«Abstract Photoautotrophy is the foundation of all ecosystems. In order to explore the vast diversity of mechanisms used by bacteria to capture light ...»
The utilization of varying wavelengths of light by cultured and
James R. Henriksen
Microbial Diversity Course 2010, MBL
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.
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 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.
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.
Table 2: LED speciﬁcations
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.
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).