Cecilia A. Popovich

Lipid analysis in Haematococcus pluvialis to assess its potential use as a biodiesel feedstock

The lipid content and composition of Haematococcuspluvialis exposed to stress conditions were analyzed to assess the potential of this microalga as a biodiesel feedstock. The total lipid content of control cells was 15.61% dw, whereas that of cells exposed to continuous high light intensity with nitrogen-sufficient medium (A-stress condition) or under continuous high light intensity with nitrogen-deprivation medium (B-stress condition) was 34.85% dw and 32.99% dw, respectively. The fatty acid profile was similar under all conditions and indicated that the main components were palmitic, stearic, oleic, linoleic, linolenic and linolelaidic acids. The neutral lipid fraction increased about 2-fold under both stress conditions. The percentage of saturated fatty acids in the neutral lipid fraction was 30.36% and 29.62% in cultures grown under A-stress and B-stress, respectively, and 27.81% under control conditions. The monounsaturated fatty acid content was not significantly different in control and A-stress cultures (20.07% and 19.91%, respectively), but was 18.96% under B-stress. The content of polyunsaturated fatty acids was 47.23% under B-stress and 43.15% under A-stress. Growth-rate was higher under A-stress compared to B-stress. This is the first study of H.pluvialis that provides a detailed characterization of its lipid content in relation to bioenergy. The results indicate the potential of this microalga as a biodiesel feedstock; however, culture conditions still have to be improved in order to achieve an adequate energy balance in mass culture.

Neochloris oleoabundans grown in enriched natural seawater for biodiesel feedstock: evaluation of its growth and biochemical composition.

The freshwater microalga Neochloris oleoabundans was used to study algal lipid production in enriched natural seawater, in order to assess its suitability as biodiesel feedstock. Optimal and nitrogen-stress (N-stress) conditions were analyzed. Under optimal conditions, the strains growth rate was 0.73 div day(-1) and the biomass concentration was 1.5 g L(-1), while it had a maximum lipid yield under N-stress conditions (lipid content: 26% of dry weigh and lipid productivity: 56 mg L(-1) day(-1)). Lipid accumulation was mainly due to a significant increase of triacylglycerol content. Neutral lipids were characterized by a dominance of monounsaturated fatty acids and displayed a fatty acid profile that is suitable for biodiesel. This work offers an interesting alternative for sustainable microalgal oil synthesis for biodiesel production without using freshwater resources. However, further studies are necessary in order to optimize the lipid productivities required for commercial biodiesel production.

Dissolved Nutrient Availability during Winter Diatom Bloom in a Turbid and Shallow Estuary (Bahía Blanca, Argentina)

Abstract The inner zone of Bahía Blanca Estuary is a shallow, well-mixed, highly turbid, and nutrient-rich temperate ecosystem. The phytoplankton annual cycle is characterized by a recurrent winter/early-spring diatom bloom. From May to August 2002 a research program aimed to describe the potential relations between the dynamics of phytoplankton and nutrients was carried out. Phytoplankton composition, abundance, chlorophyll a (Chl a), and dissolved inorganic nutrients (nitrate, nitrite, ammonium, phosphate, and silicate) were measured. The species involved were classified in blooming (including Thalassiosira spp. and Chaetoceros spp.) and nonblooming species (including other diatoms and dinoflagellates). The results show: (1) a prebloom period, which includes low biomass values and a strong decrease of NO3−; (2) an initial bloom period characterized by high diatom abundance and Chl a, a marked dominance of Thalassiosira curviseriata, and a strong decrease of NH4+; (3) an intermediate bloom period of high abundance, codominance of blooming species, and a strong decrease of PO43−; and (4) a final bloom period with a decrease in abundance of blooming species and a general nutrient recovery. Thalassiosira curviseriata seems to be a species associated with high nutrient stock and low light intensity. Its growth, mainly concurrent with DIN decrease, was related to new production in this area. The obtained data suggest that low phosphate concentrations, more than DIN and silicates, contributed to the diatom bloom collapse. According to our results, this high production during winter can be explained as a net growth of phytoplankton because of a successful low light- and temperature-acclimated species, possibly favored by low predation and high nutrient availability.

Feedstocks for Second-Generation Biodiesel: Microalgae’s Biology and Oil Composition

The solar energy is an inexhaustible source, while other energy reserves, like fossil and nuclear fuels, are limited in quantity and are depleted as years go by. Renewable energy is necessary to replace petroleum-derived fuels. The first generation biofuels, which are produced from oil seeds and crops, are a possible alternative, but they are limited in their capacity to provide all the energy demanded in the world. Therefore, new sources for the sustainable production of renewable energy are being looked for. This concern has promoted the keen interest in developing second generation biofuels, which are produced from other feedstocks, such as microalgal oils (Schenk et al., 2008; Mata et al., 2010). Some microalgal species are capable of producing biomass yields containing high percentages of oils (Aaronson et. al., 1980). In addition, microalgal systems can use low value natural resources, such as arid lands and saline water, thus offering the potential for large biomass energy contributions without competing for prime agricultural or forest land. Most microalgae grow photoautotrophically by using solar energy and mainly carbon dioxide as carbon source. Alternatively, some species can grow heterotrophically or mixotrophically using organic compounds as energy and carbon sources (Kitano et al., 1997; Hu & Gao, 2003; Xu et al., 2006; Liang et al., 2009). Some microalgae are called oleaginous because they synthesize and accumulate substantial amounts of neutral lipids, mainly as triacylglycerol (TAG), under diverse stress conditions (Bigogno et al., 2002; Hu et al., 2008; Gardner et al., 2010; Damiani et al., 2010). TAGs as storage lipids are the best substrate to produce biodiesel (Xu et al., 2006; Schenk et al., 2008). This biofuel is obtained by transesterification of oil or fat with a monohydric alcohol, yielding the corresponding mono-alkyl esters (Knothe, 2005). Since transesterification maintains the relative ratio of fatty acids present in the feedstock (Costa Neto et al., 2000), the profile of the fatty acid methyl esters is a reflection of the feedstock fatty-acid composition (Lang et al. 2001; Ferrari et al. 2005). Biodiesel production from microalgae is technically feasible (Xu et al., 2006; Patil et al., 2008; Francisco et al., 2010), but for an effective use of this renewable resource as biofuel, it is necessary to be able to modify microalgal growth conditions in order to obtain high biomass productivity and the desired lipid quantity and quality. Those interested in

Role of Nutrients in Phytoplankton Development during a Winter Diatom Bloom in a Eutrophic South American Estuary (Bah´ õa Blanca, Argentina)

ABSTRACT Spetter, C.A.; Popovich, C.A.; Arias, A.; Asteasuain, R.O.; Freije, R.H., and Marcovecchio, J.E., 2015. Role of nutrients in the phytoplankton development during a winter diatom bloom in a eutrophic South American estuary (Bahía Blanca, Argentina). The Bahía Blanca Estuary is considered highly eutrophic. Long-term studies have shown that the winter–early spring bloom can be considered the development of a diatom assemblage with Thalassiosira curviseriata as the dominant species. Since 2003, several changes have been observed in the annual pattern of nutrients and phytoplankton. To assess the availability of nutrients and their relationship with phytoplankton development, nutrient variability was studied during a winter bloom in 2003 (April 22–September 4). Nitrite, nitrate, ammonium, phosphate, silicates, chlorophyll-a, phytoplankton abundance, particulate organic matter, and physicochemical parameters were measured in surface water at Cuatreros Port. In the Sauce Chico River mouth, we determined the concentration of nutrients to estimate the input of this river. The results of this study were compared with those of a previous usual diatom bloom from 2002. In 2003, the bloom was dominated by the Cyclotella sp. with high chlorophyll-a concentrations (26.5–40.4 μg L−1). Thalassiosira curviseriata was present only at three sampling dates, reaching up to 19% of the total abundance. Mean values of dissolved oxygen, nitrate, oxygen saturation percentage, and salinity were significantly higher in 2003 than in 2002, while N:P ratios were significantly lower. Si was never limiting. A shift in the limiting nutrient between the years (N in 2003 and P in 2002) could have lead to a change in species dominance during the blooms. Results suggest that the nutrients levels in the coastal ecosystems at Cuatreros Port play an important role in the control of phytoplankton dynamics during the productive period and partially explain its interannual variability. RESUMEN El estuario de Bahía Blanca es considerado altamente eutrófico. Los estudios anteriores han demostrado que la floración invernal del fitoplancton se caracteriza por el desarrollo de una asociación de diatomeas con Thalassiosira curviseriata como la especie dominante. Desde el 2003, se han observado cambios en el patrón anual de nutrientes y fitoplancton. La variabilidad de los nutrientes fue estudiada en Puerto Cuatreros durante una floración invernal (22 Abril–4 Septiembre 2003) con el fin de evaluar la disponibilidad de nutrientes y su relación con el desarrollo del fitoplancton. Se determinó la concentración de nitrito, nitrato, amonio, fosfato, silicatos, clorofila a, materia orgánica particulada, la abundancia del fitoplancton y los parámetros fisicoquímicos en agua superficial. Para estimar el aporte del Río Sauce Chico se determinaron las concentraciones de los nutrientes en su desembocadura. Los resultados de este estudio se compararon con los de una floración típica previamente estudiada durante el 2002. En 2003, la floración estuvo dominada por Cyclotella sp. presentando altas concentraciones de clorofila a (26.5–40.4 μg L−1). Thalassiosira curviseriata alcanzó el 19% de la abundancia total y se detectó solo en tres fechas del muestreo. Los valores promedios de oxígeno disuelto, nitrato, porcentaje de saturación de oxígeno, y salinidad fueron significativamente mayores en 2003 que en 2002, mientras que la relación N:P fue significativamente menor. El Si nunca fue limitante. Se sugiere que un cambio en el nutriente limitante (N en 2003 y P en 2002) puede haber conducido a un cambio en la dominancia de la especies durante la floración invernal. Los resultados evidencian que los niveles de nutrientes en sistemas costeros como Puerto Cuatreros cumplen un rol importante en el control de la dinámica del fitoplancton durante un período productivo y podrían explicar en parte su variabilidad interanual.

Phytoplankton-aloricate ciliate community in the Bahía Blanca Estuary (Argentina): seasonal patterns and trophic groups

O padrao sazonal da comunidade de fitoplâncton-ciliados aloricados e um indicador adequado das relacoes troficas e reciclagem de nutrientes nos ambientes costeiros. A abundância, biomassa e composicao da comunidade de fitoplâncton-ciliados foram determinadas por classes de tamanho durante um ciclo anual no estuario de Bahia Blanca. Alem disso, foram estimadas tambem a producao e as taxas de excrecao de ciliados. Uma floracao de diatomaceas no periodo de fim de inverno e inicio de primavera foi o acontecimento mais importante do ciclo anual. Para avaliar a significância do tamanho do fitoplâncton em relacao ao tamanho dos ciliados, foram determinadas as fracoes de clorofila de 25 µm. A fracao 105µm3foram indicadoras dapresenca dediferentes grupos troficos e, consequentemente, da variedade das suas presas potenciais. Os ciliados pequenos dominaram em abundância e os maiores em biomassa. A abundância total de ciliados variou de 0,2 a 5,2 x 103 celulas L-1 e de 0,55 a 83,9 µg C L-1. A concentracao de clorofila a total de ate 40,4 µg L -1 foi superior as registradas em outros sistemas de estuarios em regioes temperadas. Os ciliados menores foram importantes como regeneradores de nitrogenio no verao e na primavera, quando a sua producao estimada foi mais alta. A producao potencial anual de 3 mg C L-1 representa um importante parâmetro de referencia na qual possiveis mudancas do status trofico deste ambiente podem ser previstas.

Tidal time-scale variation of inorganic nutrients and organic matter in Bahía Blanca mesotidal estuary, Argentina

Land-derived materials are regulated by coastal and shelf environmental conditions before being transported to the open sea. It is of great concern to understand the processes and to establish the extent in which they modify terrestrial compound fluxes, such as nutrients, that end up in the oceans. At present, one of the topics that arouses the highest interest within environmental coastal studies is the direction and magnitude of inorganic nutrients and the exchange fluxes of organic matter between the water column and the associated tidal plains during the daily tidal cycle. These processes, together with the local hydrographic conditions, define the key role of this type of environment: its function as a nutrient and organic matter reservoir and/or as a source. A research programme directed to understand this mechanism within mesotidal estuaries was developed in the Bahía Blanca estuary, on the coast of Buenos Aires (Argentina). On a tidal time-scale basis, levels of DIN (nitrate + nitrite + ammonium), DIP, DISi, and organic matter were measured in the estuarine water column and tidal plain porewater, for two years. Results showed no significant variations during the tidal cycle, even though the temporal variation of these compounds was clearly identified. In addition, the biological production of the estuary was considered and taken into account, so as to understand the organic matter cycle within the system. Particular conditions of the environment (sediment characteristics, porewater chemical environment, hydrodynamics, anthropogenic sources, etc.) were also considered to help fully understand the results.

Short-term variability in the phytoplankton and physico-chemical variables in a high-tidal regime, Bahía Blanca Estuary, Argentina

Corresponding autor: vguinder@criba.edu.ar Hydrological processes such as advection, resuspension, estuarine circulation, internal waves, tidal stirring, vertical mixing and stratification are the main physical forces affecting the phytoplankton distribution in estuaries (DENMAN; GARGETT, 1983; CLOERN et al., 1989; CUSHING 1989; DE JONGE; VAN BEUSEKOM, 1992). Vertical and horizontal transport of phytoplankton in coastal areas could be induced by winds, water mass intrusions from the open sea, turbulence, freshwater inflow and tides (CLOERN et al., 1989; LI; SMAYDA, 2001; BRUNET; LIZON, 2003; HAGY III et al., 2005; POPOVICH; MARCOVECCHIO, 2008; POPOVICH et al., 2008b) and these sorts of transport are related to biomass accumulation and dispersion. Many studies have revealed intratidal variations in phytoplankton dynamics (e.g. DEMERS; LEGENDRE, 1979; DEMERS et al., 1986; DUSTAN; PINCKNEY, 1989; BRUNET; LIZON, 2003) and even differences in biomass between micro and macrotidal estuaries (MONBET, 1992). Moreover, phytoplankton patchiness in estuaries has been associated with the physical influence of tides (DUSTAN; PINCKNEY, 1989). In estuaries characterized by high-tidal energy, a steady phytoplankton composition over short time periods might be expected owing to the intense mixing processes continuously occurring in the water column. However, heterogeneous distributions of phytoplankton cells in the water column on different temporal and spatial scales have been observed (e.g. CUSHING, 1989; BRUNET; LIZON, 2003). Many studies have used the biomass (estimated by the chlorophyll a concentration) as the main intrinsic property to describe the phytoplankton community dynamics during tidal cycles (DENMAN, 1977; FRECHETTE; LEGENDRE, 1982, CLOERN et al., 1989; DUSTAN; PINCKNEY, 1989; MONBET 1992), without taking into consideration the species-specific patterns related to these regular short-time events. Hence, studies based only on phytoplankton biomass variability throughout tidal cycles may overlook the short-term temporal changes in the species composition. The Bahia Blanca Estuary constitutes a mesotidal system with a semidiurnal cycle, the tides being the single most important input of energy (PERILLO; PICCOLO, 1991; PERILLO et al., 2004). The temporal and spatial dynamics of the phytoplankton in the estuary have been extensively studied (GAYOSO, 1998, 1999; POPOVICH; MARCOVECCHIO, 2008; POPOVICH et al. 2008a, 2008b) but none of these works has considered the short-term variability associated with the tidal cycle. The main goal of this study was to describe the temporal phytoplankton and physico-chemical dynamics over a short-time period at a fixed station under strong tidal influence. This study presents the first integrated description of daily phytoplankton-community dynamics together with physical and chemical variables in the Bahia Blanca Estuary. Beside its local relevance, it is also a contribution to the understanding of the short-term variability in estuaries characterized by high tidal energy. Bahia Blanca is a coastal plain estuary (38°45´S; 62°22´W) on the Atlantic coast of Argentina (Fig. 1). Tidal currents are reversible with maximum surface velocities of about 1.3 m s

In vivo measurements to estimate culture status and neutral lipid accumulation in Nannochloropsis oculata CCALA 978: implications for biodiesel oil studies

The development of effi cient, rapid and species-specifi c techniques is indispensable for assessing growth and neutral lipid yield in microalga cultures for biodiesel oil production. Nannochloropsis oculata is a small microalgae with a thick cell wall. In vivo techniques to es- timate cell density, chlorophyll a and neutral lipids are reported. A calibration curve of cell density versus optical density was obtained and validated at 540 nm, under different growth phases. Intracellular neutral lipid storage was evaluated with fl uorometry and epifl uorescent microscopy employing fl uorochrome Nile Red. The addition of 5 % dimethyl sulfoxide en- hanced 12.5 times the fl uorescence signal effi ciency. In situ fl uorescence measurements al- lowed estimating the neutral lipid content (NR-FI). Besides, no signifi cant differences were found in the lipid neutral content between gravimetric and triolein methods. The relationship between NR-FI and chlorophyll fl uorescence signals was used as a neutral lipid accumulation index, which is useful in order to establish the optimum harvesting time. Thus, these procedures may be applied for a better monitoring mode of growth and neutral lipid accumulation in N. oculatas cultures at commercial scale.

Phenological Changes of Blooming Diatoms Promoted by Compound Bottom-Up and Top-Down Controls

Understanding phytoplankton species-specific responses to multiple biotic and abiotic stressors is fundamental to assess phenological and structural shifts at the community level. Here, we present the case of Thalassiosira curviseriata, a winter-blooming diatom in the Bahía Blanca Estuary, Argentina, which displayed a noticeable decrease in the past decade along with conspicuous changes in phenology. We compiled interannual field data to assess compound effects of environmental variations and grazing by the invasive copepod Eurytemora americana. The two species displayed opposite trends over the period examined. The diatom decreased toward the last years, mainly during the winters, and remained relatively constant over the other seasons, while the copepod increased toward the last years, with an occurrence restricted to winter and early spring. A quantitative assessment by structural equation modeling unveiled that the observed long-term trend of T. curviseriata resulted from the synergistic effects of environmental changes driven by water temperature, salinity, and grazing. These results suggest that the shift in the abundance distribution of T. curviseriata toward higher annual ranges of temperature and salinity—as displayed by habitat association curves—constitutes a functional response to avoid seasonal overlapping with its predator in late winters. The observed changes in the timing and abundance of the blooming species resulted in conspicuous shifts in primary production pulses. Our results provide insights on mechanistic processes shaping the phenology and structure of phytoplankton blooms.