofilm when light was provided
from the material side or the water-side of the biofilm. The total incident photon flux density
was 100 µmol/m2/s. ...................................................................................................................... 81
Figure 16. Photon flux densities in the bulk medium of the flat photobioreactors when biofilms are
irradiated from the materials side, the water side, and both the materials and water side. A very
sharp attenuation of light was observed with time when biofilms were irradiated from materials
side only, with only a fraction of incident light being transmitted across the biofilm within the
first few days of growth. .............................................................................................................. 82
Figure 17. Algal biofilm biomass production rates following 7 days of cell recruitment and re-growth
of biofilms. Throughout the experimental run shear rates were maintained at 4 s-1, pH at 7 +/-
0.2, temperature at 25°C +/- 2°C, CO2 concentration at 2%, and total photon flux densities at
100 µmol/m2/s. Light direction was as indicated, and with enhanced suspended cell
concentrations. Different letters denote differences at the 95% confidence interval. The 95%
confidence intervals are shown as the error bars. ........................................................................ 83
Figure 18: Schematic diagram of the algae biofilm culturing system used to study the affects of
photon flux density on algae biofilm growth kinetics and internal lipid concentrations. ............ 99
Figure 19: Algal biofilm growth kinetics determined at 50 µmol/m2/s (A), 150 µmol/m2/s (B), 300
µmol/m2/s (C), and 600 µmol/m2/s (D) of red LED light (620-640 nm). Throughout each
experimental run shear rates were maintained at 4 s-1, pH at 7 +/- 0.2, CO concentration at 6%,
2
and temperature at 25°C +/- 2°C. Each data point is a mean of triplicate samples taken on a
particular day, and the error bars are the standard error of the mean. Each experimental run
represents an independent biological replicate. Inoculum was a mixed culture of Scenedsmus
obliquus, Chlorella vulgaris, and Clamydomonas reinhardtii. ................................................. 104
Figure 20: Algal biofilm biomass productivities at various PFDs of red light (620-640nm) as an
average of all biological replicate runs at each PFD. Throughout each experimental run shear
rates were maintained at 4 s-1, pH at 7 +/- 0.2, CO concentration at 6%, and temperature at
2
25°C +/- 2°C. The error bars represent the 95% confidence intervals of the linear regression.
Different letters denote statistically significant differences of biofilm biomass productivities
according to the 95% confidence interval limits of their linear regressions. ............................. 105
Figure 21: Fatty acid methyl ester (FAME) concentrations within algal biofilm biomass grown at
various red light PFDs . The error bars represent 95% confidence interals, and the different
letters denote statistically significant differences between growth conditions according to their
95% confidence interval limits. .................................................................................................. 111
Figure 22: Overall fatty acid productivities of algal biofilms grown at various red light intensities.
The error bars represent 95% confidence interals, and the different letters denote statistically
x