The utilization of varying wavelengths of light by cultured and
environmental cyanobacteria
James R. Henriksen
Microbial Diversity Course 2010, MBL
Abstract
Photoautotrophy is the foundation of all ecosystems. In order to explore the vast diversity of mechanisms
used by bacteria to capture light energy, two methods were developed: a high-throughput method for
physiological investigations and selective enrichments of specific organisms based on varying wavelength and
intensity of light, and a culture independent, microscopic method based on autofluorescence for probing
the light utilization and components of microbial photosystems. These were applied in an investigation
of cyanobacterial light harvesting apparatus in cultured isolates and environmental samples that provided
insights into cellular variation and life cycle in isolated cyanobacterial cultures and the ecological physiology
and selection in a “Green Berry” cyanobacteria-dominated microbial consortium.
Background
Microorganisms in the environment, particularly prokaryotes, are major drivers in all biogeochemical cycles,
and are critical for providing ecosystem functions (Madigan 2008). The vast diversity of physiologies and
characteristics are the very fabric of life on this planet, and may increase the stability of the biosphere. An
understanding of the the vectors in Hutchinsonian niche space that lead to specalisation and evolutionary
pressures, and an understanding of the mechanisms of utilising these diverse resources are the major pursuits
of environmental microbiology (Holt 2009). One such vector at the base of all foodchains are the wavelength
and amount of light present for photosynthesis.
Oxagenic photosynthesis drives almost all energy cycling and carbon fixation on the planet. Even the 0.8%
of global carbon fixation that is carried out by anoxic autotrophy is dependent on oxagenic photosynthesis,
as the oxidant and reductants in all modern food webs, even deep sea hydrothermal vents, originate from
oxagenic photosynthesis (Raven 2009). Oxagenic photosynthesis evolved only in the cyanobacterial lineage,
probably from a horizontal gene transfer that resulted in the combination of the photosystems from green and
purple bacteria (Madigan 2008). Cyanobacteria are responsible for 50% of the global primary productivity
and the majority of carbon that is rapidly cycled, with the remainder being provided by the cyanobacterial
endosymbionts (chloroplasts) of eukaryotes (Phillips 2009).
The major components needed for the growth of oxygenic diazotrophic photoautotrophs are available in
abundance. These organisms require only light, water, common gases found in the atmosphere, and trace
elements. It is instructive to consider the amounts of these components found in nature. All values for
the following are calculated from information in the Bionumbers database (Phillips 2009) and the CRC
Handbook of Chemistry and Physics (Haynes 2010). Water is the most common electron donor available
that is accessible to life, at concentrations of ∼55 M (the molarity of pure elemental water) over the majority
of the plants surface. Dinitrogen gas is the most abundant atmospheric gas, present at 3.5 moles/L air at
sea level, but only at ∼45 µM in seawater, due to it’s poor solubility. However, this concentration is enough
to meet the nitrogen demand of ∼107 bacterial cells/ml of seawater, even without taking into account a net
flux from the atmosphere. The carbon dioxide that is the sole source of carbon for autotrophs is present at
17mmols/L in the atmosphere at sealevel, and at average of ∼2mM in seawater, sufficient for ∼108 bacterial
cells/ml of seawater. Trace elements needed by oxigenic diazotrophic photoautotrophs are only needed at
low levels, but may be limiting in parts of the ocean where dust inputs are low.
1
Light, the final requirement of oxigenic diazotrophic photoautotrophs, and their sole source of energy, is
abundant at the surface of the planet. If an organism has the machinery to access it, light is an excellent
source of energy. The energy available in light varies with its wavelength (sometimes termed quality, measured
in nm or in inverse frequency in Hz) and the number of photons (intensity or quantity measured in mols
of photons, or Einsteins, per second per area). Light with wavelengths of 400 to 1200nm provides -350 to
-100 kJ/mol (Overmann 2000), and in direct sunlight photons across the spectrum are provided at 2000
µEinsteins/m2 /sec, close to the maximum flux used in photosynthesis (Phillips 2009). For a cell with a
photosynthetic cross section of 10µm, this would correspond to 1010 photons per sec. For comparison, E.
coli growing aerobically with a doubling time of 20 minutes use glucose at -2870 kJ/mol at a turnover rate
of 106 mol/sec (Phillips 2009). Therefore, light provides less energy per electron transferred, but is available
at a high rate.
The wavelength and intensity of light available varies in the environment (particularly with depth, as
shorter wavelengths of light penetrate deeper in bodies of water). The wavelength and intensity of light that
can be utilised or tolerated between organisms also varies. The the minimum energy and intensity of light
thought necessary to support life are 117 KJ/mol (corresponding to a wavelength of 1020nm, Overmann
2000) and 10-5 µE/m2 /s (Beatty 2005) respectively. The maximum energy of light is assumed to be limited
by the toxisity of far UV light. The major groups of phototrophs (PSB, PNS, GNS, cyanobacteria, green
and red algae, and diatoms) utilize different spectra of light (termed the action spectra) and use different
photosynthetic apparatus. This may be based on the molecular characteristics of light absorption water, as
well as light characteristics of different bodies of water (Stomp 2007).
Cyanobacteria are unique among oxygenic photoautotrophs in their ability to capture light across the
visible range, including the green light that is not utilised by eukaryotic phototrophs. Cyanobacteria, like
many phototrophic groups, contain organisms that have specific characteristic wavelengths of light that
they utilize. However cyanobacteria are unique in that some organisms can change the wavelength of light
that can be captured by producing different phycobiliproteins, a process termed complementary chromatic
adaptation.
Cyanobacteria use a diverse set of phycobiliproteins in various combinations in a phycobilisome antenna
complex. All cyanobacteria produce the phycobiliproteins allophycocyanin and phycocyanin, while some
also have phycoerythrin or phycoerythrocyanin. These proteins contain chromophore tetrapyrroles known
as phycobilins that along with the protein and modifications to the structure determine the wavelength of
light captured. Different phycobiliproteins contain different combinations of phycobilins (see Table 1). All
cyanobacteria produce phycobilins phycocyanobilin and phycoerythrobilin, and some can produce phycobiliviolin (also known as phycoviolobilin or cryptoviolin) or phycourobilin (see Table 1). Some cyanobacteria
constitutively express all phycobiliproteins they can produce, while others can regulate some or all of their
phycobiliproteins. A number of factors can change the amounts, ratios and types phycobiliproteins and
chlorophyll reaction centers. Of these the best characterised in complementary chromatic adaptation species
is the available amount and wavelength of light. Other factors that might influence the light absorbed include
the characteristics of the organisms, nitrogen and CO2 availability, and cellular stress.
Cyanobacterial photosynthesis is composed of a z scheme where a photon of light excites antenna pigments in the phycobilisomes. If the organisms is producing them, lower wavelength (higher energy) photons
are absorbed by other phycobiliproteins and their energy is transferred to phycocyanin and then to allophycocyanin. The energy from the phycobilisome is passed PSII where charge separation traps the energy in
electron potential and splits water to O2 . This energy is then passed to through a electron transport chain
(generating ATP) to PSI, which, when excited by another photon, passes the energy along another electron
transport chain to either the chain of PSII in cyclic electron flow or to form NADH in a non-cyclic manner. Besides this two-photosystem system, all cyanobacteria can carry out PSI-based sulfate oxidation by
degrading PSII and the phycobiliproteins. There are some observations that suggest that phycobiliproteins
may in some cases pass energy to PSI, but this is controversial.
The excitation peaks of the phycobiliproteins are broad and often show significant tailing and variation
between sub-types in different organisms. The characteristic maxima of the spectral curves of the major
components of the Cyanobacterial photosynthetic machinery are listed in Table 1. The florescent emission
spectra of any compound is characteristic for a molecule. If the absorbed light energy causes fluorescence,
the absorbance peak is also the fluorescent excitation maxima. If energy is passed from one component to
another (usually from phycobiliproteins to chlorophil), the observed fluorescence emission will be that of
2
Table 1: Absorbance and fluorescence values for cyanobacterial photosystems. Values are from whole cells
and peptide-bound billins, not extracts or pure compounds. Note that the exact shape of the absorbance
and the specific fluorescence maxima can be determined, and are diagnostic of a compound in a specific
intracellular environment. Ranges are compiled from Overman 2010, Fay 1987, Platt 1986 and Glazer 1988.
Photopigment
Components
Absorbance (nm)
R-phycoerythrin
B-phycoerythrin
C-phycoerythrin
phycoerythrocyanin
R-phycocyanin
C-phycocyanin
allophycocyanin
chlorophyll a
phycoerythrobilin, phycourobilin
phycoerythrobilin, phycourobilin
phycoerythrobilin
phycocyanobilin, phycobiliviolin
phycocyanobilin, phycoerythrobilin
phycocyanobilin
phycocyanobilin
chlorophil a
455-575
475-575
494–575
568-590
533-615
615–630
650–655
400, 660-700
Florescent
emission
(nm)
538-581
538-581
538-581
568-625
640-650
617-645
650-680
680-720
chlorophil, while the excitation maxima will be the absorption maxima of the initial absorbing component.
The transfer of energy between components is very efficient, with more than 98% of the energy being captured
delivered to chlorophil before it can be re-emited as heat or fluorescence. Energy is usually only lost at the
chlorophil molecule, causing fluorescence. The quantum yield of the photosynthetic reaction complex is high,
and the autoflourecence seen from these systems are within the range for fluorophores used in fluorescent
microscopy. The pattern of excitation and emission is characteristic of the presence of the various parts of
the components in the photochain.
Cyanobacteria occur in many environments, and are responsible for large amounts of primary productivity
in the open ocean. One coastal ecosystem where macroscopic aggregates of cyanobacteria are visually
dominant is in pools found in Sippiwisit Marsh that are filled with small (approximately 1-5mm) spherical
“green berries”. For previous work by a Microbial Diversity Course student on the macroscale dark green
cyanobacterial aggregates, see Gentile 2002.
Materials and Methods
Cultures
The unicellular marine cyanobacteria Cyanothece ATCC51142 and Synechococcus PCC7335, and the unicellular freshwater, low-light adapted Gloeothece PCC7109 were kindly provided by John Waterbury. Anabena
PCC7120 was kindly provided by Bob Haselkorn.
Environmental samples
Macroscale dark green cyanobacterial aggregates (“green berries”) were kindly collected by Parris Humphrey,
Ulli Jaekel, and Lizzy Wilibanks from pools in the Lesser Sippiwisit Marsh, a salt-water marsh north of Woods
Hole, Massachusetts where they co-occur with purple bacterial aggregates. Three berries were homogenised
with a microcentrifuge pestle and resuspended in media by vortexing.
Culture conditions
Cultures were grown on SN or BN media (Waterbury 2006) for marine and freshwater strains respectively at
30C inoculated from liquid stock cultures. All pre-cultures were grown for one week under broad-spectrum
fluorescent lights at 75 µEinsteins/m2 /s unless otherwise noted. All strains were checked for nitrogen fixation
in media lacking a nitrogen source, and examined microscopically for morphology.
3
Table 2: LED specifications
Luminous
Flux at
Current
(mlm)
Current
(A)
Forward
Voltage
(Vf)
Viewing
Angle
100mA
3.5
∼45◦
495
20mA
3.4
85◦
525
1110
20mA
3.4
85◦
Amber-Yellow
592
278
20mA
2
30◦
Orange-Yellow
605
278
20mA
2
30◦
Orange-Red
615
278
20mA
2
30◦
Red
623
941
20mA
2.2
85◦
IR
850
not
reported
not reported
1.5
26◦
900K White
broad
3750
20mA
3.2
110◦
Color / Name
Wavelength
(nm)
UV
380
Blue
470
Green
Supplier and
Part #
All Electronics LED-910
Digi-Key
365-1201-ND
Digi-Key
365-1202-ND
Digi-Key
160-1503-ND
Digi-Key
160-1502-ND
Digi-Key
160-1501-ND
Digi-Key
365-1203-ND
Digi-Key
475-1460-ND
Digi-Key
C535AWJNCS0V0151ND
Unit
Price
(US$)
0.5
0.52
0.58
0.25
0.29
0.29
0.3
0.3
0.43
Multi-spectral LED culturing plates electronics
A led lightbox was prepared with cheap commercially available 5mm through-hole LEDs arrayed in the wells
of a black polystyrene 96 well plate (see Table 2). Each row consisted of one of each LED type wired in
series so as to provide a constant voltage drop. These were connected in parallel with a 100W high wattage
resister to limit current spikes and driven by a regulated power supply in series with a digital multimeter to
measure current. At a current through the LEDs of 150mA (slightly less than the test current for the LEDs
of 20mA times 8 parallel LED circuits), the voltage across the LED array was close to the 19.86 predicted
by Ohm’s law and the typical forward voltage of the LEDs reported on their specification sheets. The power
supply was set so as to be voltage limiting, but any increase in current would cause it to become current
limited. These precautions should prevent any problems with burning out of LEDs. The LED wavelengths
(from specification sheets and confirmed with a handheld diffraction grating spectrometer) and intensities
are shown in Table 3. Typical LEDs have spectral half-max widths of 5-10nm.
Multi-spectral LED culturing plates
Stacks of sterile clear-bottomed and clear-lidded, black-walled polystyrene 96 well plates were placed on top
of the lightbox, such such that each well received light from one LED. Stack of plates of 200ul of cultures were
interspaced with 96 well 50% neutral density plates. The neutral density plates were also clear-bottomed
96 well plates with 200 µl of a dilution of 0.12g activated charcoal in 50% saturated sucrose. Linear and
logarithmic dilutions of this suspension were made on a 96 well plate and measured with a spectrophotometer
to determine the dilution that archived 50% transmittance.
4
Table 3: LED spectral characteristics
Color / Name
Wavelength
(nm)
UV
Blue
Green
Amber-Yellow
Orange-Yellow
Orange-Red
Red
IR
900K White
380
470
525
592
605
615
623
850
broad
Calculated
photon
energy
(KJ/E)
288.42
233.19
208.76
185.14
181.16
178.21
175.92
128.94
Approximate
Pyranometer
Flux
measurements
(µE/m2 /s)
Quantum
sensor
measurements
3.9
43.35
19.55
2.7
3.3
8.1
15.3
100
150
100
75
210
180
101.25
550
450
200
300
200
450
1500
150
60
96 well light culturing
High throughput LED culturing chambers were used to expose multiple cultures (up to 8 per plate) to each of
the different wavelengths of light, at different intensities (using the charcoal-glycerol neutral density filters).
Wells in three 96 well plates were filled with 100ul of an appropriate medium (SN or BN). All wells in a row
were inoculated with one of the pure cultures or consortia, or left uninoculated as a sterility control. Since
the different light conditions were arrayed in columns on the plate, each inocula was exposed to one of the
10 different wavelengths, at three intensities (100%, 50%, 25% of values listed in Table 3), as well as multiple
dark controls. During use the LED light array was operated with a timer for 16h light / 8h dark and the
cultures kept at 30‰in an incubator Absorbance and fluorescence were measured daily.
Absorbance and fluorescence
A Molecular devices M5 plate reading flouromiter was used for all well absorbance (at 600nm and the
Chlorophyll absorbance maximum) and fluorescence measurements. For fluorescence, the following excitation
wavelengths were used, corresponding to the LED wavelengths: 380, 470, 525, 592, 605, 615, 623, and 850nm.
For each, the closest filter set was used, and all emission scans were started at least 50nm from the excitation
source to limit ramen scattering and other spurious effects. An automated program was used to collect
absorbance and each of the fluorescent emission spectral scans. Please contact the author for copies of the
program that automates the multi-spectral scanning of the 96 well plates.
Light intensity
Intensities were measured by a Li-Cor photometer (model LI-185-B) with a PAR photosensor (LI200SB
pyranometer) or a quantum sensor (LI 190S). The meters were not corrected for spectral sensitivity, but
were calibrated at at 150 mA µE/m2 /s.
Microscopic slide preparation
For microscopic spectral determination, 10ul samples scraped from the bottom of a well were placed on agar
coated slides, covered with a coverslip, and sealed with clear fingernail polish. The agar slides were prepared
by coating a clean glass slide with hot 1% agar dissolved in water and allowing them to dry for 24h.
Spectral confocal scanning laser microscopy (spectral-CSLM)
A Ziess 710 spectral confocal scanning laser inverted microscope was used for all autoflourecence spectral
microscopy. For each 2nm bandwidth excitation laser (405, 458, 488, 514, 561, 594, and 633nm), the percent
5
Table 4: Table 4: 710 lasers and settings
Laser (nm)
405
458
488
514
561
594
633
Laser power (mW)
30
2.63
13.16
6.58
15
2
5
%T setting
6.67
76
15.2
30.4
13.33
100
40
transmittance was adjusted to provide a maximum laser output of 2mW (see Table 4). The PMT in the
710 varies linearly from 30% quantum efficiency at 400nm, to 10% at 700, and the detectors are tuned to
deliver a near-linear response across the full wavelength (Zeiss representative, personal communication). The
automated capabilities of Zeiss ZEN software were used to automate the multi-spectral imaging and scans
that collected z-stack, tiled image, or timelapse data. Please contact the author for copies of the settings
files, program, and scripts that automate the multi-spectral scanning.
Data analysis and visualisation
A script in the R programming language (R Core Development Team 2010, Gentlemen 2008) was used to
extract data from comma separated values files exported from the M9 and Zeiss ZEN software, and generate
tiled plots using a grammar of graphics-based package (Wikinson 1999, Wickam 2009). Please contact the
author for copies of script.
Results and Discussion
While autoflourecence, or the fluorescence from phototropic microorganisms, is usually viewed as a nuisance
by microscopists wishing to observe their own introduced fluorophore markers (such as FISH), there is
information available in the excitation and emission spectra of this autoflourecence. While photobleaching
might indicate maximum usable fluxes, the most accessible information is the wavelength of light that
causes fluorescence, and the wavelength of the emitted fluorescence In particular, if light energy is absorbed
by phycobiliproteins and emitted by chlorophyll, the types of phycobiliproteins and their coupling to the
photosystem can be deduced. The functioning is much like a tightly coupled, multiple- fluorophore FRET
using the coupled antenna and photosystem chain instead of two fluorescent molecules in close proximity. This
is the basis of the autoflourecence spectral microscopy technique developed in this work. After developing
this technique, two papers with similar methods were found (Polerecky 2009, Roldán 2004). To the author’s
knowledge this work is the first to utilise this method in cyanobacterial macro-consortia, the first to utilise
CSLM and multiple wavelength lasers, and the first to couple it to enrichment based on wavelength of light.
Four pure cultures and a consortium (Figure 1) were grown under three intensities and eight monochromatic LEDs, as well as broad-spectrum white LED and dark controls. Growth densities were followed by
absorbance for 7 days (example absorbance data shown in Figure 2). The culture or enrichment bulk fluorescence was measured in each 96 well plate. An example of the fluorescence data is shown in Figure 3.
While little or no growth was seen for many of the organisms under many of the light regimes, some culture
increased in absorbance under some light regimes. Also note that for cultures with low growth, the bulk
fluorescence is not informative.
Stock cultures grown for more than one month with diffuse sunlight were sampled and their photosytems
examined using spectral-CSLM analysis of their photosystems autoflourecence. The different fluorescence
spectra acquired are displayed in Figures 4, 5, 6 and 7 and initial results are discussed below. While
initial data was collected for the pure cultures grown under different wavelengths in the multi-spectral LED
culturing plates, a lack of several doubling in many of the cultures due to insufficient growth time prevents
6
the interpretation of the autoflourecence state of the cells. Instead, the results interpreted below will focus
on the death phase stock cultures and the changes to the “Green Berries” consortia enriched with various
wavelengths on multi-spectral LED culturing plates.
Cyanothece cells displayed the most restricted excitation spectra (Figure 6), with typical 650nm maximum chlorophil fluorescence observed only with 561, 594, and 633nm excitation. This indicates that under
these conditions the predominant phycobiliproteins present are not the blue-shifted phycoerythrins or phycoerythrocyanin, as nothing with their absorbance pattern transferred energy to chlorophyll, nor were their
uncoupled fluorescence observed. All Cyanothece cells gave similar autoflourecence emission / excitation
spectra. This data corresponds well to the 96 well bulk culture fluorescence.
In the Anabena cultures, there was a strong coupling of 380nm excitation to 675nm maximal fluorescence,
probably from a the lower absorbance band of chlorophil. There was a lower emission from across all the
longer laser wavelengths. Heterocysts (region 23 in Figure 7) gave reduced fluorescence, and in other cultures
decoupling of phycobiliproteins was observed during the dismantling of photosystem I during heterocyst
development (data not shown).
Gloeothece cells displayed two different autoflourecence emission / excitation spectra. Most cells in the
culture (Figure 4), like actively growing cultures (data not shown) had a fluorescence maxima at either 650
or 725nm or both, depending on the excitation wavelength. This indicates multiple fluorophores, either
coupled to different chlorophylls or longer-wavelength emitting phycobiliprotein that is not coupled to any
chlorophil. Occasional cells were observed that had fewer intracellular vacuoles and a lower density in the
DIC micrograph which corresponded to a fainter fluorescence with less fluorescence shift (Regions 16 to 20 in
Figure 4). This was probably due to the degradation of portions of the photosynthetic apparatus. Likewise,
there was a presumed degradation of some Synechococcus cells.
While preliminary, this analysis of the spectral-CSLM analysis of their photosystems autoflourecence indicates that differences in single-cell autoflourecence can reveal details of photosystem function. For instance,
Gloeothece grown under these conditions would seem to be a photo-generalist, while Cyanothece seems to
utilize a more restricted wavelength of photons. In addition, spectral-CSLM analysis of autoflourecence
shows a decoupling and degradation of photosystem components during the death phase of cultures.
The homogenised “Green Berry” consortia grown in wells on the highest light exposure plate and exposed
to 4 different light conditions are displayed in Figures 8, 9, 10, 11, and 12. The spectral patterns and cellular
abundances found in the white LED enrichments and initial micrographs are most smiler to the cultures
enriched at 470nm LED (Figure 8 and 9, respectively). The dominant Gloeothece-morphology organisms
(Gentile 2002) enriched with all light treatments were particularly prevalent at 470nm LED light enrichment
(Regions 2 to 4 in Figures 8, 10 11, 12 and 13 as well as Regions 2 to 16 in Figure 9). These Gloeothecemorphology organisms grown at 470nm display a similar pattern to the pure culture Gloeothece grown in
sunlight, in that they have a strong emission maximum at 725nm with excitation at 594 and 633nm, and
a shorter wavelength emission with broad excitation. However, the exact emission spectra were slightly
shifted at the middle frequencies. In all other enrichments, the show a different pattern, fluorescing less
below 514nm. Filamentous phototrophs were observed as minor organisms is lower-wavelength enrichments
(Regions 8-10 in Figure 8, Regions 11 to 13 in Figure 10 and Regions 8-13 in Figure 11) with two strikingly
different emission fluorescence spectra (compare Regions 8 to 10 and 11 to 13 on Figure 11). The Blue-shifted
fluorescence seen in many of the cultures is very unusual, and does not correspond to known chlorophyll
emission spectra. Diatoms with morphologies similar to stramenopiles and dinophyceae were found in many
micrograph of the Green Berries, and all had the expected patterns of fluorescence (see Regions 5 to 7 in
Figure 8, Regions 17-25 in Figure 9, regions 5 to 7 in Figure 10, and Regions 5 to 10 in Figure 12). They
were particularly abundant in enrichments at 561, 594, and 633 nm (Figure 12 and data not shown).
Enrichments based on the wavelength of light provided by multi-spectral LED 96 well culturing plates
impacted the morphological makeup of the consortia, with diatoms out-competing the cyanobacteria at
higher wavelength. Using the autoflourecence of naturally coupled photosystems with spectral CSLM is a
powerful technique to investigate both single-cell heterogeneity, development, and ageing in pure culture, and
the photosystems and light utilization of not-yet cultured organisms. Green Berries contain organisms that
can utilize diverse wavelengths of light, and the dominant Gloeothece-morphology organisms may photoadapt
at 470nm. Interesting diversity, even within one filamentous morphology in the same light enrichment was
seen, and potentially novel photosystem were observed using this technique.
7
Future work
High throughput isolation of organisms with varying light conditions is possible with the Multi-spectral
LED culturing plates developed for this work. Sequencing of communities exposed to different LED light
treatments would allow more in-depth measure of the enrichment effects, and potentially tie organisms to
observed spectral patterns. Investigations of complex photosynthetic communities could be pursued with the
autoflourecence spectral microscopy technique developed here. Further conformation, as well as extractions of
pigments (with both water and acetone) could lead to more precise understanding of uncultivated organisms’
photosystems, particularly photoadaptation with phycobilins and novel chlorophylls. Microrespirometry of
wells lit with a single wavelength LED would connect the absorbance measurements to the efficiency of energy
capture at various wavelengths. Pigments that are not involved in photosystems, such as that produces by
Pseudomonads in biofilms could also be used with the autoflourecence spectral microscopy technique to act
as a spectral fingerprint. A full calibration and propagation of spectral sensitivities with this technique could
also provide a quantification of light utilization. Extending this technique with time resolved and polarised
florescence could provide data on the the dynamics and the bound state of the fluorophores respectively.
Acknowledgements
I would like to acknowledge Steven Zinder, Dan Buckly, Bill Metcalf and all of my classmates and instructors
at the 2010 Microbial Diversity Course at MBL for many hours of discussion and friendship. These weeks have
been the most intense, interesting, and exhausting of my academic life. Thanks for the great companionship
and great times.
I wish to thank John Waterbury and Bob Haselcorn, and Team Berry (Parris, Ulli, and Lizzy) for cultures
and advice. I would like to thank Rudi Rottenfusser, Chris Reikin and Alex Valm for fruitful discussions
about quantitative CSLM. In addition, I wish to thank my mentor, Yosko Fujita, for her generous help and
support in attending this course.
This work was supported by grants from Idaho National Laboratory Division Initiative and Biological
Systems Departmental Grants, The Gordon & Betty Moore Foundation, and the National Aeronautic and
Space Administration.
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9
Cyanothece
ATCC51142
Gloeothece
PCC7109
Anabena
PCC7120
Synechococcus
PCC7335
Green Berries
20µm
Figure 1: DIC micrographs of the Cyanobacterial cultures and
consortium used in this study. All figures are at the same scale, see
scale bar in lower right corner.
Figure 2: OD at multiple wavelengths (see legend) indicating growth over time (x axis) for
a blank and 8 strains (upper grey tabs, JW=original stock cultures >1month old, HL=precultures grown under high intensity florescent lights), grown at three light intensity levels
(right grey tabs).
Figure 3: Fluorescence of culture wells in 96well plate. Each of the 66 tiles in this array
represents measurements on one well. The grey tabs along the top indicate the light
exposure (0=dark, 1=white LED, and NA=empty well blank, all others are wavelength in nm),
the abbreviations along the grey boxes at the right indicate the organism (blank =
uninoculated, Ana=Anabaena, Cya=Cyanothece, GB=green berries, Glo=Gloeothece,
Syn=Synechococcus). Each tile displays the intensity of the fluorescence emission (legend)
and it's wavelength (y axis) detected at one of the seven laser excitation wavelengths (x
axis). The intensity value of red color indicates the relative florescent units.
Figure 4: Gloeothece cultures grown with diffuse sunlight. Each of the tiles in this array
represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 5: Synechococcus cultures grown with diffuse sunlight. Each of the tiles in this
array represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 6: Cyanothece cultures grown with diffuse sunlight. Each of the tiles in this array
represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 7: Anabaena cultures grown with diffuse sunlight. Each of the tiles in this array
represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 8: Green Berries grown with white LED enrichment. Each of the tiles in this array
represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 9: Green Berries grown with 470nm LED enrichment. Each of the tiles in this array
represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 10: Green Berries grown with 521nm LED enrichment. Each of the tiles in this array
represents a spectral-CSLM auto fluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 11: Green Berries grown with 521nm LED enrichment. Note blue-shifted
fluorescence emission of the filaments in the upper right (Regions 11-13). See previous
Figure legends for a description of the representation of the data in the figure.
Figure 12: Green Berries grown with 595nm LED enrichment. Each of the tiles in this
array represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).
Figure 13: Green Berries grown with 380nm LED enrichment. Each of the tiles in this
array represents a spectral-CSLM autofluorescence scan of a circular area (one cell) in the
micrograph. The color indicates the intensity of the fluorescence emission (legend) and it's
wavelength (y axis) detected at one of the seven laser excitation wavelengths (x axis).