DOI: 10.1002/cssc.201500332 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers Gas Transfer Controls Carbon Limitation During Biomass Production by Marine Algae Bojan Tamburic,*[a] Christian R. Evenhuis,[a] David J. Suggett,[a] Anthony W. D. Larkum,[a] John A. Raven,[a, b] and Peter J. Ralph[a] This study presents the first in-depth analysis of CO2 limitation on the biomass productivity of the biofuel candidate marine microalga Nannochloropsis oculata. Net photosynthesis decreased by 60 % from 125 to 50 mmolO2 L1 h1 over a 12 h light cycle as a direct result of carbon limitation. Continuous dissolved O2 and pH measurements were used to develop a detailed diurnal mechanism for the interaction between photosynthesis, gas exchange and carbonate chemistry in the photo-bioreactor. Gas exchange determined the degree of carbon limitation experienced by the algae. Carbon limitation was confirmed by delivering more CO2, which increased net photosynthesis back to its steady-state maximum. This study highlights the importance of maintaining replete carbon concentrations in photo-bioreactors and other culturing facilities, either by constant pH operation or preferably by designing a feedback loop based on the dissolved O2 concentration. Introduction Biofuel from microalgae croalga Nannochloropsis oculata (N. oculata), which is known for its high lipid productivity.[7] To develop production systems and processes that maximise biofuel output, it is first necessary to gain a deep understanding of photosynthetic controls for a given algal species. &&please provide academic titles (Prof., Dr.) for all authors.& & Microalgae have the potential to provide sustainable and affordable transport fuels in the future. These unicellular photosynthetic organisms use light energy to fix atmospheric CO2 and synthesise biomass. Algal biomass is then processed chemically to either biodiesel by transesterification or to biocrude oil by hydrothermal liquefaction.[1] Algal biofuels are of particular interest in Australia as many algae can be grown in hostile conditions, such as saltwater ponds on arid coastal terrain.[2] Algae cultivation that covers 1 million hectares of land (roughly the size of Sydney) would, by a conservative estimate,[3] produce enough bio-crude to replace 40 billion litres of crude oil per annum, which is equal to the entire crude oil demand of Australia, 85 % of which is currently imported.[4] It has been estimated that there are over 350 000 extant species of algae, which range across the most commonly recognised green, brown, red and golden brown algae to other forms that have been discovered only recently.[5] More than half of the ~ 20 microalgal species that are currently used in biofuel production are green algae (Chlorophyta), but there is a huge biodiversity of algae yet to be explored for bio-energy applications.[6] This includes the biofuel candidate marine mi- Limits to biomass production Resource availability fundamentally governs algal growth rates, the rate of biomass accumulation and hence the maximum achievable yield of biofuel.[8] Photosynthesis is driven by light energy, photochemistry and downstream energy transformations, which generate a reductant and adenosine triphosphate (ATP) that fuel metabolic reactions and consume inorganic nutrients (for example, carbon, nitrogen and phosphorus) to construct complex, energy-rich organic macromolecular compounds. The dependence of both the growth rate and biomass yield on the availability of these resources can be described mathematically using threshold and co-limitation theory, with taxonomic-specific variations that determine whether light availability and nutrient concentrations are limiting, saturating or excessive.[9] The algal cell size, in particular, influences the effectiveness of resource acquisition; for example, larger cells with a smaller surface-area-to-volume quotient exhibit lower light harvesting efficiency caused by enhanced pigment packaging as well as a lower inorganic nutrient uptake.[10] The external nutrient concentration and the intracellular demand for inorganic nutrients both dictate the elemental stoichiometry of an algal cell, described most commonly by the “Redfield Ratio”.[11] Consequently, a large number of factors, which can ultimately operate in complex positive or negative feedback loops, influence how rapidly microalgal cells drive photosynthesis and subsequently accrue biomass. [a] B. Tamburic, C. R. Evenhuis, D. J. Suggett, A. W. D. Larkum, J. A. Raven, P. J. Ralph Plant Functional Biology and Climate Change Cluster University of Technology Sydney Ultimo, New South Wales ### (Australia) [b] J. A. Raven Division of Plant Sciences University of Dundee at the James Hutton Institute Invergowrie ### (United Kingdom) Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/cssc.201500332. ChemSusChem 0000, 00, 0 – 0 These are not the final page numbers! ÞÞ 1  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 If cells are in exponential growth, the allocation of energy, reductant and assimilated inorganic nutrients are near optimal. Under these ideal conditions, the cells are acclimated to a steady-state supply of resources, regardless of whether the resources are available in excess or limiting.[8, 9] In contrast, conditions in large-scale biofuel cultivation facilities (and in nature in general) are highly variable so that an algal culture can experience different limitations depending on the time of day, the age of the culture and the location within the culture vessel.[12] In addition to maximising biomass production rate, algal cells also allocate resources to fitness-increasing traits, such as defence against grazers, parasitoids and viruses. Under variable environmental conditions, algal cells are in a dynamic state and the instantaneous growth rate depends on what limit is already imposed. This is a fundamental challenge for algae producers looking to increase algal biofuel yield, as the insights of how algae grow under steady-state conditions in the laboratory may not be relevant to large-scale production conditions. For example, light availability in an algal pond decreases exponentially with depth and thus small shifts in cell location caused by mixing or turbulence result in the algal cells experiencing a highly stochastic light field.[13] Microalgae have, therefore, evolved numerous mechanisms to cope with environmental variability, which can operate on scales of microseconds to days. For example, the reaction centres continually balance the excitation pressure required to convert energy photochemically and produce reductant and ATP;[14] similarly, cells regulate the supply of inorganic nutrients, which includes carbon.[15] All such regulatory mechanisms incur costs to the cells and reduce the efficiency of biomass accumulation, that is, growth.[16] It is, therefore, important to be aware of the regulatory costs upon maximisation of the biomass yield of a microalgal culture system. Recently, advanced molecular biology tools have become increasingly used to identify lipid production pathways[17] as well as genetic markers of algal stress.[18] These studies produce intriguing results, which are only reproducible and valid if physical parameters such as light, temperature and nutrient availability are measured and controlled. Photo-bioreactors (PBRs) are devices used to grow microalgae on a laboratory scale. They are used to maintain precise control over the algal growth environment, such as light and gas supply, and continuously measure key parameters, which include the dissolved oxygen concentration and pH. PBR studies facilitate a deeper understanding of the effect of dynamic fluctuations on conditions and limitations in biomass production and they generate more reproducible experimental results than open systems. PBRs exist in multiple geometries, which include flat-panel, tubular, annular and helical systems.[19] The design of a PBR influences the limitations that the culture experiences. For example, an important consideration in any PBR design is the provision of appropriate illumination to algal cells.[20] Light is absorbed and scattered by algal cells, which results in the formation of an attenuation gradient normal to the PBR surface. Another important factor is the PBR surface-tovolume quotient: a relatively large surface area increases the effectiveness of light absorption but may lead to photoChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org damage in algae, whereas a relatively large volume makes better use of space but requires additional energy for mixing, gas exchange and aeration. Fluid dynamics control the circulation and movement of individual algal cells within this light gradient, and the photochemical reactions of photosynthesis and respiration add further complexity to the fluid dynamics of gas exchange.[21] Carbon availability and limitation Carbon is an essential element for the biosynthesis of organic molecules within a cell; algae have developed (or inherited) a series of metabolic mechanisms that harvest and fix inorganic carbon. Microalgal photosynthesis fixes inorganic carbon into sugars, which then act as substrates for more complex molecules, such as starch and lipids. CO2 is fixed by the Calvin– Benson cycle by the enzyme ribulose bisphosphate carboxylase oxygenase (RuBisCO) using ATP as energy and nicotinamide adenine dinucleotide phosphate (NADPH) as the reductant. Importantly, O2 competes continuously with CO2 for RuBisCO, and the process of O2 consumption by RuBisCO is known as photo-respiration.[22] Photo-respiration reduces CO2 fixation efficiency by 20 to 30 %, and the rate of photo-respiration increases under a carbon-limited environment.[23, 24] Microalgae have evolved a series of carbon-concentrating mechanisms (CCMs) independently that increase the concentration of CO2 available at the RuBisCO active site and thereby inhibit photo-respiration competitively.[25] Many of these CCMs actively pump bicarbonate (HCO3) into algal cells, in which it is dehydrated to CO2 by the carbonic anhydrase enzyme, which elevates intracellular CO2 that, in turn, decreases photo-respiration and potentially increases the energetic efficiency of photosynthesis.[16] The rate of photosynthesis in the PBR can be calculated immediately from changes in oxygen concentration.[26] In an algal monoculture, the concentration of dissolved O2 and CO2 in a PBR is in dynamic equilibrium with air that is bubbled through the PBR (Figure 1). The relationship for oxygen may be described by Fick’s Law &&which one is meant?& &[Eq. (1)]: d½O2  dt ¼ ðP  RÞ  kL að½O2   ½O2 eq Þ ð1Þ in which [O2] is the dissolved O2 concentration; (PR) is the rate of net photosynthetic O2 production, that is, the rate of photosynthesis P minus the rate of dark (mitochondrial) respiration R; kLa is the gas exchange (or mass transfer) coefficient; and [O2]eq is the equilibrium or Henry’s Law O2 concentration. Dissolved O2 concentrations can be measured directly by using an optode or an electrode. A similar mathematical expression can be written for CO2.[27] Although the mass transfer coefficient is scaled by the ratio of molecular masses, there is an additional complication in that dissolved CO2 is in equilibrium with HCO3 and carbonate ions (CO32 ; Figure 1). The addition of CO2 to seawater increases the concentration of HCO3 but decreases the concentration of CO32.[28] The formation of HCO3 leads to the dissociation of 2  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ÝÝ These are not the final page numbers! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 internal conversion to CO2 and a leak of some of the CO2 back to the medium.[34] Importantly, the conversion of bicarbonate to CO2 involves the production of equal quantities of hydroxyl ions or the uptake of equal quantities of protons. Intracellular acid–base regulation involves the efflux of these hydroxyl ions or protons, so that the effect on the medium acid–base and equilibrium concentrations of inorganic carbon species is the same for bicarbonate entry as would have been the case for CO2 uptake.[26, 36] Therefore, the pH increases during the photo-period in the diurnal light cycle because the consumption of bicarbonate, and the corresponding influx of protons and/or efflux of hydroxyl ions by photosynthesis is faster than the re-supply of inorganic carbon as CO2 from the gas stream. Some of this pH increase is also the result of nitrate assimilation if nitrate is the nitrogen source.[36, 37] There are strong experimental indications that inorganic carbon may limit algal growth across the diurnal photo-period.[38, 39] The concept of using a constant pH system, or pH-stat, to control carbon availability in a PBR has been used extensively.[40–43] The pH-stat solves many of the challenges associated with managing carbonate chemistry in largescale culturing facilities, however, there are some advantages in alternative methods. Firstly, if CO2 addition was managed more precisely, it may be possible to reduce running costs or to improve yields of algal biomass production. Secondly, if the pH (or equivalently CO2) is controlled, the rate of carbon fixation cannot be estimated from changes in pH. Figure 1. The major continuous inputs to a gas-bubbled PBR (light, O2, CO2) and the main processes [P: photosynthesis; R: respiration; kLa: gas exchange; carbonate chemistry including bicarbonate ions (HCO3), carbonate ions (CO32) and protons (H+)]. protons, which reduces the pH of the solution in the PBR; however, this pH shift is buffered by the corresponding decrease in CO32.[29] This dampened pH response in saltwater cultures calls for greater measurement precision to resolve changes in carbonate chemistry. The various dissociation constants of carbonate chemistry have been measured as a function of temperature, salinity and alkalinity[26, 28] and can be used to calculate the concentrations of carbonate species in solution. The equations can be solved conveniently using the spreadsheet “CO2 sys”.[30] Hence, pH is an indirect measure of the quantity of inorganic carbon available in a stable marine environment. However, once biology becomes involved, photosynthesis (P) and dark respiration (R) processes alter the pH value. Algal photosynthesis fixes CO2 and produces O2 as a byproduct, whereas respiration is essentially the elemental reversal of this photosynthesis (Figure 1). So, if pH and [O2] are monitored continuously and provided that gas exchange and carbonate chemistry are measured, it is possible disentangle the rate of photosynthesis from the dynamics of gas transfer in a PBR.[24, 31] The carbonate chemistry of seawater equilibrated with air at the ambient CO2 concentration of 400 ppm indicates that the predominant inorganic carbon species is bicarbonate, with carbonate next most abundant and CO2 even less abundant.[26, 27] The CO2 concentration in air-equilibrated seawater is approximately the same as that in air; however, the mass transfer coefficient of CO2 is 10 000-fold lower in seawater, therefore, the supply of CO2 to the surface of photosynthesising cells in seawater is much slower than that in air.[32] This makes bicarbonate, present at  200 times the concentration of CO2, a clear candidate for transport across the plasmalemma by the CCM present in most marine microalgae, which include many candidate biofuel species.[33–35] The Nannochloropsis CCM is based on an active bicarbonate influx across the plasmalemma with ChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org These are not the final page numbers! ÞÞ Aims and objectives The aims of this study are to determine the carbon availability in PBRs using measurements of dissolved oxygen concentration [O2] and pH, and to investigate the effects of carbon limitation on the green microalga N. oculata. The objectives are to determine when N. oculata cells become carbon limited during the diurnal light cycle, to quantify to what extent this carbon limitation affects their photosynthetic activity and to examine whether CO2 addition can alleviate the situation. Results and Discussion Diurnal [O2] and pH variations Nannochloropsis oculata cells were cultivated to an exponential growth phase under a 12:12 h diurnal light/dark cycle in two PBRs. The dissolved oxygen concentration [O2] and pH were measured continuously, and the cell density was measured by automated cell counts (complete results profile available in the Supporting Information, page 1). Diurnal [O2] and pH profiles consistently follow the characteristic and reproducible trends shown on page 2 in the Supporting Information. To investigate this phenomenon in more detail, a single representative light/ dark period is shown in Figure 2; five distinct phases (A, B, C, D and E) have been assigned to represent the variations in [O2] and pH value. During the dark cycle (phases D and E) the [O2] profile remains steady at, or near, the [O2] minimum of 218 mmol L1 3  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 cantly slower than the corresponding [O2] response (Figure 2 a). The rate of pH increase slows gradually, and the pH reaches a maximum value of 9.5 near the end of the light cycle (phase C). During the dark cycle (phases D and E), the pH gradually returns to its &&dark&& minimum of 8.0. [O2] as a measure of net photosynthesis The net rate of photosynthetic O2 production or consumption can be calculated from the dissolved oxygen concentration.[26] In this experiment, the “spikes” in the [O2] profile (Figure 2 a) are artefacts that were introduced deliberately to measure the rate of photosynthesis and gas exchange at specific time points.[38, 44] The spikes were created by switching off air bubbling for approximately 15 min before turning it back on again (Figure 3). Initially, there is a dynamic equilibrium between gas exchange and the biological processes, which gives an [O2] of [O2]de.[32] If the gas supply is turned off, the gas transfer coefficient is negligible and Equation (1) can be simplified to Equations (2) and (3): Figure 2. Representative a) [O2] and b) pH profiles over a single day (12:12 h light/dark cycle) for N. oculata in linear growth phase; both profiles show evidence of carbon limitation that occurs towards the end of the light cycle. Spikes in [O2] were used for oxygen evolution measurements (see Figure 3). d½O2  dt ð2Þ ¼PR solution : ½O2  ¼ ðP  RÞt þ ½O2 de ð3Þ Net photosynthesis (PR) is, therefore, equal to the gradient (Figure 2 a). This [O2] minimum is slightly lower than the [O2] of of the linear increase in [O2]. Once gas exchange is turned seawater in equilibrium with air (221 mmol L1 at 20 8C), calcuback on, Equation (1) can now be solved because the start lated from Henry’s Law[32] because of the consumption of point [O2] and the end point [O2]eq are both known [Eq. (4)]: oxygen by N. oculata cells by dark respiration. If the light is turned on, photosynthetic O2 production starts up within ð4Þ solution : ½O2  ¼ D½O2 ekL a t þ ½O2 de a matter of minutes, and [O2] reaches its maximum of 252 mmol L1 within the first hour (phase A). At this point, [O2] The decrease in [O2] is described as an exponential function, reflects the maximum rate of photosynthesis achievable and the gas exchange coefficient (kLa) determines the half-life through the steady-state acclimation of N. oculata to the averof this exponential decay.[26] The gas exchange coefficient is age photon availability within the PBR, if we assume replete nitrate and phosphate concentrations (approximately 300 mg L1 of nitrate and 40 mg L1 of phosphate). Following this steady-state maximum, [O2] begins to decrease (phase B) and approaches a new plateau of 236 mmol L1, the “terminal [O2]”, near the end of the light cycle (phase C). This decrease in [O2] may be attributed to a decrease in the rate of photosynthesis and/or an increase in the rate of respiration over the light cycle. As the light intensity does not change, this behaviour indicates either the presence of nutrient limitation or photo-inhibition. If the light is turned off, [O2] returns quickly to its dark cycle minimum of 218 mmol L1. The pH is at its minimum value of 8.0 at the end of the dark cycle (Figure 2 b). This is lower than the equilibrium pH of 8.2 because of CO2 production by dark respiration during the dark cycle. The pH increase during the light cycle (phases A, B and C) is an indication of photosynthetic carbon consumption by Figure 3. A typical spike in the [O2] profile, which was used to calculate the net photoN. oculata cells. Notably, the pH response is signifi- synthesis (PR) and the gas exchange coefficient (kLa) at a specific point in time. ChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org 4  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ÝÝ These are not the final page numbers! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 strongly dependent on temperature, pressure, salinity and bubble size,[30] but these are all assumed to be constant throughout the experiment. Once kLa is known, it is possible to calculate (PR) directly from the [O2] profile without performing a spike by using the expected dissolved oxygen concentration calculated from Henry’s Law ([O2]eq) and considering Fick’s Law solution under steady-state conditions [Eq. (5)]: solution : ½O2 de ¼ ½O2 eq þ PR kL a based system. Net photosynthesis (PR) is at its steady-state maximum of 125 mmolO2 L1 h1 &&at the start of the light cycle&& and decreases to the terminal rate of 50 mmolO2 L1 h1 by the end of the light cycle. This corresponds to a 60 % decrease in net photosynthesis by the end of the light cycle, or a ~ 30 % decrease in net productivity. pH as a measure of carbon availability ð5Þ As outlined in the Introduction (Figure 1), dissolved CO2 increases the concentration of HCO3 ions and decreases the concentration of CO32 ions in seawater; pH can be used as an indirect measure of carbon availability in a PBR.[47] The increase in pH over the light cycle (Figure 2 b) can be attributed to the removal of dissolved inorganic carbon (DIC) by N. oculata cells, which shifts the equilibrium of the carbonate system. Dissolved CO2 molecules have little direct influence on N. oculata photosynthesis as bicarbonate dominates the extracellular carbon pool (Figure 5) and is also the inorganic carbon species taken up by the CCM. At the minimum pH of 8.0, the bicarbonate pool is almost full at ~ 2500 mmol L1, whereas at the maximum pH of 9.5, the bicarbonate concentration is reduced to ~ 300 mmol L1 and carbonate is the dominant carbon species (Figure 5). The pH of the medium normally increases with time if algae are grown with nitrate because of the excretion of OH anions,[37, 48] but this does not occur here as the pH returns to the same dark cycle minimum after 24 h. This is consistent with the fact that N. oculata uses an acid–base regulation mechanism to synthesise organic acids to neutralise excess OH and store the resulting organic anion salts in vacuoles.[49] N. oculata cells, like most algae, cannot use carbonate as a carbon source.[14, 25] As bicarbonate is used up and becomes a limiting resource, so the rate of pH increase decreases. N. oculata cells find it more difficult to obtain carbon, and the rate of photosynthesis decreases consequently (Figure 4). The pH maximum plateau at the end of the light cycle (Figure 2 b), therefore, represents the point at which the carbon-limited rate of photosynthesis matches the input of carbon from gas exchange. The reduced afternoon [O2] terminal corresponds to the equilibrium between the rate at which carbon is removed from solution by photosynthesis and the rate at which carbon is added to solution by gas exchange (bubbling). This equilibrium will depend on the factors that affect both photosynthesis (light level, cell number, temperature, nutrient levels) and gas exchange (gas bubbling rate, CO2 concentration, bubble size, gas residence time).[5, 47] Equations (3)–(5) were fitted to the [O2] measurements before bubbling was interrupted [Eq. (5)], during the interruption [Eq.( 3)] and after [Eq. (4)] using the least squares fitting procedure in gnuplot. A more precise way to calculate net photosynthesis is to remove the complication of gas exchange by measuring [O2] in a traditional cuvette-based respirometer setup.[44, 45] By using this system, a 2 mL culture sample was extracted from the PBR, dark-adapted for 10 min and the oxygen production rate was measured by performing a rapid light curve over five irradiances. The use of a sealed cuvette ensures that photosynthesis rate measurements are free of interference from atmospheric gas exchange. However, it is difficult to relate the light field that cells experience in the PBR to the light field in the respirometer cuvette. Other bulk properties, such as mixing, will also be different. For these reasons, the rate of photosynthesis measured in the PBR and cuvette are not compared directly, and different ordinate scales are used in Figure 4. A similar diurnal pattern is observed in the PBRs and in the respirometer setup, which implies that [O2] can be used with confidence as proxy for net photosynthesis provided that the gas exchange is controlled and stable[46] (Figure 4). Dark respiration (R) measurements of 20 mmolO2 L1 h1 are consistent across the diurnal cycle in the PBR and against the cuvette- Mechanism Figure 4. A comparison between different types of photosynthesis rate measurements and calculations over two days of PBR operation: red pluses, calculated using [O2] spikes; red line, calculated directly from [O2] profile using Fick’s Law &&which one?&& and assuming constant gas exchange; blue dots (in the light) and blue squares (in the dark), measured using the cuvette-based respirometer setup. ChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org These are not the final page numbers! ÞÞ 5 The asymptotic diurnal [O2] trend cannot be attributed to photo-inhibition as the light availability in a culture decreases monotonically with age; hence photoinhibition is more likely in dilute cultures at early stages of growth. Therefore, this afternoon decrease in photosynthesis is likely caused by a resource limi 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 conversion of CO2 plus hydroxyl ions and bicarbonate in the culture,[50] as well as internal buffering by N. oculata cells caused by fluxes of the pH-influencing species across the cell plasmalemma.[29, 49] The extracellular carbon pool continues to empty over a timescale of hours, and the inflection in the pH rise indicates that bicarbonate ions are starting to become limiting. This limitation results in a reduction in the photosynthesis rate (phase B). Finally, near the end of the light period, the extracellular carbon pool is almost empty of bicarbonate ions and photosynthesis becomes fully carbon limited (phase C). Photosynthesis is maintained at a new equilibrium between carbon demand and the delivery of additional usable carbon by gas exchange. Gas exchange, therefore, controls the degree of carbon limitation experienced by the algal culture as the light cycle progresses.[51] At the beginning of the dark cycle, photosynthesis is immediately switched off, and [O2] rapidly returns to its dark minimum (phase D). The pH gradually returns to its minimum value during the dark cycle as the extracellular carbon pool is refilled slowly by CO2 delivered by gas exchange and dark respiration (phase E). This process continues as long as the N. oculata culture remains in the exponential growth phase and no additional nutrient limitation is present.[9] It has been shown that photo-respiration increases linearly with [O2] at O2 saturation levels greater than 75 % (165 mmol L1 at 20 8C and salinity of 33&&unit?&&), which is within the range of this experiment.[52] The initial (phase A) increase of [O2] from 218 to 252 mmol L1, therefore, increases photo-respiration by ~ 16 %. This increase in photo-respiration likely triggers the initial decrease in [O2] (phase B), which provides another explanation for the quick [O2] response compared to the pH response. Following on from phase A, the photo-respiration rate stabilises and any further decreases in [O2] may be attributed directly to carbon limitation. Figure 5. Carbonate chemistry of seawater shows a predominance of bicarbonate at the equilibrium pH of 8.2. Consumption of bicarbonate and influx of protons and/or efflux of hydroxyl ions by photosynthesis is faster than inorganic carbon is resupplied as CO2 from the gas stream, which causes a decrease in total inorganic carbon; CO2 and bicarbonate decrease, whereas carbonate and pH increase. The inorganic carbon pool is refilled during the night as part of the diurnal cycle. Phases A–E correspond to the classification in Figure 2. tation. In these batch cultures, the total concentration of inorganic nutrients, such as phosphorous and nitrogen, also decreases monotonically and could not be responsible for the diurnal pattern. This leaves DIC as the most likely candidate for resource limitation. The concentration of DIC and O2 in a culture vessel are determined by gas exchange. It is now possible to propose a mechanism for the carbon limitation of N. oculata in PBRs (Figure 6). At the end of the dark cycle, [O2] and pH are both at their minimum levels (phase E). Dissolved O2 and CO2 are in equilibrium with the air supply, and the gas exchange system is in a steady state. The extracellular DIC pool, which contains carbonate and bicarbonate ions, is at its highest level in the light/ dark cycle and the proton concentration is high. A small amount of O2 is used up by dark respiration at a constant rate. Once the light is turned on, photosynthesis is activated rapidly and the steady-state [O2] maximum is attained (phase A). At this point, net photosynthesis is limited only by photon availability.[38] Some O2 is used up by dark respiration and photo-respiration, but the majority of O2 departs the system by gas exchange as the O2 concentration is now significantly higher than that under air equilibrium. The carbon pool begins to decrease slowly as N. oculata cells take up bicarbonate ions, the conversion of which to CO2 supplies photosynthesis and limits photo-respiration.[16] As the carbon pool is large relative to the oxygen pool, the pH response is significantly slower than the [O2] response. The slow pH response also reflects the buffering capacity of seawater and the slow uncatalysed interChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org Confirmation To further verify whether carbon really limits N. oculata growth, additional CO2 was introduced into the PBR and the [O2] and pH profiles were measured. The results are shown in Figures 7 and 8. The initial proof-of-concept experiment involved a single CO2 injection (Figure 7). The control PBR displays the typical diurnal [O2] and pH variation over a four-day cycle (Figure 7 a). The second PBR follows the same pattern until CO2 is injected at the presumed carbon-limited period (before the end of phase C) on day 8 of the experiment, and the light cycle changed from a diel pattern to continuous illumination. If the N. oculata cells are carbon limited, the addition of more carbon should increase the rate of photosynthesis. Alternatively, if the cells are photo-inhibited, additional CO2 should have a negligible effect.[16] This large CO2 injection, 12 min in duration, saturated the extracellular carbon pool rapidly back to its pre-dawn maximum, as evidenced by a rapid decrease in the pH (Figure 7 b). 6  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ÝÝ These are not the final page numbers! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 6. Mechanism for carbon limitation of N. oculata in a PBR. Changes in arrow thickness indicate changes in the rate of photosynthesis, respiration and gas exchange. Changes in the red and white ovals indicate changes in extracellular DIC and proton (H+) pool sizes respectively. At the same time, [O2] returned to its maximum value, which indicates that steady-state photosynthesis is possible in a carbon-replete system at any point during the light cycle. Following CO2 addition, the light cycle was extended for an additional 10 h (Figure 7 b). Over this time, the [O2] reduction corresponded directly to carbon pool depletion (increased pH) with no evidence of photo-inhibition, circadian cycles or any other light-related effects. A second experiment was performed to determine whether [O2] could be maintained at its carbon-replete maximum by periodic CO2 injection (Figure 8). In the control PBR, the typical diurnal carbon limitation pattern was observed over a period of five days (Figure 8 a). In the second PBR, a brief CO2 injection of 10 s in duration was applied whenever [O2] began to fall beneath the light-limited maximum (Figure 8 b). The maximum daily [O2] was maintained across five consecutive light cycles by applying a set of 5–8 such CO2 injections to maintain a carbon-replete environment. One way to ensure that the PBR remains carbon replete is to operate the PBR as a pH-stat at the night cycle minimum pH ChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org These are not the final page numbers! ÞÞ of 8.0. There are two disadvantages to this approach: 1) the pH response time from the carbonate chemistry is slow, especially in the absence of external carbonic anhydrase in Nannochloropsis spp., and 2) carbonate chemistry is complex and pH is only a proxy for carbon availability. Salinity and dissolved nutrient concentrations play a significant role to determine the relationship between carbonate chemistry and pH.[29] In a pHstat system, the control parameter is separated by numerous chemical and metabolic steps from the desired algal physiological response, that is, maximum photosynthesis rate. pH is one of many inputs of photosynthesis, which include irradiance, temperature, inorganic nutrient availability and cell density. A more precise control system could instead utilise [O2], which is a direct output of photosynthesis. An example of one such “[O2]-stat” system in operation is given in Figure 8. As the [O2] response is more dynamic and faster, it would result in more efficient CO2 delivery, but it will carry additional challenges in maintaining a steady equilibrium. 7  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 7. Effects of a single CO2 injection on diurnal [O2] and pH profiles. a) The control experiment shows consistent [O2] and pH profiles over a four-day period. b) A single, large CO2 injection at terminal [O2] on day 8 of the experiment fills up the carbon pool and returns [O2] back to its steady-state maximum, which effectively replicates DIC replenishment during a night cycle. thesis was limited by the depletion of usable carbon, or more specifically bicarbonate ions, in the system. A mechanism that involves photosynthesis, respiration and carbonate chemistry can explain this phenomenon. The addition of CO2 was used to recover the steady-state maximum photosynthesis rate and, therefore, prove that N. oculata cells were carbon limited under the environmental conditions provided by the photo-bioreactor. This ability to identify and analyse carbon limitation is of critical importance to both experimental design and commercial algal biomass production. The dissolved oxygen concentration [O2] proved to be an effective proxy for photosynthesis rate. In future, a feedback loop will be used to maintain [O2] at its maximum level by gradually increasing the CO2 concentration in the gas bubbling mix across the light cycle. This approach would alleviate carbon limitation and maximise photosynthesis, which would also maximise N. oculata biomass production in the absence of other limiting factors, such as nitrogen or phosphorus depletion. As the CO2 molar fraction of the gas phase and the gas exchange coefficient are both known, it will also be possible to calculate the CO2 sequestration rate by N. oculata cells. Once set up, this system could be applied to any photo-bioreactor geometry or indeed to any algal species. Experimental Section Figure 8. The use of periodic CO2 injections to maintain [O2] at its carbon-replete maximum throughout the light cycle. Algae are increasingly carbon limited across the light cycle in a) the control experiment, whereas a higher rate of net photosynthesis could be consistently maintained with b) periodic CO2 injection across five consecutive light cycles. The microalga Nannochloropsis oculata (Droop) Green (Australian National Algae Culture Collection; strain CS-179) was used. Taxonomically, N. oculata is in the class Eustigmatophyceae of the phylum Heterokontophyta. N. oculata was grown in f/2 seawater medium: artificial seawater (salinity 33) enriched with 54.7 mg L1 NO3, 4.5 mg L1 PO4 and micronutrients. Stock cultures were maintained in 200 mL Erlenmeyer flasks at Conclusions The green alga N. oculata was cultured in an environmental photo-bioreactor, and a significant reduction in the rate of photosynthesis was observed during the light cycle. PhotosynChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org Algae and stock culture 8  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ÝÝ These are not the final page numbers! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Full Papers 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 20 8C under cool-white fluorescent light with an irradiance of (50  5) &&mmolphoton m2 s1&& and a 12:12 h light/dark cycle in an incubator (Labec Temperature Cycling Chamber incubator, Labec Pty Ltd, Australia). Cultures were diluted seven days before the experiment to ensure that N. oculata cells were in the exponential growth phase. PBRs were inoculated with a 1:10 v/v algal dilution in f/2 to give a starting concentration of approximately 106 cells mL1. 10 min before the experiment. The temperature was controlled at 20 8C in an optical cuvette holder (ED-101US/MD, Heinz Walz GmbH, Effeltrich, Germany) with integrated magnetic stirring. White actinic light was provided by the multi-colour pulse-amplitude-modulated fluorometer (MC PAM; Heinz Walz GmbH, Effeltrich, Germany). A [O2] fixed needle-type minisensor probe with optical isolation (OXF1100-OI; PyroScience, Aachen, Germany) was used to measure photosynthesis and respiration rates. PBRs Oxygen production Experiments were performed in environmental photo-bioreactors (ePBR, Phenometrics, Lensing, MI, USA) with an inverted conical frustum geometry and a working volume of 500 mL.[38] Two simultaneous ePBR experimental replicates were used. The algae were mixed by magnetic stirring. The temperature was maintained at 20 8C using a Peltier heater-cooler connected to a water jacket. Cool-white LED illumination was delivered from above; the path length through the algal culture was 20 cm. The algae were illuminated with a photon flux density of 500 mmolphoton m2 s1 over a 12:12 h light/dark cycle during a six-day adaptation period. Once acclimation was complete, that is, once N. oculata cells had reached the exponential growth phase, irradiance was increased to 2000 mmolphoton m2 s1 (12:12 h light/dark cycle), which corresponds to the day-time maximum irradiance at outdoor culturing facilities. Air bubbling was also turned on at this time. Oil-free laboratory air was humidified by passing it through distilled water after which it was filtered through a 0.2 mm filter and then delivered through a 0.45 mm sterile needle placed near the bottom of the ePBR vessel. Oxygen production was measured several times per day in the ePBR. The air supply was turned off for approximately 15 min to measure the rate of oxygen production (modelled by a linear increase) associated with net photosynthesis. Once the air supply was turned back on, the oxygen concentration returned to its baseline as a result of gas exchange (modelled by an exponential decay). Rapid light curves were used to measure oxygen production in the cuvette. Five consecutive illumination steps were used: 44 mmolphoton m2 s1 for 8 min; 122 mmolphoton m2 s1 for 4 min; 258 mmolphoton m2 s1 for 2 min; 499 mmolphoton m2 s1 for 2 min; 831 mmolphoton m2 s1 for 2 min. Carbon addition Two mass flow controllers were used to mix pure CO2 at a flow rate of 20 mL min1 with air at a flow rate of 70 mL min1 to give a CO2 molar gas fraction of 22.3 %. This CO2 was delivered into one of the ePBRs at the end of the light cycle on day 10 of the experiment (Figure 7). It was delivered over a period of 12 min through an additional humidified gas line with a 0.45 mm sterile needle. In a second experiment, CO2 was instead delivered periodically at 10 s intervals (Figure 8). A solenoid valve was used to control CO2 injection. Continuous [O2] and pH measurements Dissolved oxygen [O2] was measured by using a 3 mm robust [O2] optical probe with optical isolation (OXROB10-OI-CL4; PyroScience, Aachen, Germany). A two-point [O2] calibration against air-saturated and nitrogen-saturated seawater was performed. A prototype pH optical probe with optical isolation (PyroScience, Aachen, Germany) was used to measure pH; it was calibrated by titration with NaOH and HCl, and the pH response was linear within the pH region of interest. Acknowledgements The authors would like to thank Dr Milan Szab for his scientific input and PyroScience GmbH for giving us access to their prototype pH optodes. Automated cell counts Keywords: biomass · carbon dioxide fixation · kinetics · photosynthesis · renewable resources Samples (1 mL) were extracted daily from each ePBR, fixed with 1 % glutaraldehyde and stored in the dark at 5 8C. 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Received: March 5, 2015 Revised: June 22, 2015 Published online on && &&, 0000 10  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ÝÝ These are not the final page numbers! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 FULL PAPERS B. Tamburic,* C. R. Evenhuis, D. J. Suggett, A. W. D. Larkum, J. A. Raven, P. J. Ralph Just grow it: We present the first indepth analysis of CO2 limitation on biomass productivity of the marine microalga Nannochloropsis oculata. Net photosynthesis decreases by 60 % over a 12 h light cycle as a direct result of carbon limitation. Continuous dissolved O2 and pH measurements are used to develop a detailed diurnal mechanism for the interaction between photosynthesis, gas exchange and carbonate chemistry in the photo-bioreactor.&&ok? text and graphics were missing.&&Tamburic et al. study CO2 limitation on #biomass productivity in a #photobioreactor ChemSusChem 0000, 00, 0 – 0 www.chemsuschem.org These are not the final page numbers! ÞÞ && – && Gas Transfer Controls Carbon Limitation During Biomass Production by Marine Algae 11  0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57