Gaussian band analysis of absorption,fluorescence and photobleaching difference spectra of D1/D2/cytb-559 complex

A study of the absorption and fluorescence characteristics of the D1/D2/cytb-559 reaction centre complex of Photosystem II has been carried out by gaussian decomposition of absorption spectra both at room temperature and 72 K and of the room temperature fluorescence spectrum. A five component fit was found in which the absorption and fluorescence sub-bands could be connected by the Stepanov relation. The photobleaching and light-activated degradation in the dark of long wavelength pigments permitted a further characterisation of the absorption bands. The absorption (fluorescence) maxima of the five bands at room temperature are 660 nm (670 nm), 669 nm (675 nm), 675 nm (681 nm), 680 nm (683 nm), 681 nm (689 nm). A novel feature of this analysis is the presence of two approximately isoenergetic absorption bands near 680 nm at room temperature. The narrower one (FWHM=12.5 nm) is attributed to pheophytin while the broader band (FWHM=23 nm) is thought to be P680. The P680 band width is discussed in terms of homogeneous and site inhomogeous band broadening. The P680 fluorescence has a large Stokes shift (9 nm) and most fluorescence in the 690–700 nm range is associated with this chromophore.The three accessory pigment bands are broad (FWHM=17–24 nm) and the 660 nm gaussian is largely temperature insensitive thus indicating significant site inhomogeneous broadening.The very slight narrowing of the D1/D2/cytb-559 Q_y absorption at crytogenic temperatures is discussed in terms of the coarse spectral inhomogeneity associated with the spectral forms and the apparently large site inhomogeneous broadening of short wavelength accessory pigments.     

Purification and characterization of the photochemical reaction center of the thermophilic purple sulfur bacterium Chromatium tepidum

Pure reaction center preparations from the thermophilic species Chromatium tepidum have been obtained by lauryldimethylamine N-oxide treatment of chromatophores. The light-induced difference spectrum in presence of 10 mM sodium ascorbate revealed the presence of two high-potential cytochrome c hemes (-band, 555 nm; γ-band, 422 nm). The dithionite-minus-oxidized difference spectrum in the -band suggests the presence of additional hemes of low potential. These hemes are associated with a single polypeptide (M_r = 36 000). The reaction center pigments, probably four bacteriochorophyll a and two bacteriopheophytin a molecules, are associated with three polypeptides of apparent molecular weights equal to 33 000, 30 000 and 22 000. A carotenoid molecule is also bound to the reaction center. The three main absorption bands of this molecule are located at 480, 510 and 530 nm at liquid helium temperature. Photochemical activity is found to be stable, even after heating for 10 min at temperatures higher than 60 °C in intact chromatophore membranes. On the other hand, isolated reaction centers or chromatophores treated with 1% lauryldimethylamine N-oxide are fully inactivated after heating at temperatures higher than 50 °C. From these results, we propose that lipid-protein interactions are of prime importance in the thermal stabilization of Chromatium tepidum reaction centers.     

Orientation of the hemes of high potential cytochromes relative to photosynthetic membranes, as shown by the linear dichroism of oriented preparations

The orientations of high potential cytochromes with respect to photosynthetic membranes was investigated in spinach chloroplasts and in Rhodopseudomonas viridis. The general approach consists in detection with polarized light of photoinduced absorbance changes related to the oxidation of the cytochromes. The orientation of cytochrome c-558 was measured at room temperature in chromatophores and whole cells of Rps. viridis, oriented on glass slides and in a magnetic field, respectively. The orientation of cytochrome b-559 of green plants was detected at 77 K in magnetically oriented chloroplasts. In both cases the dichroic ratio for the band shows that the heme plane makes an angle greater than 35°C with the membrane plane. Moreover, the dichroic ratio is not constant throughout the and β bands, for both cytochrome c-558 and b-559. Linear dichroism spectra of oriented pure horse heart cytochrome c and cytochrome c₂ of Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films show that the variations of the dichroic ratio in the and β bands can be explained by the occurrence of x- and y-polarized transitions absorbing at slightly different wavelengths.     

Analysis by exciton theory of the optical properties of the Chloroflexus aurantiacus reaction center

The optical properties of the reaction center of the filamentous green bacterium Chloroflexus aurantiacus, that contains three bacteriochlorophyll (BChl) a and three bacteriopheophytin (BPh) a molecules, were analyzed in the near-infrared region with the aid of exciton theory. The coordinates obtained from the X-ray analysis of the reaction center of Rhodopseudomonas viridis (Deisenhofer, J., Epp, O., Miki, K., Huber, R. and Michel, H. (1984) J. Mol. Biol. 180, 385–398) were used for the geometry of the reaction center of C. aurantiacus, with the replacement of one of the ‘accessory’ BChl molecules by BPh. The results were found to be in good agreement with experimental low-temperature absorption spectra, linear and circular dichroism and fluorescence polarization spectra and lead to the following conclusions. The allowed, low-energy exciton transition of the primary electron donor (P-865) is located at 887 nm and carries the dipole strength of approx. two BChl a monomers; the high-energy exciton transition, around 790 nm, is mixed with wave functions of other pigments, which explains its relatively small angle with respect to the 887 nm transition. The optical transition of the accessory BChl a molecule near 812 nm has some contribution of the BChls that constitute P-865. This can account for the experimentally observed reorientation and shift of this transition upon oxidation of P-865. Two of the BPh molecules are located on the same (probably the M) polypeptide subunit and show a clear splitting of absorption bands (11 nm) due to exciton coupling; the single BPh on the opposite branch shows hardly any exciton shift. Similar calculations for reaction centers of purple bacteria that contain four BChl a and two BPh a molecules resulted in a very low dipole strength for the high-energy transition of the primary donor due to antisymmetric mixing with both accessory BChl a wave functions and gave very little splitting of the absorption bands of BPh a. Our results indicate that the arrangement of the chromophores in reaction centers of C. aurantiacus is very similar to that in purple bacteria. The functional L-chains of the reaction centers of purple and filamentous green bacteria consist of pigments of the same type in a probably very similar arrangement.     

Flash-induced absorption changes in Photosystem I,Radical pair or triplet state formation?

Absorption changes induced in chlorophyll protein (CP 1) particles by short laser flashes have been analyzed in order to decide whether a state lasting for a few microseconds at 21°C or 800 μs at 10 K corresponds to the biradical P-700⁺ ... A⁻₁ (A₁ being a chlorophyll a) or to a triplet state produced in a submicrosecond recombination of the preceding state. At 21°C the spectrum of the flash-induced ΔA (720–870 nm) presents a flat-topped band from 740 to 820 nm, clearly different from that of P-700⁺. A saturation curve (ΔA vs. laser energy), obtained with a 2 or 10 ns laser pulse, indicates that ΔA saturates at a value 2- or 3-times smaller than that expected on the basis of the chemical oxidation of P-700. At 21°C the size of flash-induced ΔA is slightly decreased (5–15%) when the sample is subjected to a 400 G magnetic field. The kinetics of decay are not affected; they are not affected either by the oxygen concentration. At 10 K the spectrum of the flash-induced ΔA has been measured between 650 and 1700 nm. Between 650 and 720 nm, the spectrum presents only one major negative peak at 702 nm; it is quite different from that due to the chemical oxidation of P-700 (which has additional peaks at 688 and 677 nm). Between 720 and 870 nm, the spectrum is identical to that obtained at 21°C. Above 870 nm, the spectrum includes a broad band around 1250 nm, which is absent in P-700⁺. A saturation curve leads to a maximum ΔA greater than that at 21°C and which is also greater with a 1 μs dye laser flash than with a 10 ns ruby laser flash. An analysis of the spectral data indicates that these do not fit correctly with the hypothesis of a contribution of P-700⁺ and of a chlorophyll a anion radical. They fit more closely with the hypothesis of a triplet state of P-700, a hypothesis which is discussed in relation to other experimental data.     

Circular dichroism of rhodopsins and porphyropsins

Circular dichroism and absorption spectra were determined for digitonin extracts of three rhodopsins: cattle, grass frog, and pigeon; and three porphyropsins: channel catfish, bluegill sunfish, and redear sunfish. A comparison of these spectra shows the following: (1) Porphyropsins, like rhodopsins, exhibit two positive CD peaks in the spectral region 321–700 nm: an α peak at about 520 nm and a small β peak at about 355 nm. These peaks substantially diminish upon bleaching. (2) In the CD spectra the α peaks of the porphyropsins are larger than the α peaks of the rhodopsins, while the β peaks are smaller than those of the rhodopsins. This is just the opposite of the corresponding relationship between the peaks in the absorption spectra. (3) The maxima of these peaks in the CD spectra of rhodopsins and porphyropsins are consistently blue-shifted from the corresponding maxima in absorption spectra. (4) Some of the visual pigments show additional positive CD peaks in the spectral region 250–320 nm. In all the visual pigments studied, the CD spectra in this region decrease on bleaching. No reciprocal relationship is observed between any of the CD bands in the visible and near ultraviolet region of the spectrum.     

Spectral inhomogeneity of photosystem I and its influence on excitation equilibration and trapping in the cyanobacterium Synechocystis sp. PCC6803 at 77 K.

Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trapping at 77 K in the photosystem I (PSI) core antenna from the cyanobacterium Synechocystis sp. PCC 6803. Excitation of the bulk antenna at 670 and 680 nm induces a subpicosecond energy transfer process that populates the Chl a spectral form at 685--687 nm within few transfer steps (300--400 fs). On a picosecond time scale equilibration with the longest-wavelength absorbing pigments occurs within 4-6 ps, slightly slower than at room temperature. At low temperatures in the absence of uphill energy transfer the energy equilibration processes involve low-energy shifted chlorophyll spectral forms of the bulk antenna participating in a 30--50-ps process of photochemical trapping of the excitation by P(700). These spectral forms might originate from clustered pigments in the core antenna and coupled chlorophylls of the reaction center. Part of the excitation is trapped on a pool of the longest-wavelength absorbing pigments serving as deep traps at 77 K. Transient hole burning of the ground-state absorption of the PSI with excitation at 710 and 720 nm indicates heterogeneity of the red pigment absorption band with two broad homogeneous transitions at 708 nm and 714 nm (full-width at half-maximum (fwhm) approximately 200--300 cm(-1)). The origin of these two bands is attributed to the presence of two chlorophyll dimers, while the appearance of the early time bleaching bands at 683 nm and 678 nm under excitation into the red side of the absorption spectrum (>690 nm) can be explained by borrowing of the dipole strength by the ground-state absorption of the chlorophyll a monomers from the excited-state absorption of the dimeric red pigments.     

Difference picosecond spectroscopy of pigment-protein complexes of photosystem I from higher plants

Using a difference picosecond spectrophotometer with a time resolution of 10 ps, we investigated excitation energy transfer and charge separation in pigment-protein complexes of Photosystem I from bean leaves (chlorophyll/P-700 = 60). Under 20 ps excitation at 650 or 667 nm, the difference absorption spectra in the spectral region 600–720 nm were measured. They are associated with transition of antenna chlorophylls into singlet excited states and P-700 photooxidation. It was shown that the excited states in the whole inhomogeneous antenna were generated within 10 ps and deactivated with three-component kinetics, the t_1/e values being 20–45, 100–300 and over 500 ps. Formation of P-700⁺ has a rise time of 15–30 ps. The fast component of the depletion of the antenna excited states is suggested to be due to transfer of excitation energy from antenna pigments to reaction centers and its trapping. The kinetics of the fast component is independent of excitation energy and a redox state of P-700.     

Circular dichroism of halorhodopsin: comparison with bacteriorhodopsin and sensory rhodopsin I

Circular dichroic (CD) spectra of three related protein pigments from Halobacterium halobium, halorhodopsin (HR), bacteriorhodopsin (BR), and sensory rhodopsin I (SR-I), are compared. In native membranes the two light-driven ion pumps, HR and BR, exhibit bilobe circular dichroism spectra characteristic of exciton splitting in the region of retinal absorption, while the phototaxis receptor, SR-I, exhibits a single positive band centered at the SR-I absorbance maximum. This indicates specific aggregation of protein monomers of HR, as previously noted [Sugiyama, Y., & Mukohata, Y. (1984) J. Biochem. (Tokyo) 96, 413-420], similar to the well-characterized retinal/retinal exciton interaction in the purple membrane. The absence of this interaction in SR-I indicates SR-I is present in the native membrane as monomers or that interactions between the retinal chromophores are weak due to chromophore orientation or separation. Solubilization of HR and BR with nondenaturing detergents eliminates the exciton coupling, and the resulting CD spectra share similar features in all spectral regions from 250 to 700 nm. Schiff-base deprotonation of both BR and HR yields positive CD bands near 410 nm and shows similar fine structure in both pigments. Removal of detergent restores the HR native spectrum. HR differs from BR in that circular dichroic bands corresponding to both amino acid and retinal environments are much more sensitive to external salt concentration and pH. A theoretical analysis of the exciton spectra of HR and BR that provides a range of interchromophore distances and orientations is performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy transfer in the photochemical apparatus of flashed bean leaves

Fluorescence and energy transfer properties of bean leaves greened by brief, repetitive xenon flashes were studied at −196 °C. The bleaching of P-700 has no influence on the yield of fluorescence at any wavelength of emission. The light-induced fluorescence yield changes which are observed in both the 690 and 730 nm emission bands in the low temperature fluorescence spectra are due to changes in the state of the Photosystem II reaction centers. The fluorescence yield changes in the 730 nm band are attributed to energy transfer from Photosystem II to Photosystem I. Such energy transfer was also confirmed by measurements of the rate of photooxidation of P-700 at −196 °C in leaves in which the Photosystem II reaction centers were either all open or all closed. It is concluded that energy transfer from Photosystem II to Photosystem I occurs in the flashed bean leaves which lack the light-harvesting chlorophyll a/b protein.     

Anisotropy of photosynthetic membranes and the degree of fluorescence polarization

The degree of fluoresence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400–680 nm) and emission wavelengths (675–750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, P_IN and P_AN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization P_IN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Q_y (≈ 660−680 nm) absorption band of chlorophyll in vivo. With 670–680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts, P_IN is shown to be +5±1%. The excitation wavelength dependence studies of P_IN indicate that the carotenoid and chlorophyll Q_y transition moments tend to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).     

Evidence for 5- and 6-coordinated magnesium in bacteriochlorophyll a from visible absorption spectroscopy

The visible absorption spectrum of bacteriochlorophyll a (Bchl) in the 560–620 nm (yellow) region can be used to establish the coordination state of the central Mg atom. Five-coordinated Mg species absorb near 580 nm, whereas 6-coordinated Mg species are red-shifted to about 610 nm. Five-coordination is confirmed to be the principal coordination state of Mg in self-aggregated bacteriochlorophyll. The equilibrium constant for the reaction Bchl · Py + Py ai Bchl · Py₂ has been determined from computer-assisted analyses of spectral data, where Py represents pyridine. The spectral criteria for the coordination state of Mg in bacteriochlorophyll advanced here are shown to be applicable to both in vitro and in vivo systems. Similar spectral behavior is exhibited by chlorophylls a and b, and a band at 633 nm is shown to be associated with the presence of 6-coordinated Mg in chlorophyll a.     

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.     

Electronic spectra of PS I mutants: the peripheral subunits do not bind red chlorophylls in Synechocystis sp. PCC 6803

Steady-state fluorescence and absorption spectra have been obtained in the Qy spectral region (690-780 nm and 600-750 nm, respectively) for several subunit-deficient photosystem I mutants from the cyanobacterium Synechocystis sp. PCC 6803. The 77 K fluorescence spectra of the wild-type and subunit-deficient mutant photosystem I particles are all very similar, peaking at approximately 720 nm with essentially the same excitation spectrum. Because emission from far-red chlorophylls absorbing near 708 nm dominates low-temperature fluorescence in Synechocystis sp., these pigments are not coordinated to any the subunits PsaF, Psa I, PsaJ, PsaK, PsaL, or psaM. The room temperature (wild-type-mutant) absorption difference spectra for trimeric mutants lacking the PsaF/J, PsaK, and PsaM subunits suggest that these mutants are deficient in core antenna chlorophylls (Chls) absorbing near 685, 670, 675, and 700 nm, respectively. The absorption difference spectrum for the PsaF/J/I/L-deficient photosystem I complexes at 5 K reveals considerably more structure than the room-temperature spectrum. The integrated absorbance difference spectra (when normalized to the total PS I Qy spectral area) are comparable to the fractions of Chls bound by the respective (groups of) subunits, according to the 4-A density map of PS I from Synechococcus elongatus. The spectrum of the monomeric PsaL-deficient mutant suggests that this subunit may bind pigments absorbing near 700 nm.     

Variation in ultraviolet reflectance by carotenoid-bearing feathers of tanagers (Thraupini: Emberizinae: Passeriformes)

Avian visual sensitivity encompasses both the human visible range (400–700 nm) and also near‐ultraviolet (UV) wavelengths (320–400 nm) invisible to normal humans. I used reflectance spectrophotometry to assess variation in UV reflectance for yellow, orange and red plumage in 67 species of tanager (Passeriformes). Previous chemical studies, and my analysis of reflectance minima, suggest that carotenoids are the dominant pigments in yellow, orange and red tanager plumage. Spectra recorded over the range of wavelengths to which birds are sensitive (320–700 nm) were invariably bimodal, with both a plateau of high reflectance at longer (> 500 nm) wavelengths and a distinct secondary peak at UV (< 400 nm) wavelengths. Within this overall framework, variation in UV reflectance was expressed within well‐defined quantitative limits: (1) peak reflectance was always lower than the corresponding plateau of reflectance at longer visible wavelengths; (2) the intensity of peak reflectance declined steadily below 350 nm; (3) wavelengths of peak reflectance clustered between 350 and 370 nm. Significant correlations were detected between various measures of total reflectance in the UV and visible wavebands, but not between various measures of spectral location of UV and visible reflectance. I propose that the strong absorption band at short visible wavelengths (～ 380–550 nm) responsible for bimodal spectra of carotenoids in vitro is also responsible for bimodal reflectance by carotenoid‐based plumage colours. The construction of the UV and visible reflectance bands from different sides of this same absorbance band provides a mechanism for the observed covariation between UV and visible wavelengths. Lack of an association between the spectral locations of the UV and visible reflectance bands may result from the limited variation in spectral location of the UV band. These patterns suggest that plumage colours are subject to constraints, just as are more traditional morphological characters. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84 , 243–257.     

Properties of partially purified photosynthetic reaction centers from scenedesmus mutant 6E and Anabaena variabilis grown in the presence of diphenylamine

When membrane fragments of Anabaena variabilis grown in the presence of diphenylamine (designated diphenylamine-Anabaena) are treated with Triton X-100 and subjected to sucrose density gradient centrifugation, a bluish-green membrane fragment enirched in P700 is obtained. This high-P700 fragment, denoted HP700, contains three P700 molecules per 100 chlorophyll a molecules and reduces NADP at a rate that is approximately nine times higher than that of HP700 fragments prepared from normally cultured Anabaena by the use of Triton X-100 following extraction with organic solvents. An HP700 fragment has also been isolated from a carotenoidless Scenedesmus mutant 6E, by the use of Triton X-100 and sucrose density gradient centrifugation.

Both HP700 fragments show the characteristic rapid absorbance changes of P700 upon illumination. The fluorescence properties of the HP700 fragments at −196° are different from those of the original membrane fragments. At −196° the long wavelength fluorescence peak is located at a shorter wavelength (724 mμ) in the diphenylamine-Anabaena HP700 fragment and is lower in intensity than that observed with the membrane fragment. Long wavelength fluorescence at −196° is low in the flurorescence spectra of the membrane fragments of Scenedesmus mutant 6E and is barely observable in the HP700 fragment. The fluorescence spectra of the HP700 fragments of both diphenylamine-Anabaena and Scenedesmus mutant 6E at −196° show a shoulder or peak at 700 nm. The data on fluorescence properties of the HP700 fragments suggest that 730 nm fluorescence does not originate from P700.     

Commercial production of microalgae: ponds, tanks, tubes and fermenters

The commercial culture of microalgae is now over 30 years old with the main microalgal species grown being Chlorella and Spirulina for health food, Dunaliella salina for β-carotene, Haematococcus pluvialis for astaxanthin and several species for aquaculture. The culture systems currently used to grow these algae are generally fairly unsophisticated. For example, Dunaliella salina is cultured in large (up to approx. 250 ha) shallow open-air ponds with no artificial mixing. Similarly, Chlorella and Spirulina also are grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors in 20–40 l carboys or in large plastic bags of up to approximately 1000 l in volume. More recently, a helical tubular photobioreactor system, the BIOCOIL™, has been developed which allows these algae to be grown reliably outdoors at high cell densities in semi-continuous culture. Other closed photobioreactors such as flat panels are also being developed. The main problem facing the commercialisation of new microalgae and microalgal products is the need for closed culture systems and the fact that these are very capital intensive. The high cost of microalgal culture systems relates to the need for light and the relatively slow growth rate of the algae. Although this problem has been avoided in some instances by growing the algae heterotrophically, not all algae or algal products can be produced this way.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550–650 and 700–950 nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as Δa_v/Δa_h, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810 nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm, and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the “special pair” that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the “light-dark” difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.