Morphophysiological analyses of Neochloris oleoabundans (Chlorophyta) grown mixotrophically in a carbon-rich waste product

Neochloris oleoabundans is considered one of the most promising oil-rich microalgae because of its ability to store lipids under nitrogen starvation. However, high biomass densities, required for applications on medium to large scale, are not reached in this condition of growth. As previous studies on other microalgae have shown that mixotrophy allows to obtain higher biomass in comparison to autotrophic cultures, we performed morphophysiological analyses in order to test the mixotrophic growth capability of N. oleoabundans. A carbon-rich manure derived from the apple vinegar production (AWP) was added to the medium. Cells were also cultivated under nutrient starvation (tap water), to observe the expected lipids accumulation, and combining AWP to water, to test the potential of this waste in a low-cost culture system. The results highlighted that AWP in the medium allowed to obtain the highest final cell density. Moreover, starch granules were stored inside chloroplast at the beginning of the experiment. The presence of AWP did not induce variations on light harvesting complex II (LHCII)–photosystem II (PSII) assembly, even if an interesting promotion of pigment synthesis in cells was observed. On the other hand, in starved cells, chloroplast degeneration, pigment content decrease, altered LHCII–PSII assembly and accumulation of high amount of lipid globules were observed, irrespective of the presence of AWP. The results suggest that mixotrophy promotes growth in N. oleoabundans and open up the possibility of using waste products from agri-food industries for this purpose. After growth, cells could be transferred under nutrient starvation to induce lipid accumulation.     

Growth, morphology and photosynthetic responses of Neochloris oleoabundans during cultivation in a mixotrophic brackish medium and subsequent starvation

The green microalga Neochloris oleoabundans is able to grow in both low and high salinity media and is largely studied for its capability to accumulate lipids under starvation. Moreover, N. oleoabundans is a mixotrophic alga, and then organic carbon addition can promote its growth. This research aims to study the morpho-physiological aspects, with a particular attention on the photosynthetic response, both during mixotrophic growth and starvation in brackish media, more sustainable than freshwater cultivation. In the first step, the alga was cultivated mixotrophically in a brackish medium added with an apple waste product; in the second one, cells were starved also to verify lipid induction. Results indicate that growth is highly promoted during the first week of mixotrophic cultivation, while photosynthetic pigments and lipids are over-produced during the following three weeks of cultivation. In parallel, in mixotrophic cultures the maximum PSII quantum yield was enhanced during the exponential phase of growth. Interesting changes affected the mixotrophic cultures with respect to the partitioning of absorbed light energy. Starvation of both 7-day-grown mixotrophic and autotrophic cultures caused growth inhibition, pigments and photosynthesis downshifting, and concomitantly promoted evident lipid synthesis.     

A microscale approach for simple and rapid monitoring of cell growth and lipid accumulation in Neochloris oleoabundans

Bioprocess and Biosystems Engineering - Due to the increasing environmental problems caused by the use of fossil fuels, microalgae have been spotlighted as renewable resources to produce biomass...     

Dual purpose system that treats anaerobic effluents from pig waste and produce Neochloris oleoabundans as lipid rich biomass

Dual purpose systems that treat wastewater and produce lipid rich microalgae biomass have been indicated as an option with great potential for production of biodiesel at a competitive cost. The aim of the present work was to develop a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing Neochloris oleoabundans and to evaluate its growth, lipid content and lipid profile of the harvested biomass and the removal of nutrients from the media. Cultures of N. oleoabundans were established in 4 L flat plate photobioreactors using diluted effluents from two different types of anaerobic filters, one packed with ceramic material (D1) and another one packed with volcanic gravel (D2). Maximum biomass concentration in D1 was 0.63 g L⁻¹ which was significantly higher than the one found in D2 (0.55 g L⁻¹). Cultures were very efficient at nutrient removal: 98% for NNH₄⁺ and 98% for PO₄³⁻. Regarding total lipid content, diluted eflluents from D2 promoted a biomass containing 27.4% (dry weight) and D1 a biomass containing 22.4% (dry weight). Maximum lipid productivity was also higher in D2 compared to D1 (6.27 ± 0.62 mg L⁻¹ d⁻¹ vs. 5.12 ± 0.12 mg L⁻¹ d⁻¹). Concerning the FAMEs profile in diluted effluents, the most abundant one was C18:1, followed by C18:2 and C16:0. The profile in D2 contained less C18:3 (linolenic acid) than the one in D1 (4.37% vs. 5.55%). In conclusion, this is the first report demonstrating that cultures of N. oleoabundans treating anaerobic effluents from pig waste are very efficient at nutrient removal and a biomass rich in lipids can be recovered. The maximum total lipid content and the most convenient FAMEs profile were obtained using effluents from a digester packed with volcanic gravel.     

Multiplex fluorometric assessment of nutrient limitation as a strategy for enhanced lipid enrichment and harvesting of Neochloris oleoabundans

Detailed in this study are the results of fluorometric assays used to assess the impact of gradual nutrient limitation versus punctuated nitrate limitation on the lipid content and morphology of Neochloris oleoabundans cells in batch culture. Punctuated nitrate limitation was imposed during pre‐log, log, late‐log, stationary, and senescent growth phases, and the cells were analyzed by bulk fluorescence emission, flow cytometry, and hyperspectral fluorescence imaging. In addition to intrinsic spectroscopic signatures provided by scatter and endogenous fluorescence, Nile Red staining was employed to monitor relative changes in lipid concentration. Analysis of the fluorescence images and temporal data sets was performed using multivariate curve resolution and fitting to logistic growth models to extract parameters of interest. The spectral components independently isolated from the image and temporal data sets showed close agreement with one another, especially relating to chlorophylls and Nile Red in polar and neutral lipid fractions, respectively. The fastest accumulation and highest total neutral lipid per cell and per chlorophyll were obtained with punctuated nitrate limitation during log phase growth on day 4 of culture. The presence of unbound chlorophyll in the resulting lipid bodies supports a membrane recycling TAG accumulation mechanism mediated by chloropolast–ER lipid exchange. Furthermore, an increase in cell size, indicated by forward scatter, was also found to correlate with increased neutral lipid, providing a size selection mechanism for passive harvest of algal cells at peak lipid enrichment. Biotechnol. Bioeng. 2012; 109: 2503–2512. © 2012 Wiley Periodicals, Inc.     

13C metabolic flux analysis (MFA) to find out the metabolic fluxes of biomass production and lipid accumulation in Neochloris oleoabundans UTEX 1185

Journal of Applied Phycology - Neochloris oleoabundans UTEX 1185 is a green microalga that is considered as a promising feedstock for microalgal biodiesel production as it contains high amount of...     

In situ biological CO2 fixation and wastewater nutrient removal with Neochloris oleoabundans in batch photobioreactor

Bioprocess and Biosystems Engineering - Microalgae cultivation in wastewater media in phototrophic condition is a promising approach for integrated CO2 biofixation and wastewater treatment. For...     

Biomass production and nitrogen and phosphorus removal by the green alga Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent

Biomass productivity of 350 mg DCW L⁻¹ day⁻¹ with a final biomass concentration of 3.15 g DCW L⁻¹ was obtained with Neochloris oleoabundans grown in artificial wastewater at sodium nitrate and phosphate concentrations of 140 and 47 mg L⁻¹, respectively, with undetectable levels of residual N and P in effluents. In secondary municipal wastewater effluents enriched with 70 mg N L⁻¹, the alga achieved a final biomass concentration of 2.1 g DCW L⁻¹ and a biomass productivity of 233.3 mg DCW L⁻¹ day⁻¹. While N removal was very sensitive to N:P ratio, P removal was independent of N:P ratio in the tested range. These results indicate that N. oleoabundans could potentially be employed for combined biofuel production and wastewater treatment.     

Resolving the identity of commercially cultivated Ulva (Ulvaceae,Chlorophyta) in integrated seaweed-abalone aquaculture farms in South Africa

Species of Ulva have a wide range of commercial applications and are increasingly being recognized as promising candidates for integrated aquaculture. In South Africa, Ulva has been commercially cultivated in integrated seaweed-abalone aquaculture farms since 2002, with more than 2000 tonnes of biomass cultivated per annum in land-based paddle raceways. However, the identity of the species of Ulva grown on these farms remains uncertain. We therefore characterized samples of Ulva cultivated in five integrated multi-trophic aquaculture farms (IMTA) across a wide geographical range and compared them with foliose Ulva specimens from neighboring seashores. The molecular markers employed for this study were the chloroplast-encoded Ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL), the Internal Transcribed Spacer (ITS) of the nuclear, and the chloroplast elongation factor tufA. All currently cultivated specimens of Ulva were molecularly resolved as a single species, U. lacinulata. The same species has been cultivated for over a decade, although a few specimens of two other species were also present in early South African IMTA systems. The name Ulva uncialis is adopted for the Ulva “Species A” by Fort et al. (2021), Molecular Ecology Resources, 22, 86) significantly extending the distribution range for this species. A comparison with wild Ulva on seashores close to the farms resulted in five new distribution records for South Africa (U. lacinulata, U. ohnoi, U. australis, U. stenophylloides, and U. aragoënsis), the first report of a foliose form of U. compressa in the region, and one new distribution record for Namibia (U. australis). This study reiterates the need for DNA confirmation, especially when identifying morphologically simple macroalgae with potential commercial applications.     

Mitochondrial uncoupling protein 2 (UCP2) in glucose and lipid metabolism

Nutrient availability is critical for the physiological functions of all tissues. By contrast, an excess of nutrients such as carbohydrate and fats impair health and shorten life due by stimulating chronic diseases, including diabetes, cancer and neurodegeneration. The control of circulating glucose and lipid levels involve mitochondria in both central and peripheral mechanisms of metabolism regulation. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in physiological and pathological processes related to glucose and lipid metabolism, and in this review we discuss the latest data on the relationships between UCP2 and glucose and lipid sensing from the perspective of specific hypothalamic neuronal circuits and peripheral tissue functions. The goal is to provide a framework for discussion of future therapeutic strategies for metabolism-related chronic diseases.     

Micronutrient Requirements for Growth and Hydrocarbon Production in the Oil Producing Green Alga Botryococcus braunii (Chlorophyta)

The requirements of micronutrients for biomass and hydrocarbon production in Botryococcus braunii UTEX 572 were studied using response surface methodology. The concentrations of four micronutrients (iron, manganese, molybdenum, and nickel) were manipulated to achieve the best performance of B. braunii in laboratory conditions. The responses of algal biomass and hydrocarbon to the concentration variations of the four micronutrients were estimated by a second order quadratic regression model. Genetic algorithm calculations showed that the optimal level of micronutrients for algal biomass were 0.266 μM iron, 0.707 μM manganese, 0.624 μM molybdenum and 3.38 μM nickel. The maximum hydrocarbon content could be achieved when the culture media contained 10.43 μM iron, 6.53 μM manganese, 0.012 μM molybdenum and 1.73 μM nickel. The validation through an independent test in a photobioreactor suggests that the modified media with optimised concentrations of trace elements can increase algal biomass by 34.5% and hydrocarbon by 27.4%. This study indicates that micronutrients play significant roles in regulating algal growth and hydrocarbon production, and the response surface methodology can be used to optimise the composition of culture medium in algal culture.     

Growth and photosynthetic responses of Ulva lactuca (Ulvales,Chlorophyta) germlings to different pH levels

In ocean ecosystems, fluctuations in seawater pH affect CO₂ fluxes, fundamentally influencing the metabolism of marine algae, especially during the early stages of macroalgal development. In this study, short-term exposure tests (minutes) and prolonged culture experiments (eight days) were performed at different pH levels to investigate the growth and photosynthetic responses of Ulva lactuca (Ulvales, Chlorophyta) germlings. Both acidified and alkalized seawater significantly depressed algal photosynthesis during short-term exposure tests. Prolonged culture in acidified or alkalized seawater also notably decreased photosynthesis rates and growth rates of U. lactuca germlings, but increased energy consumption and lipid peroxidation, indicating damage to the germlings. Our results suggested that both lowered and increased pH levels of seawater exert significant physiological stress on U. lactuca germlings.     

Long-term outdoor growth and lipid productivity of Tetraselmis suecica, Dunaliella tertiolecta and Chlorella sp (Chlorophyta) in bag photobioreactors

There has been considerable interest on cultivation of green microalgae (Chlorophyta) as a source of lipid that can alternatively be converted to biodiesel. The ideal microalga characteristics are that it must grow well even under high cell density and under varying outdoor environmental conditions and be able to have a high biomass productivity and contain a high oil content (~25–30 %). The main advantage of Chlorophyta is that their fatty acid profile is suitable for biodiesel conversion. Tetraselmis suecica CS-187 and Chlorella sp. were grown semi-continuously in bag photobioreactors (120 L, W?×?L?=?40?×?380 cm) over a period of 11 months in Melbourne, Victoria, Australia. Monthly biomass productivity of T. suecica CS-187 and Chlorella sp. was strongly correlated to available solar irradiance. The total dry weight productivity of T. suecica and Chlorella sp. was 110 and 140 mg L^?1 d^?1, respectively, with minimum 25 % lipid content for both strains. Both strains were able to tolerate a wide range of shear produced by mixing. Operating cultures at lower cell density resulted in increasing specific growth rates of T. suecica and Chlorella sp. but did not affect their overall biomass productivity. On the other hand, self shading sets the upper limit of operational maximum cell density. Several attempts in cultivating Dunaliella tertiolecta CS-175 under the same climatic conditions were unsuccessful.     

Zoospore ultrastructure in species ofTrebouxia andPseudotrebouxia (Chlorophyta)

Zoospore ultrastructure (incl. flagellar apparatus) has been investigated in three species ofTrebouxia (T. glomerata, T. erici, T. pyriformis) and one species ofPseudotrebouxia (P. impressa) using an absolute configuration analysis. Zoospores in all taxa studied are nearly identical in ultrastructure and exhibit a very distinctive disposition of cell organelles: cells are naked, biflagellate and considerably flattened along the plane of flagellar beat, the single contractile vacuole is located anteriorly in the ventral region of the cell, the nucleus is anteriorly to centrally located in the dorsal region of the cell. A single dictyosome is located close to the anterior, ventral edge of the nucleus. The chloroplast occupies a posterior position in the cell and usually has an anterior profile in the left region of the cell. There are two branched mitochondria per cell or a single mitochondrial reticulum with profiles anterior to the nucleus (in the dorsal region of the cell), and posterior to the nucleus. In zoospores ofTrebouxia spp. the posterior mitochondrial profile is associated with a microbody, inP. impressa zoospores the anterior mitochondrial profiles are associated with a microbody. The zoospores contain a distinctive system of three ER-cisternae: one system links to both basal bodies and extends to the nucleus, the other two systems subtend the plasmamembrane on the left and right broad cell surfaces and extend to the posterior region of the cell. The flagellar apparatus is structurally identical to that previously described for zoospores ofFriedmannia israelensis and exhibits basal body displacement by one basal body diameter into the 11/5 o'clock direction, a non-striated distal connecting fiber, a cruciate microtubular root system lacking system I fibers and presence of a single system II fiber which connects the basal bodies with the nucleus and runs parallel to one of the ER-strands. The left flagellar roots (X-roots) are subtended by a complex set of amorphous and striated material that connects each left root with both basal bodies.—This study demonstrates the close systematic relationship between the phycobiontsTrebouxia andPseudotrebouxia and the generaFriedmannia, Pleurastrum, andMicrothamnion and supports recent classification schemes which place all these taxa into a single order separate from otherChlorophyta. Dedicated to Prof. DrElisabeth Tschermak-Woess on the occasion of her 70th birthday.     

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta)

In order to broaden our understanding of the eukaryotic CO₂-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO₂-concentrating mechanism when grown under limiting CO₂ conditions. Using mass-spectrometric measurements of ¹⁸O exchange from doubly labelled CO₂, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex. Received: 13 May 2000 / Accepted: 16 August 2000     

Growth and neutral lipid synthesis in green microalgae: A mathematical model

Many green microalgae significantly increased their cellular neutral lipid content when cultured in nitrogen limited or high light conditions. Due to their lipid production potential, these algae have been suggested as promising feedstocks for biofuel production. However, no models for algal lipid synthesis with respect to nutrient and light have been developed to predict lipid production and to help improve the production process. A mathematical model is derived describing the growth dynamics and neutral lipid production of green microalgae grown in batch cultures. The model assumed that as the nitrogen was depleted, photosynthesis became uncoupled from growth, resulting in the synthesis and accumulation of neutral lipids. Simulation results were compared with experimental data for the green microalgae Pseudochlorococcum sp. For growth media with low nitrogen concentration, the model agreed closely with the data; however, with high nitrogen concentration the model overestimated the biomass. It is likely that additional limiting factors besides nitrogen could be responsible for this discrepancy.