Challenges in microalgal biofuel production: A perspective on techno economic feasibility under biorefinery stratagem.

Rapidly exhausting fossil fuels combined with the ever-increasing demand for energy led to an ongoing search for alternative energy sources to meet the transportation, manufacturing, domestic and other energy demands of the grown population. Microalgae are at the forefront of alternative energy research due to their significant potential as a renewable feedstock for biofuels. However, microalgae platforms have not found a way into industrial-scale bioenergy production due to various technical and economic constraints. The present review provides a detailed overview of the challenges in microalgae production processes for bioenergy purposes with supporting techno-economic assessments related to microalgae cultivation, harvesting and downstream processes required for crude oil or biofuel production. In addition, biorefinery approaches that can valorize the by-products or co-products in microalgae production and enhance the techno-economics of the production process are discussed.

Carbon streaming in microalgae: extraction and analysis methods for high value compounds.

There is a growing recognition that carbon-neutral biofuels and microalgae are eco-friendly options because of their high CO2 sequestering capability and ability to grow in wastewater/sea water and non-arable land. Also the intrinsic properties of microalgal systems can be exploited for high value compounds such as carbohydrates, lipids, pigments and proteins. This article provides a comprehensive review of various microalgae cultivation practices utilizing organic and inorganic carbon sources. The merits and demerits of the various extraction and analytical procedures have also been discussed in detail.

Effect of moderate and high light on photosystem II function in Arabidopsis thaliana depleted in digalactosyl-diacylglycerol.

The response of the heat-sensitive dgd1-2 and dgd1-3 Arabidopsis mutants depleted in the galactolipid DGDG to photoinhibition of chloroplasts photosystem II was studied to verify if there is a relationship between heat stress vulnerability due to depletion in DGDG and the susceptibility to photoinhibitory damage. Non-photochemical quenching (NPQ) is known to dissipate excessive absorbed light energy as heat to protect plants against photodamage. The main component of NPQ is dependent of the transthylakoid pH gradient and is modulated by zeaxanthin (Zx) synthesis. These processes together with chlorophyll fluorescence induction were used to characterize the response of the genotypes. The mutants were more sensitive to photoinhibition to a small extent but this was more severe for dgd1-3 especially at high light intensity. It was deduced that DGDG was not a main factor to influence photoinhibition but other lipid components could affect PSII sensitivity towards photoinhibition in relation to the physical properties of the thylakoid membrane. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Inhibition of photosystems I and II activities in salt stress-exposed Fenugreek (Trigonella foenum graecum).

Fenugreek (Trigonella foenum graecum) seedlings were exposed to increasing NaCl concentrations in the growth medium to examine the effect of salt stress on the electron transport reactions of photosynthesis. Activities of both photosystem II (PSII), measured by chlorophyll fluorescence, and photosystem I (PSI), measured by P700 photooxidation, were decreased by salt stress. The inhibition proceeded in a two step manner. At the lower salt concentrations used and shorter exposition periods, electron transfer between the quinone acceptors of PSII, Q(A) and Q(B), was strongly retarded as shown by an increased amplitude of the OJ phase of the OJIP chlorophyll fluorescence induction traces and slowed chlorophyll fluorescence relaxation kinetics following a single turn-over flash. The above indicated a disturbance of the Q(B) binding site likely associated with the first step of photoinhibition. In the second step, strong photoinhibition was observed as manifested by increased F(0) values, declined F(v)/F(0) and loss of photoactive P700.

Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol.

Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5min) mild (40°C) or strong (44°C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.

Inhibition of photosynthetic oxygen evolution and electron transfer from the quinone acceptor QA- to QB by iron deficiency.

The effect of iron deficiency on photosynthetic electron transport in Photosystem II (PS II) was studied in leaves and thylakoid membranes of lettuce (Lactuca sativa, Romaine variety) plants. PS II electron transport was characterized by oxygen evolution and chlorophyll fluorescence parameters. Iron deficiency in the culture medium was shown to affect water oxidation and the advancement of the S-states. A decrease of maximal quantum yield of PS II and an increase of fluorescence intensity at step J and I of OJIP kinetics were also observed. Thermoluminescence measurements revealed that charge recombination between the quinone acceptor of PS II, Q(B), and the S(2) state of the Mn-cluster was strongly perturbed. Also the dark decay of Chl fluorescence after a single turnover white flash was greatly retarded indicating a slower rate of Q(A)(-) reoxidation.

Abolition of photosystem I cyclic electron flow in Arabidopsis thaliana following thermal-stress.

Heat tolerance of Arabidopsis thaliana (WT) and its mutants, crr2-2, lacking NADPH-dehydrogenase (Ndh-pathway), and pgr5, deficient in proton gradient regulation and/or ferredoxin-quinone-reductase (FQR-pathway), was studied from 30 to 46°C. Chlorophyll fluorescence revealed that thermal damage to photosystem II (PSII) was maximal in WT plants following short-term exposure of leaves to moderate or high temperature stress. Thermal stress impaired the photosynthetic electron flow at oxidizing and reducing sides of PSII. This was deduced from the transformation of temperature dependent OJIP to OKP patterns, changes in the relative amplitudes of K-step fluorescence rise and F(v)/F(o) ratio. The amplitude of the K-peak that corresponds to the magnitude of damage to the oxygen evolving complex (OEC) in crr2-2 mutants was about 50% of that observed in WT plants exposed to 46°C. The damage to OEC in pgr5 mutants was relatively smaller and thus their PSII complexes were more heat tolerant. P700 oxidation-reduction kinetics following heat-stress revealed that photosystem I (PSI) complexes remained oxidizable either with 10-ms multiple turn-over flashes or far-red illumination but the complementary cyclic electron flow around PSI (CEF) was abolished in both mutants. With further increase in incubation temperature, CEF was fully suppressed even in WT. Thus, P700 turn-over was not enhanced following thermal stress. Furthermore, the experimental data predicts the onset of pseudocyclic electron transport with molecular oxygen as terminal acceptor in crr2-2 and pgr5 mutants but not in wild type Arabidopsis subjected to severe thermal-stress.

Photosystem II reconstitution into proteoliposomes and methodologies for structure-function characterization.

This chapter discusses the photosystem II (PSII) reconstitution into proteoliposomes. In the first part of the chapter, protocols are outlined for the preparation of lipid bilayer vesicles (liposomes) constituted of individual thylakoid lipids or their mixtures, for the preparation of PSII particles, and for the incorporation of the PSII particles into the liposomes. In the second part of the chapter, methodologies are described for the structure-function characterization of the PSII-lipid complexes (proteoliposomes). This includes the sodium dodecylsulfate-polyacrylamide gel electrophoresis determination of the PSII proteins, the measurement of oxygen-evolving activity of PSII in the proteoliposomes, the study of structural changes of the PSII proteins upon their incorporation into the lipid bilayers by Fourier transform infrared (FT-IR) spectroscopy, and the characterization of the PSII activity by fluorescence induction.

Changes in the mode of electron flow to photosystem I following chilling-induced photoinhibition in a C3 plant, Cucumis sativus L.

This study provides evidence for enhanced electron flow from the stromal compartment of the photosynthetic membranes to P700+ via the cytochrome b6/f complex (Cyt b6/f) in leaves of Cucumis sativus L. submitted to chilling-induced photoinhibition. The above is deduced from the P700 oxidation-reduction kinetics studied in the absence of linear electron transport from water to NADP+, cyclic electron transfer mediated through the Q-cycle of Cyt b6/f and charge recombination in photosystem I (PSI). The segregation of these pathways for P700+ rereduction were achieved by the use of a 50-ms multiple turnover white flash or a strong pulse of white or far-red illumination together with inhibitors. In cucumber leaves, chilling-induced photoinhibition resulted in approximately 20% loss of photo-oxidizible P700. The measurement of P700+ was greatly limited by the turnover of cyclic processes in the absence of the linear mode of electron transport as electrons were rapidly transferred to the smaller pool of P700+. The above is explained by integrating the recent model of the cyclic electron flow in C3 plants based on the Cyt b6/f structural data [Joliot and Joliot (2006) Biochim Biophys Acta 1757:362-368] and a photoprotective function elicited by a low NADP+/NAD(P)H ratio [Rajagopal et al. (2003) Biochemistry 42:11839-11845]. Over-reduction of the photosynthetic apparatus results in the accumulation of NAD(P)H in vivo to prevent NADP+-induced reversible conformational changes in PSI and its extensive damage. As the ferredoxin:NADP reductase is fully reduced under these conditions, even in the absence of PSII electron transport, the reduced ferredoxin generated during illumination binds at the stromal openings in the Cyt b6/f complex and activates cyclic electron flow. On the other hand, the excess electrons from the NAD(P)H pool are routed via the Ndh complex in a slow process to maintain moderate reduction of the plastoquinone pool and redox poise required for the operation of ferredoxin:plastoquinone reductase mediated cyclic flow.

Inhibition of the oxygen-evolving complex of photosystem II and depletion of extrinsic polypeptides by nickel.

The toxic effect of Ni(2+) on photosynthetic electron transport was studied in a photosystem II submembrane fraction. It was shown that Ni(2+) strongly inhibits oxygen evolution in the millimolar range of concentration. The inhibition was insensitive to NaCl but significantly decreased in the presence of CaCl(2). Maximal chlorophyll fluorescence, together with variable fluorescence, maximal quantum yield of photosystem II, and flash-induced fluorescence decays were all significantly declined by Ni(2+). Further, the extrinsic polypeptides of 16 and 24 kDa associated with the oxygen-evolving complex of photosystem II were depleted following Ni(2+) treatment. It was deduced that interaction of Ni(2+) with these polypeptides caused a conformational change that induced their release together with Ca(2+) from the oxygen-evolving complex of photosystem II with consequent inhibition of the electron transport activity.

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680+ and the quinone acceptors QA- to QB.

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, QA and QB, and the S2 state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of QA- reoxidation.

Interaction of N,N,N',N'-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the Q(B) niche.

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (muM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S(2)Q(B)(-) and S(3)Q(B)(-) (B-band), S(2)Q(A)(-) (Q-band), and Y(D)(+)Q(A)(-) (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y(n)) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q(B) confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q(B) niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q(B)-site and to reduce the higher S-states of the Mn cluster.

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes.

N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I-P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I-P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b6/f complex, could prevent the quenching of I-P rise by TMPD. In control thylakoids, MV suppressed the I-P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I-P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O2 during re-oxidation of TMPD by PS I contributes towards an early decline in the I-P step of the OJIP fluorescence rise.

Changes in polyphasic chlorophyll a fluorescence induction curve upon inhibition of donor or acceptor side of photosystem II in isolated thylakoids.

The action of various inhibitors affecting the donor and acceptor sides of photosystem II (PSII) on the polyphasic rise of chlorophyll (Chl) fluorescence was studied in thylakoids isolated from pea leaves. Low concentrations of diuron and stigmatellin increased the magnitude of J-level of the Chl fluorescence rise. These concentrations barely affected electron transfer from PSII to PSI as revealed by the unchanged magnitude of the fast component (t(1/2) = 24 ms) of P700+ dark reduction. Higher concentrations of diuron and stigmatellin suppressed electron transport from PSII to PSI, which corresponded to the loss of thermal phase, the Chl fluorescence rise from J-level to the maximal, P-level. The effect of various concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP), which abolishes S-state cycle and binds at the plastoquinone site on QB, the secondary quinone acceptor PSII, on the Chl fluorescence rise was very similar to that of diuron and stigmatellin. Low concentrations of diuron, stigmatellin, or CCCP given on the background of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), which is shown to initiate the appearance of a distinct I-peak in the kinetics of Chl fluorescence rise measured in isolated thylakoids [BBA 1607 (2003) 91], increased J-step yield to I-step level and retarded Chl fluorescence rise from I-step to P-step. The increased J-step fluorescence rise caused by these three types of inhibitors is attributed to the suppression of the non-photochemical quenching of Chl fluorescence by [S2+ S3] states of the oxygen-evolving complex and oxidized P680, the primary donor of PSII reaction centers. In the contrary, the decreased fluorescence yield at P step (J-P, passing through I) is related to the persistence of a "plastoquinone"-type quenching owing to the limited availability of photochemically generated electron equivalents to reduce PQ pool in PSII centers where the S-state cycle of the donor side is modified by the inhibitor treatments.

Photosystem II inhibition by moderate light under low temperature in intact leaves of chilling-sensitive and -tolerant plants.

Photosystem II (PSII) activity was examsined in leaves of chilling-sensitive cucumber (Cucumis sativus L.), tomato (Lycopersicum esculentum L.), and maize (Zea mays L.), and in chilling-tolerant barley (Hordeum vulgare L.) illuminated with moderate white light (300 micro mol m(-2) s(-1)) at 4 degrees C using chlorophyll a fluorescence measurements. PSII activity was inhibited in leaves of all the four plants as suggested by the decline in F(v)/F(m), 1/F(o) - 1/F(m), and F(v)/F(o) values. The changes in initial fluorescence level (F(o)), F(v)/F(m), 1/F(o) - /1/F(m), and F(v)/F(o) ratios indicate a stronger PSII inhibition in cucumber, maize and tomato plants. The kinetics of chlorophyll a fluorescence rise showed complex changes in the magnitudes and rise of O-J, J-I, and I-P phases caused by photoinhibition. The selective suppression of the J-I phase of fluorescence rise kinetics provides evidence for weakened electron donation from the oxidizing side, whereas the accumulation of reduced Q(A) suggests damage to the acceptor side of PSII. These findings imply that the process of chilling-induced photoinhibition involves damage to more than one site in the PSII complexes. Furthermore, comparative analyses of the decline in F(v)/F(o) and photooxidation of P700 explicitly show that the extent of photoinhibitory damage to PSII and photosystem I is similar in leaves of cucumber plants grown at a low irradiance level.

Recovery of photosystem I and II activities during re-hydration of lichen Hypogymnia physodes thalli.

Photochemical efficiencies of photosystem I (PSI) and photosystem II (PSII) were studied in dry thalli of the lichen Hypogymnia physodes and during their re-hydration. In dry thalli, PSII reaction centers are photochemically inactive, as evidenced by the absence of variable chlorophyll (Chl) fluorescence, whereas the primary electron donor of PSI, P700, exhibits irreversible oxidation under continuous light. Upon application of multiple- and, particularly, single-turnover pulses in dry lichen, P700 oxidation partially reversed, which indicated recombination between P700(+) and the reduced acceptor F(X) of PSI. Re-wetting of air-dried H. physodes initiated the gradual restoration of reversible light-induced redox reactions in both PSII and PSI, but the recovery was faster in PSI. Two slow components of P700(+) reduction occurred after irradiation of partially and completely hydrated thalli with strong white light. In contrast, no slow component was found in the kinetics of re-oxidation of Q(A)(-), the reduced primary acceptor of PSII, after exposure of such thalli to white light. This finding indicated the inability of PSII in H. physodes to provide the reduction of the plastoquinone pool to significant levels. It is concluded that slow alternative electron transport routes may contribute to the energetics of photosynthesis to a larger extent in H. physodes than in higher plants.

Enhanced rates of P700(+) dark-reduction in leaves of Cucumis sativus L photoinhibited at chilling temperature.

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 micromol photons m(-2) s(-1)) at 4 degrees C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (deltaA(820)), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of deltaA(820) changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700(+) reduction after far-red light excitation as the half-times of the two exponential components of P700(+) decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700(+) reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700(+) re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.

N,N,N',N'-tetramethyl-p-phenylenediamine initiates the appearance of a well-resolved I peak in the kinetics of chlorophyll fluorescence rise in isolated thylakoids.

Addition of N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) to thylakoid membranes isolated from pea leaves initiates the appearance of peak I in the polyphasic rise of chlorophyll (Chl) fluorescence observed during strong illumination, making it similar to that observed in leaves or intact chloroplasts. This effect depends on TMPD concentration and incubation period of isolated thylakoids with TMPD. The resolution of I-peak in the presence of weak concentrations of TMPD which reduced the overlap between I- and P-peaks, resulted from a decreased reduction of both fast and slow plastoquinone (PQ) pools of the granal and stromal thylakoids, respectively, as TMPD effectively accepts electrons from reduced PQ. High concentrations of TMPD markedly decreased the J-I-P phase of fluorescence rise and greatly retarded the I-P step rise. Accumulation of oxidized TMPD in the thylakoid lumen accelerated the re-oxidation of the acceptor side of Photosystem II (PSII) as illustrated by a two-fold increase in the magnitude of the fast component and complete suppression of the middle component of the variable Chl fluorescence (F(v)) decay in the dark. Evidently, exogenous addition of high concentrations of TMPD prevented the light-induced reduction of the slow PQ pool.