Media engineering in marine diatom Phaeodactylum tricornutum employing cost-effective substrates for sustainable production of high-value renewables.

Phaeodactylum tricornutum is a marine diatom, rich in omega-3 polyunsaturated fatty acids especially eicosapentaenoic acid (EPA) and brown pigment, that is, fucoxanthin. These high-value renewables (HVRs) have a high commercial and nutritional relevance. In this study, our focus was to enhance the productivities of such renewables by employing media engineering strategy via., photoautotrophic (P1, P2, P3) and mixotrophic (M1, M2, M3, M4) modes of cultivation with varying substrate combinations of carbon (glycerol: 0.1 m) and nitrogen (urea: 441 mm and/or sodium nitrate: 882 mm). Our results demonstrate that mixotrophic [M4] condition supplemented with glycerol (0.1 m) and urea (441 mm) feed enhanced productivities (mg L-1  day-1 ) as follows: biomass (770.0), total proteins (36.0), total lipids (22.0), total carbohydrates (23.0) with fatty acid methyl esters (9.6), EPA (2.7), and fucoxanthin (1.1), respectively. The overall yield of EPA represents 28% of total fatty acids in the mixotrophic [M4] condition. In conclusion, our improved strategy of feeding urea to a glycerol-supplemented medium defines a new efficient biomass valorization paradigm with cost-effective substrates for the production of HVRs in oleaginous diatoms P. tricornutum.

Assessment on the oil accumulation by knockdown of triacylglycerol lipase in the oleaginous diatom Fistulifera solaris.

Microalgae are promising producers of biofuel due to higher accumulation of triacylglycerol (TAG). However, further improvement of the lipid metabolism is critical for feasible application of microalgae in industrial production of biofuel. Suppression of lipid degradation pathways is a promising way to remarkably increase the lipid production in model diatoms. In this study, we established an antisense-based knockdown (KD) technique in the marine oleaginous diatom, Fistulifera solaris. This species has a capability to accumulate high content of lipids. Tgl1 KD showed positive impact on cell growth and lipid accumulation in conventional culture in f/2 medium, resulting in higher oil contents compared to wild type strain. However, these impacts of Tgl1 KD were slight when the cells were subjected to the two-stage growth system. The Tgl1 KD resulted in slight change of fatty acid composition; increasing in C14:0, C16:0 and C16:1, and decreasing in C20:5. This study indicates that, although Tgl1 played a certain role in lipid degradation in F. solaris, suppression of only a single type of TAG lipase was not significantly effective to improve the lipid production. Comprehensive understanding of the lipid catabolism in this microalga is essential to further improve the lipid production.

Multifunctionalizing the marine diatom Phaeodactylum tricornutum for sustainable co-production of omega-3 long chain polyunsaturated fatty acids and recombinant phytase.

There is an urgent requirement for sustainable sources of food and feed due to world population growth. Aquaculture relies heavily on the fish meal and fish oils derived from capture fisheries, challenging sustainability of the production system. Furthermore, substitution of fish oil with vegetable oil and fish meal with plant seed meals in aquaculture feeds reduces the levels of valuable omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, and lowers the nutritional value due to the presence of phytate. Addition of exogenous phytase to fish feed is beneficial for enhancing animal health and reducing phosphorus pollution. We have engineered the marine diatom Phaeodactylum tricornutum, accumulating high levels of EPA and DHA together with recombinant proteins: the fungal Aspergillus niger PhyA or the bacterial Escherichia coli AppA phytases. The removal of the N-terminal signal peptide further increased phytase activity. Strains engineered with fcpA and CIP1 promoters showed the highest level of phytase activity. The best engineered strain achieved up to 40,000 phytase activity units (FTU) per gram of soluble protein, thus demonstrating the feasibility of development of multifunctionalized microalgae to simultaneously produce industrially useful proteins and fatty acids to meet the demand of intensive fish farming activity.

Risk assessment of excessive CO2 emission on diatom heavy metal consumption.

Diatoms are the dominant group of phytoplankton in the modern ocean, accounting for approximately 40% of oceanic primary productivity and critical foundation of coastal food web. Rising dissolution of anthropogenic CO2 in seawater may directly/indirectly cause ocean acidification and desalination. However, little is known about dietary diatom-associated changes, especially for diatom heavy metal consumption sensitivity to these processes, which is important for seafood safety and nutrition assessment. Here we show some links between ocean acidification/desalination and heavy metal consumption by Thalassiosira weissflogii. Excitingly, under desalination stress, the relationships between Cu, Zn, and Cd were all positively correlated, especially between Cu and Zn (r=0.989, total intracellular concentration) and between Zn and Cd (r=0.962, single-cell intracellular concentration). Heavy metal consumption activity in decreasing order was acidification

The nitrogen costs of photosynthesis in a diatom under current and future pCO2.

With each cellular generation, oxygenic photoautotrophs must accumulate abundant protein complexes that mediate light capture, photosynthetic electron transport and carbon fixation. In addition to this net synthesis, oxygenic photoautotrophs must counter the light-dependent photoinactivation of Photosystem II (PSII), using metabolically expensive proteolysis, disassembly, resynthesis and re-assembly of protein subunits. We used growth rates, elemental analyses and protein quantitations to estimate the nitrogen (N) metabolism costs to both accumulate the photosynthetic system and to maintain PSII function in the diatom Thalassiosira pseudonana, growing at two pCO2 levels across a range of light levels. The photosynthetic system contains c. 15-25% of total cellular N. Under low growth light, N (re)cycling through PSII repair is only c. 1% of the cellular N assimilation rate. As growth light increases to inhibitory levels, N metabolite cycling through PSII repair increases to c. 14% of the cellular N assimilation rate. Cells growing under the assumed future 750 ppmv pCO2 show higher growth rates under optimal light, coinciding with a lowered N metabolic cost to maintain photosynthesis, but then suffer greater photoinhibition of growth under excess light, coincident with rising costs to maintain photosynthesis. We predict this quantitative trait response to light will vary across taxa.

Assessment of growth rate, chlorophyll a fluorescence, lipid peroxidation and antioxidant enzyme activity in Aphanizomenon flos-aquae, Pediastrum simplex and Synedra acus exposed to cadmium.

In this study, the effects of cadmium on the cyanobacterium Aphanizomenon flos-aquae, the green alga Pediastrum simplex and the diatom Synedra acus was evaluated on the basis of growth rate, chlorophyll a fluorescence, lipid peroxidation and antioxidant enzyme activity. The EC50 values (effective concentration inducing 50 % of growth inhibition) of cadmium in A. flos-aquae, P. simplex and S. acus were 1.18 ± 0.044, 4.32 ± 0.068 and 3.7 ± 0.055 mg/L, respectively. The results suggested that cadmium stress decreases growth rate and chlorophyll a concentration. The normalized chlorophyll a fluorescence transients significantly increased at cadmium concentrations of 5.0, 10.0 and 20.0 mg/L, but slightly decreased at concentrations of 0.2, 0.5 and 1.0 mg/L. The chlorophyll fluorescence parameters showed considerable variation among the three species, while lipid peroxidation and antioxidant enzyme activities showed a significant increase. Our results demonstrated that blockage of electron transport on the acceptor side of photosystem II is the mechanism responsible for cadmium toxicity in freshwater microalgae, and that the tolerance of the three species to cadmium was in the order green alga P. simplex > diatom S. acus > cyanobacterium A. flos-aquae.

Sources and resources: importance of nutrients, resource allocation, and ecology in microalgal cultivation for lipid accumulation.

Regardless of current market conditions and availability of conventional petroleum sources, alternatives are needed to circumvent future economic and environmental impacts from continued exploration and harvesting of conventional hydrocarbons. Diatoms and green algae (microalgae) are eukaryotic photoautotrophs that can utilize inorganic carbon (e.g., CO2) as a carbon source and sunlight as an energy source, and many microalgae can store carbon and energy in the form of neutral lipids. In addition to accumulating useful precursors for biofuels and chemical feed stocks, the use of autotrophic microorganisms can further contribute to reduced CO2 emissions through utilization of atmospheric CO2. Because of the inherent connection between carbon, nitrogen, and phosphorus in biological systems, macronutrient deprivation has been proven to significantly enhance lipid accumulation in different diatom and algae species. However, much work is needed to understand the link between carbon, nitrogen, and phosphorus in controlling resource allocation at different levels of biological resolution (cellular versus ecological). An improved understanding of the relationship between the effects of N, P, and micronutrient availability on carbon resource allocation (cell growth versus lipid storage) in microalgae is needed in conjunction with life cycle analysis. This mini-review will briefly discuss the current literature on the use of nutrient deprivation and other conditions to control and optimize microalgal growth in the context of cell and lipid accumulation for scale-up processes.

Risk assessment of nitrate and petroleum-derived hydrocarbon addition on Contricriba weissflogii biomass, lifetime, and nutritional value.

Coastal diatoms are often exposed to both petroleum-derived hydrocarbon pollution and eutrophication. How these exposures influence on algal biomass, lifetime, and nutritional value are unknown. To examine a more accurate risk assessment of the pollutants on the role of diatoms in coastal ecosystem functions, Conticribra weissflogii was maintained at different concentrations of nitrate (N) and/or water-soluble fractions of No.0 diesel oil (WSF). Algal density, cell growth cycle, protein, chlorophyll a, superoxide dismutase (SOD) activity, and malonaldehyde (MDA) were determined for the assessment of algal biomass, lifetime, nutritional value, photosynthesis and respiration, antioxidant capacity, and lipid peroxidation, respectively.When N addition was combined with WSF pollution, the cell growth cycles were shortened by 27-44%; SOD activities were decreased by 1-64%; algal density, the concentrations of chlorophyll a, protein, and MDA were varied between 38 and 310%, 62 and 712%, 4 and 124%, and 19 and 233% of the values observed in N addition experiments, respectively. Coastal ecosystem functions were severely weakened by N and WSF additions, and the influence was increased in the order: N

Rapid, colorimetric quantification of lipid from algal cultures.

Algae have significant potential as a source of biomass for the production of biofuels, due to their high growth rates and high cellular lipid content. Studies that address the use of algae as biofuels often require the frequent measurement of algal lipid content. Traditional methods for the quantification of lipid are, however, costly if sub-contracted, or involve the use of expensive analytical equipment that is not available in many labs. This study describes a simple, colorimetric method for the quantification of algal lipid from small amounts of culture. The technique is derived from a method for the quantification of fatty acids dissolved in chloroform. Algal lipids are saponified to fatty acids and then mixed with a copper reagent. Chloroform-extractable copper soaps of long-chain fatty acids are then colorimetrically measured by the addition of diethyldithiocarbamate to develop a yellow colored product. Linear responses for fatty acids in the range of C10:0 to C16:0 were observed for a concentration range between 0.025 and 1 micromol of fatty acid per 200 microL of sample. Fatty acids with chain lengths of less than twelve carbons produced significantly reduced signal. Decenoic acid yielded a slightly, but significantly lower signal than decanoic acid indicating that the assay underestimates the presence of unsaturated fatty acids. Lipid contents of Phaeodactylum tricornutum and Chlorella vulgaris CM2 were monitored for eight days during exponential growth to demonstrate the feasibility of the technique as a monitoring methodology. Overall, the method allowed reliable detection and quantification of fatty acid content from 1 to 2 mL of algal culture. Adaptation of the technique to micro-centrifuge format allows assaying 30 samples in less than 2h. Considering reagents and time, the total cost per assay was estimated at less than $5, representing a significant cost savings over traditional lipid quantification procedures.

Sediment integrative assessment of the Bay of Cádiz (Spain): an ecotoxicological and chemical approach.

This study consisted of the sediment toxicity assessment of the Bay of Cádiz based on two endpoints: growth inhibition for Cylindrotheca closterium (benthic microalgae) and fecundity inhibition for Tisbe battagliai (harpacticoid copepod). A new methodology to eliminate (but not as storage technique) the autochthonous biota present in the sediment samples by immersing them in liquid nitrogen (-196 degrees C) was also assessed. Sediment toxicity data showed different toxicity levels for both organisms. In general, T. battagliai was more sensitive; however a good correlation (r=0.75; p<0.05) between sediment toxicity results for both species was found. Data in pore water (pH, redox potential, and toxicity for microalgae and copepod) and sediment (pH, redox potential, organic carbon, and metal concentrations) demonstrated that ultra-freezing did not alter sample characteristics; thus, this technique can be adopted as a pre-treatment in whole-sediment toxicity tests in order to avoid misleading results due to presence of autochthonous biota. Multivariate statistical analysis such as cluster and principal component analysis using chemical and ecotoxicological data were employed. Silt and organic matter percentage and lead concentration were found to be the factors that explain about 77% of sediment toxicity in the Bay of Cádiz. Assay methodology determined in this study for both assayed species is considered adequate to be used in sediment toxicity monitoring programs. Results obtained using both species show that the Bay of Cádiz can be considered a moderately polluted zone.

A phytochelatin-based bioassay in marine diatoms useful for the assessment of bioavailability of heavy metals released by polluted sediments.

The aim of the present work was to develop a new bioassay involving the presence of phytochelatins (PCs), detoxifying intracellular metal-binding peptides, in microalgae as response to metal bioavailability in re-suspensions of metal-polluted marine sediments. For this purpose, the synthesis of PCs has been studied in laboratory cultures of three marine diatoms, namely Phaeodactylum tricornutum, Thalassiosira weissflogii and Skeletonema costatum, exposed to elutriates of sediments collected in a polluted coastal area in the province of Pisa (Tuscany, Italy). Short- and long-term incubations in the elutriates of two marine sediments (named A and B) exhibited an increase of PCs synthesis in all the phytoplanktonic species examined, when the elutriate concentration increased from 0% to 100%. Elutriate B, which was mainly contaminated by Cd, was shown to be more effective to induce PCs than elutriate A, which was richer in Cu and TOC. The results show that the PCs response, in the microalgae examined, was species-specific. Our data also show that the PCs synthesis occurred before the growth rate was affected, thereby indicating that PCs can be considered as an early warning response of metal exposure. The PCs response in exponentially growing cultures of T. weissflogii, which was found to be the most sensitive alga, increased when the initial cellular density decreased. Finally, the positive relationship obtained between the cellular PCs concentration in T. weissflogii and the degree of metal contamination of the elutriates from twelve sediments collected in a metal-polluted coastal area, confirmed that the PCs-induction test can be applied in field studies. In conclusion, the response of these marine microalgae suggests that these cellular peptides could represent an excellent biomarker of metal exposure, which is useful for the assessment of sediment toxicity, by carrying out PCs-induction tests on sediment elutriates.

A complete energy balance from photons to new biomass reveals a light- and nutrient-dependent variability in the metabolic costs of carbon assimilation.

The energy balance of Phaeodactylum tricornutum cells from photon to biomass have been analysed under nutrient-replete and N-limiting conditions in combination with fluctuating (FL) and non-fluctuating (SL) dynamic light. For this purpose, the amount of photons absorbed has been related to electrons transported by photosystem II, to gas exchange rates, and to the newly formed biomass differentially resolved into carbohydrates, proteins, and lipids measured by means of Fourier transform infrared (FTIR) spectroscopy. Under high nutrient conditions, the quantum efficiency of carbon-related biomass production (Phi(C)) and the metabolic costs of carbon (C) production were found to be strongly controlled by the light climate. Under N-limited conditions, the light climate was less important for the efficieny of primary production. Thus, the largest range of Phi(C) dependent on the nutrient status of the cells was observed under non-fluctuating light conditions which are comparable with stratified conditions in the natural environment. It is evident that N limitation induced pronounced changes in the composition of macromolecular compounds and, thus, influenced the degree of reduction of the biomass as well as the metabolic costs of C production. However, Phi(C) and the metabolic costs are not predictable from the photosynthesis rates. In consequence, the results clearly show that bio-optical methods as well as gas exchange measurements during the light phase can severely mismatch the true energy storage in the biomass especially under high nutrient in combination with non-fluctuating light conditions.

Single cell level microalgal ecotoxicity assessment by confocal microscopy and digital image analysis.

In ecotoxicological studies involving environmental contaminants, rapid and multi-parametric optical detection based methods have definite advantages over traditional growth inhibition assays. In this context, a confocal laser scanning microscopy (CLSM) based method to assess ecotoxicity arising out of biocide insult to marine microalgae is reported. Using this technique, the effect of in-use concentrations of chlorine (an oxidizing biocide) on a marine diatom (Cocconeis scutellum Ehrenb) was determined based on inhibition of chlorophyll autofluorescence and esterase activity (probed by fluorescein diacetate (FDA) staining). Determination of mean fluorescence intensity (MFI) per cell by collecting auto-fluorescence from single cells in x, y and z dimensions permitted reproducible toxicity evaluation at single-cell level. Chlorine-induced inhibition of autofluorescence in laboratory cultures was dose-dependent. Additional data on metabolic activity of the diatom cells following chlorine exposure was collected by FDA staining. Our results demonstrate that chlorine, an antifouling biocide commonly used in cooling water systems, causes significant reduction in chlorophyll autofluorescence and esterase activity in diatoms in short-term exposure experiments. Tests employing multiple organisms and multiple toxicity endpoints are superior to standard algal growth inhibition assays for they provide a better understanding of algal-algal interactions and real impact in the environment. The combined autofluorescence-FDAtechnique described here is rapid and has clear advantages in terms of using environmentally relevant toxicant and cell concentrations. Additional microalgal species and toxicity end points can be employed in order to develop multi-species and multiparameter bioassay using confocal microscopy.

Enhanced productivity of Chaetoceros calcitrans in airlift photobioreactors.

The various modes of cultivation of Chaetoceros calcitrans in airlift photobioreactors (ALPBRs) were examined. The batch system illustrated that the airlift configuration was superior to the bubble column as the airlift supported the circulation of the cell within the system, leading to a better light utilization. The cultivations in both semi-continuous and continuous systems resulted in a high cell productivity, although the steady state cell concentrations in both systems were lower than that obtained from the batch system. The behavior of the large-scale airlift system was not significantly different from the conventional bubble column where the diatom could only be produced at low cell density. Despite this, among all of the systems investigated in this work, the large-scale system gave the highest productivity. The main limiting factor for the large-scale airlift culture was the availability of light. Based on economical analysis, the continuous cultivation in the 2.8L ALPBR with a medium feed rate of 3 mL min(-1) was most attractive where the operation cost could be maintained at a minimum of approx. 7.95 x 10(-4)THBL(-1)h(-1). However, this continuous small-scale system still suffered from relatively low cell productivity (8.10 x 10(4)cellss(-1)).

Assessment of the production of 13C labeled compounds from phototrophic microalgae at laboratory scale.

An integrated process for the indoor production of 13C labeled polyunsaturated fatty acids (PUFAs) from Phaeodactylum tricornutum is presented. The core of the process is a bubble column photobioreactor operating with recirculation of the exhaust gas using a low-pressure compressor. Oxygen accumulation in the system is avoided by bubbling the exhaust gas from the reactor in a sodium sulfite solution before returning to it. To achieve a high 13C enrichment in the biomass obtained, the culture medium is initially stripped of carbon, and labeled 13CO(2) is automatically injected on-demand during operation for pH control and carbon supply. The reactor was operated in both batch and semicontinuous modes. In semicontinuous mode, the reactor was operated at a dilution rate of 0.01 h(-1), resulting in a biomass productivity of 0.1 g l(-1) per day. The elemental analysis of the inlet and outlet flows of the reactor showed that 64.9% of carbon was turned into microalgal biomass, 34.9% remained in the supernatant mainly as inorganic compounds. Only 3.8% of injected carbon was effectively fixed as the target labeled product (EPA). Regarding the isotopic composition of fatty acids, results showed that fatty acids were not labeled in the same proportion, the higher the number of carbons the lower the percentage of 13C. Isotopic composition of EPA ranged from 36.5 to 53.5%, as a function of the methodology used (GC-MS, EA-IRMS or gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS)). The low carbon uptake efficiency combined with the high cost of 13CO(2) make necessary to redefine the designed culture system to increase the efficiency of the conversion of 13CO(2) into the target product. Therefore, the possibility of removing 12C from the fresh medium, and recovering and recirculating the inorganic carbon in the supernatant and the organic carbon from the EPA depleted biomass was studied. The inorganic carbon of the fresh medium was removed by acidification and stripping with N(2). The inorganic carbon of the supernatant was recovered also by acidification and subsequent stripping with N(2). The operating conditions of this step were optimized for gas flow rate and type of contactor. A carbon recovery step for the depleted biomass was designed based on the catalytic oxidation to CO(2) using CuO (10 wt.%) as catalyst with an oxygen enriched atmosphere (80% O(2) partial pressure). In this way, the carbon losses reduced an 80.2% and the efficiency of the conversion of carbon in EPA was increased to 19.5%, which is close to the theoretical maximum. Further increase in 13CO(2) use efficiency is only possible by additionally recovering other labeled by-products present in the biomass: proteins, carbohydrates, lipids, and pigments.

Determination of cadmium partitioning in microalgae and oysters: contribution to the assessment of trophic transfer.

Alternative methodologies have been applied to the study of cadmium transfer in a food chain: water, microalgae (Skeletonema costatum and Tetraselmis suecica), oysters (Crassostrea gigas). The potential bioavailability of Cd in organisms was assessed through partitioning at the cell or tissue levels, and the predictive value of this method was evaluated by determining directly the metal transfer in an experimental food chain model. Cd concentrations were lower in S. costatum than T. suecica, in controls as well as in contaminated algae. In both algal species, Cd was firmly bound to the cell wall or had entered the cell. Cytosolic Cd was bound to intracellular ligands, the biochemical characteristics of which were not consistent with the hypothesis of detoxification via phytochelatins. In both algal species, Cd was predominantly present in the insoluble fraction, but at pHs such as those existing in the digestive tract of bivalves, it was easily extracted from the cells. Thus, exposure to Cd through phytoplanktonic food induced a significant uptake of this metal in soft tissues of bivalves. Due to the difference in Cd accumulation in algae, Cd doses associated with S. costatum were lower than those bound to T. suecica. Moreover, oysters retained a lower percentage of the metal associated with S. costatum compared to T. suecica (9 and 20%, respectively, after 21 days of exposure). Cd doses potentially available to oysters exposed directly in sea water were considerably higher, and direct uptake induced the highest levels of Cd incorporation but only 2% of dissolved Cd was actually retained by oysters over 21 days of exposure. In the soft tissues of oysters, Cd was distributed equally between soluble and insoluble fractions. Cytosolic Cd was present predominantly in the heat-stable fraction and mainly bound to compounds of molecular weight equal to 13.5 kDa. Moreover, a positive correlation was observed between metallothionein-like protein (MTLP) levels and gross concentrations of Cd in the soft tissues of oysters. These data are consistent with the hypothesis of an important role of metallothioneins in Cd metabolism in oysters and suggest a potential availability of MT-bound fraction of Cd to the consumers. These data are in agreement with the response of oysters exposed to Cd in the field.

Domoic acid-producing diatom blooms in Monterey Bay, California: 1991-1993.

During the autumn of 1991, numerous seabird fatalities in Monterey Bay, California, led to the discovery of a new domoic acid-producing diatom, Pseudonitzschia australis. Since this initial event, sizable populations of P. australis, as well as other likely toxin producers, P. pungens f. multiseries and P. pseudodelicatissima, have occurred biannually in Monterey Bay. Using the highly sensitive FMOC-HPLC method, we detected domoic acid whenever Pseudonitzschia australis was found in the plankton, even at densities as low as 4.0 x 10(3) cells/L. Based on correlations of domoic acid and P. australis abundances and the overwhelming biovolume dominance of P. australis, we conclude that P. australis has been the major domoic acid producer during the period of our study. Our study suggests that P. australis cells may always be toxic in natural populations and that toxin concentrations on a per cell basis have no statistically significant relationship to population density or to nutrient concentrations other than silicate. Cellular levels of domoic acid were positively correlated with silicate concentrations, which is at variance with reports from prior culture experiments. These conclusions must be tentative because of the limited extent of our sampling. Nevertheless, these preliminary data indicate that further investigations of environmental conditions affiliated with cell growth and toxin production in P. australis are warranted. As a practical matter, domoic acid in the pelagic environment cannot be reliably or consistently detected by monitoring domoic acid levels in intertidal mussels. Direct measurement of domoic acid using sensitive HPLC methods is probably the most cost-effective and accurate approach for an ongoing phycotoxin monitoring program.