Investigation of lipid production by nitrogen-starved <Emphasis Type="Italic">Parachlorella kessleri</Emphasis> under continuous illumination and day/night cycles for biodiesel application

This study aims to investigate the triacylglycerol (TAG) productivity of Parachlorella kessleri grown under continuous illumination and to investigate its metabolism in simulated day/night cycles in order to estimate the feasibility of a large-scale production in outdoor solar photobioreactors. The strain was chosen for its ability to accumulate large amounts of triacylglycerol during nitrogen starvation. Several protocols of nitrogen starvation were tested in continuous illumination as well as in simulated day/night cycles. Sudden and progressive nitrogen starvation conditions have enhanced the TAG concentration and productivity of P. kessleri reaching up to 48 dry wt% and 4.4 × 10^?3 kg m^?2 day^?1, respectively. Microalgal cell metabolism was significantly affected by the day/night illumination cycles. The energy-rich compounds (TAGs and carbohydrates) were accumulated by P. kessleri during the photoperiods and partly consumed during the dark to sustain the microalgae vitality. This TAG oxidation ultimately led to a 26% decrease in TAG productivity in cultures exposed to day/night cycles compared to ones exposed to continuous illumination of equal 24-h average photon flux density. The results can dictate the optimal time for harvesting cells for recovering the largest amount of TAGs.     

Comparative feeding ecology of invasive Ponto-Caspian gobies

Invasions of Ponto-Caspian gobiid fishes are suspected to cause regime shifts in freshwater ecosystems. This study compared the trophic niche differentiations of Neogobius melanostomus and Ponticola kessleri in the upper Danube River using stable isotope analyses (δ¹³C and δ¹⁵N), gut content analyses and morphometric analyses of the digestive tract. Both species were identified as predacious omnivores with high dietary overlap and a generalistic feeding strategy. Amphipods (especially invasive Dikerogammarus spp.) contributed 2/3 to the index of food importance. δ¹⁵N-signatures of N. melanostomus revealed an ontogenetic diet shift and significantly exceeded those in P. kessleri by ~1.5‰, indicating a niche separation of half a trophic level. P. kessleri had shorter uncoiled intestinal tracts than N. melanostomus, indicating a narrower niche and adaptation to animal food. Trophic niches in both species expanded during the growth period with increasing intraguild predation and cannibalism in P. kessleri and increasing molluscivory in N. melanostomus. P. kessleri showed a higher degree of specialization and more stable feeding patterns across seasons, whereas N. melanostomus adapted its diet according to the natural prey availability. The feeding patterns of both species observed in the upper Danube River strongly differ from those in their native ranges, underlining their great plasticity. Both goby species consumed mainly other non-native species (~92% of gut contents) and seemed to benefit from previous invasions of prey species like Dikerogammarus villosus. The invasive success of gobies and their prey mirror fundamental ecological changes in large European freshwater ecosystems.     

Trehalose phosphate synthase overexpression in Parachlorella kessleri improves growth and photosynthetic performance under high light conditions

Parachlorella kessleri is a promising oil-bearing marine alga which shows decreased growth under high light stress. Osmolytes are known to relieve stress by protecting the cell membrane, proteins, and enzymes. Enhanced production of osmolyte (trehalose) was thus used to relieve stress in P. kessleri by overexpression of trehalose phosphate synthase (TPS) gene. Transformed P. kessleri was grown under different light regimes to study the effect of trehalose overproduction on growth. Study of one of the TPS transformants showed increased trehalose as well as increased biomass and decreased pigments, reactive oxygen species, and lipid peroxidation of cell membrane. The improved photosynthetic performance of the transformant was also signified by pulse-amplitude-modulated fluorometric analysis. All of these factors reveal improved stress tolerance under high light conditions by increased trehalose accumulation due to TPS overexpression in P. kessleri.     

Detection of intracellular neutral lipid content in the marine microalgae Prorocentrum micans and Phaeodactylum tricornutum using Nile red and BODIPY 505/515

Diatoms and dinoflagellates not only have extensive distribution and a huge biomass in marine ecosystems, but also have high lipid accumulation in nature or after physiological and genetic modification, which indicates that these organisms may be optimal candidate algal strains for biodiesel production. In this study, we determined the content of intracellular neutral lipids (triacylglycerol [TAG]) in the dinoflagellate Prorocentrum micans and in the diatom Phaeodactylum tricornutum using NR and BODIPY 505/515 staining. The freshwater green alga Scenedesmus obliquus was used as a control. Optimum concentrations of 1.000 and 1.500 μg mL^?1 were determined for neutral lipid Nile red (NR) staining in P. micans and P. tricornutum. Unlike NR staining, the optimal concentrations of BODIPY 505/515 staining in P. micans and P. tricornutum were lower, at 0.100 and 0.075 μg mL^?1, respectively. High correlation coefficients of R ²?=?0.990 and R ²?=?0.989 were obtained for P. micans and P. tricornutum intracellular neutral lipid content and the relative fluorescence intensity with NR staining, while the reference alga, S. obliquus, had a relatively low correlation coefficient of R ²?=?0.908 when stained with NR. The neutral lipid content determined by thin-layer chromatography-flame ionization detector matched the analytical data from fluorescence measurements. These results indicated that NR and BODIPY 505/515 staining can be used as an excellent high-throughput approach to screen marine diatoms and dinoflagellates.     

Kinetic exploration of nitrate-accumulating microalgae for nutrient recovery

Within sustainable resource management, the recovery of nitrogen and phosphorus nutrients from waste streams is becoming increasingly important. Although the use of microalgae has been described extensively in environmental biotechnology, the potential of nitrate-accumulating microalgae for nutrient recovery has not been investigated yet. The ability of these marine microorganisms to concentrate environmental nitrate within their biomass is remarkable. The aim of this study was to investigate the application potential of nitrate-accumulating diatoms for nutrient recovery from marine wastewaters. The intracellular nitrate storage capacity was quantified for six marine diatom strains in synthetic wastewater. Amphora coffeaeformis and Phaeodactylum tricornutum stored the highest amount of nitrate with respectively 3.15 and 2.10 g N L^?1 of cell volume, which accounted for 17.3 and 4.6 %, respectively, of the total nitrogen content. The growth and nitrate and phosphate uptake of both diatoms were further analyzed and based on these features P. tricornutum showed the highest potential for nutrient recovery. A mathematical model was developed which included intracellular nitrate storage and the kinetic parameters were derived for P. tricornutum. Furthermore, a simulation study was performed to compare the performance of a proposed microalgal nutrient recovery unit with a conventional denitrification system for marine wastewater treatment. Overall, this study demonstrates the potential application of P. tricornutum for saline wastewater treatment with concurrent nitrogen and phosphorus recycling.     

Evaluating new isolates of microalgae from Kazakhstan for biodiesel production

New microalgal strains that are native to South-East Kazakhstan were isolated and characterized with a view to identifying suitable candidates for biodiesel production. Six strains of chlorophyte algae (named K1–K6) were recovered from environmental samples as axenic cultures, and molecular analysis revealed that five (K1–K5) are strains of Parachlorella kessleri, whereas K6 is a strain of Chlorella vulgaris. A third isolate from Uzbekistan (termed UZ) was also identified as a separate strain of P. kessleri. All strains show high growth rates and an ability to utilize acetate as an exogenous source of fixed carbon. Furthermore, under conditions of nitrogen depletion, all three strains showed a significant accumulation of neutral lipids (triacylglycerides). P. kessleri K5 and C. vulgaris K6 therefore represent promising autochthon strains for large-scale cultivation and biodiesel production in Kazakhstan.     

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K _m and V _max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.     

Physicochemical and structural characterisation of marine algae Kappaphycus alvarezii and the ability of its dietary fibres to bind mutagenic amines

The physicochemical and structural characteristics of the bioactive components of the marine algae, Kappaphycus alvarezii, were determined, and the ability of its dietary fibres to bind mutagens is reported. Swelling capacity (15.0 mL g^?1) and water retention capacity (10.4 g g^?1) were observed to be high and were influenced by dietary fibre fraction. Presence of sulphur is a unique characteristic of the marine dietary fibre. Insoluble fraction contained galactose (29.9 %) and glucose (21.1 %), and high Klason lignin (10.6 g (100 g)^?1); whereas, galactose formed the single monomeric unit in the soluble fractions. Binding of mutagenic amines, benzo alpha pyrene (BαP) and 3-amino-1-methyl-pyrido(4,3-b)indole (Trp-P-2) to insoluble fraction were comparatively high, while soluble fraction showed greater binding to BαP and 2-amino-9H-pyrido(2,3-b)indole (AαC). The hydrophilic-lipophilic partition coefficient (Log P) and the polar surface area of the amines were positively and negatively correlated to the dietary fibre components, respectively. Klason lignin content significantly affected the binding capacity. A statistically significant multi-linear model was developed relating these properties to the binding capacity. Chemical analysis also indicated high amounts of amino acids, tyrosine and arginine (2.6 and 1.5 g (100 g)^?1, respectively), and polyunsaturated fatty acids, eicosapentaenoic and docosahexaenoic acid (13.6 %). The current study indicates the uniqueness of the marine algae as valuable food and dietary supplement.     

Purification and characterization of a laccase from Coprinopsis cinerea in Pichia pastoris

A modified laccase gene, CcLCC6, from Coprinopsis cinerea was chemically synthesized according to the yeast codon bias and expressed in Pichia pastoris. The main properties of laccase, effects of ions and inhibitors, and optimal condition for decolouring malachite green (MG) were investigated in this study. The optimal pH level and temperature of laccase are 3.0 and 40 °C, respectively. The metal ions Mn²⁺, Zn²⁺, Fe³⁺ and Al³⁺ could inhibit laccase activity, as well as 1 mM of sodium dodecyl sulphate and sodium thiosulphate. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as a mediator, was necessary in decolorizing MG. The optimal pH and temperature for MG decolorization were 3.0 and 50 °C, respectively. Approximately 0.02 μM recombinant laccase could effectively decolour 0.05 mM of MG in 1 h. CcLCC6I could inhibit the toxicity of MG to P. pastoris. This is the first report on the successful expression in P. pastoris of CcLCC6I and its enzymatic property. Laccase can also be considered as a candidate for treating industrial effluent containing MG.     

Phenotypic Switching in Biofilm-Forming Marine Bacterium Paenibacillus lautus NE3B01

Biofilm-forming marine bacterium Paenibacillus lautus NE3B01 was isolated from a mangrove ecosystem, Odisha, India. This isolate formed a swarming type of colony pattern on the solid culture medium with 0.5–2 % agar. Phase contrast microscopy study of a growing colony of P. lautus on solid media and swarming pattern revealed the existence of two phenotypically distinct cells (i.e. cocci and rods) across the colonies. However, in actively growing planktonic culture, only rod-shaped cells were observed. Biofilm growth studies (crystal violet assay) with the isolate showed significant biofilm formation by 6 h, and the detachment phase was observed after 18 h. Biofilm parameters (such as total biomass, roughness coefficient, biofilm thickness, etc.) of 24-h-old P. lautus biofilm were studied by confocal scanning laser microscopy (CSLM). The CSLM study showed that P. lautus formed a biofilm with an average thickness of 14.8 ± 2.6 μm, a high roughness coefficient (0.379 ± 0.103) and surface to bio-volume ratio (4.59 ± 1.12 μm²/μm³), indicating a highly uneven topography of the biofilm. This also indicates that the 24-h-old biofilm is in dispersal phase. Scanning electron microphotographs of P. lautus also supported the existence of two distinct phenotypes of P. lautus. The current findings suggest that P. lautus has two vegetative phenotypes and to decongest the overcrowded biofilm the bacterium can switch over to motile rods from nonmotile cocci and vice versa.     

The genetic structure of a marine teleost, <Emphasis Type="Italic">Chrysophrys auratus,</Emphasis> in a large,heterogeneous marine embayment

There is considerable interest in the processes that generate genetic divergence in marine species and the spatial and temporal scales over which these processes operate. Shark Bay, a large embayment (~13,000 km²) on the arid west coast of Australia, has been described as a focal point for genetic divergence in marine species due to its heterogeneous environment. This study represents the first DNA-based analysis of the genetic structure of a marine species with pelagic early life stages (ELS) in this region. Twelve microsatellite loci were used to compare the genetic composition of the teleost Chrysophrys auratus from five areas within Shark Bay, and from near-by shelf waters approximately 250 km to the south. The results suggest that the microsatellite composition of C. auratus is homogeneous across most of Shark Bay and near-by shelf waters. This genetic homogeneity is probably maintained via small amounts of contemporary gene flow, which may reduce the potential for C. auratus to locally adapt to the Shark Bay environment. A weakly differentiated assemblage in Freycinet Estuary in the Western Gulf of Shark Bay provided an important exception to the otherwise homogeneous microsatellite composition of C. auratus in the study area. This weak differentiation probably reflects restrictions to gene flow into and/or out of this site due to the temporal isolation of breeding adults, selective mortality of immigrants and/or the presence of hydrological barriers to larval transport. Each of these factors can be linked to environmental heterogeneity in Shark Bay.     

Penicillium solitum: a mesophilic, psychrotolerant fungus present in marine sediments from Antarctica

Few studies have addressed the diversity of cultivable fungi from marine sediments, especially those from Antarctica. In the present study, we evaluated the presence and distribution of cultivable fungi in marine core sediments obtained from 100, 500, 700 and 1,100 m below the Antarctic Ocean surface. Fifty-two fungal isolates were identified as Penicillium solitum by their physiological and morphological characteristics, and the identity of 12 representative isolates was further confirmed by sequencing of the ITS1-5.8S-ITS2 and β-tubulin genes. P. solitum displayed high sequence similarity to Penicillium taxa that have been described from other marine habitats. Conidial germination of P. solitum occurred at low temperatures and high salinities. In addition, P. solitum displayed extracellular amylasic and esterasic activities. The isolation of P. solitum from marine sediments in Antarctica and its survival at low temperatures and high salt concentrations suggest that it is adapted to the cold and halophilic environment of the Antarctic oceans. Because P. solitum produces extracellular enzymes, it is an interesting eukaryotic model for the study of structure–function relationships during enzymatic biocatalysis and biotransformation under extreme conditions. Marine sediments from Antarctica may represent a unique source for obtaining extremophilic fungi. New studies using different culture media, temperatures ranges and pressure conditions as well as metagenomic techniques can assist in understanding the extremophilic fungal communities in marine sediments across the Antarctic Ocean.     

High responsiveness in <Emphasis Type="Italic">de novo</Emphasis> shoot organogenesis induction of <Emphasis Type="Italic">Passiflora cristalina</Emphasis> (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^?1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^?1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^?1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species.     

Agrobacterium-mediated transformation of the medicinal plant Podophyllum hexandrum Royle (syn. P. emodi Wall. ex Hook.f. & Thomas)

An efficient Agrobacterium-mediated genetic transformation method has been developed for the medicinal plant Podophyllum hexandrum Royle, an important source of the anticancer agent podophyllotoxin. Highly proliferating embryogenic cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA 2301, which contains npt II and gusA as selection marker and reporter genes, respectively. The transformed somatic embryos and plantlets were selected on Murashige and Skoog (MS) basal medium containing kanamycin and germination medium, respectively. GUS histochemical analysis, polymerase chain reaction and Southern blot hybridisation confirmed that gusA was successfully integrated and expressed in the P. hexandrum genome. Compared with cefotaxime, 200 mg l^?1 timentin completely arrested Agrobacterium growth and favoured somatic embryo development from embryogenic cells. Among the different Agrobacterium strains, acetosyringone concentrations and co-cultivation durations tested, embryogenic callus infected with A. tumefaciens EHA 105 and co-cultivated for 3 days on MS basal medium containing 100 μM acetosyringone proved to be optimal and produced a transformation efficiency of 29.64 % with respect to germinated GUS-positive plantlets. The Agrobacterium-mediated genetic transformation method developed in the present study facilitates the transference of desirable genes into P. hexandrum to improve the podophyllotoxin content and to enhance other useful traits.     

Long-term population genetic structure of an invasive urochordate: the ascidian Botryllus schlosseri

The accelerated pace of marine biological invasions raises questions pertaining to genetic traits and dynamics underlying the successful establishment of invasive species. Current research stresses the importance of multiple introductions and prolonged gene flow as the main sources for genetic diversity, which, along with genetic drift, affect invasive species success. We here attempt to determine the relative contribution of gene flow and mutation rates as sources of genetic variability using the invasive tunicate Botryllus schlosseri as a model. The study was performed over a 13-year period in the Santa Cruz Harbor, California. With a characteristic life history of five generations/year, the Santa Cruz Botryllus population has already experienced approximately 155 generations since the onset of its invasion. The results (278 specimens, 127 scored alleles, five microsatellite loci) support limited gene flow rate (2.89?×?10^?3) and relative genetic isolation. Furthermore, the study population was found to be influenced by both, genetic drift and a high mutation rate (2.47?×?10^?2). These findings were supported by high fluctuations in the frequencies of microsatellite alleles, the appearance of new alleles and the loss of others. The balance between genetic drift and a high mutation rate is further elucidated by the high, stable level of genetic variation. We suggest that rapid mutation rates at the microsatellite loci reflect genome-wide phenomena, helping to maintain high genetic variability in relatively isolated populations. The potential adaptability to new environments is discussed.     

Phycoremediation of municipal wastewater by microalgae to produce biofuel

Municipal wastewater (WW), if not properly remediated, poses a threat to the environment and human health by carrying significant loads of nutrients and pathogens. These contaminants pollute rivers, lakes, and natural reservoirs where they cause eutrophication and pathogen-mediated diseases. However, the high nutrient content of WW makes it an ideal environment for remediation with microalgae that require high nutrient concentrations for growth and are not susceptible to toxins and pathogens. Given that an appropriate algal strain is used for remediation, the incurred biomass can be refined for the production of biofuel. Four microalgal species (Chlamydomonas reinhardtii, Chlorella sp., Parachlorella kessleri-I, and Nannochloropsis gaditana) were screened for efficient phycoremediation of municipal WW and potential use for biodiesel production. Among the four strains tested, P. kessleri-I showed the highest growth rate and biomass production in 100% WW. It efficiently removed all major nutrients with a removal rate of up to 98% for phosphate after 10 days of growth in 100% municipal WW collected from Delhi. The growth of P. kessleri-I in WW resulted in a 50% increase of biomass and a 115% increase of lipid yield in comparison to growth in control media. The Fatty acid methyl ester (FAME), and fuel properties of lipids isolated from cells grown in WW complied with international standards. The present study provides evidence that the green alga P. kessleri-I effectively remediates municipal WW and can be used to produce biodiesel.     

Nonlabens arenilitoris sp. nov., a member of the family Flavobacteriaceae isolated from seashore sand

A Gram-staining-negative, non-spore-forming, aerobic, non-flagellated, non-gliding and rod-shaped bacterial strain, designated M-M3^T, was isolated from marine sand on the southern coast of South Korea and subjected to a polyphasic study. Strain M-M3^T was observed to grow optimally at 25–30 °C, at pH 7.0–8.0 and in the presence of 2 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain M-M3^T fell within the clade comprising Nonlabens species, joining the type strain of Nonlabens ulvanivorans, with which it exhibited the highest 16S rRNA gene sequence similarity value of 99.4 %. Sequence similarity to the type strains of the other Nonlabens species was 93.0–95.2 % and <92.2 % to those of other species used in the phylogenetic analysis. Strain M-M3^T was found to contain MK-6 as the predominant menaquinone and iso-C_15:0 and iso-C_17:0 3-OH as the major fatty acids. The major polar lipids were determined to be phosphatidylethanolamine, five unidentified lipids and one unidentified aminolipid. The DNA G+C content of strain M-M3^T was determined to be 38.2 mol% and its DNA–DNA relatedness values with N. ulvanivorans DSM 22727^T was 42 ± 6.2 %. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain M-M3^T is separate from other Nonlabens species. On the basis of the data presented, strain M-M3^T is considered to represent a novel species of the genus Nonlabens, for which the name Nonlabens arenilitoris sp. nov. is proposed. The type strain is M-M3^T (=KCTC 32109^T = CCUG 62919^T).     

Genetic variability among Pinus pinea L. provenances for survival and growth traits in Portugal

Pinus pinea L. (Mediterranean stone pine) is an important forest species not only for its economically relevant kernel production, but also for environmental protection. The detection of genetic variability is an essential issue for Mediterranean forest species for conservation and improvement programs. Based on data collected for several years at three field sites from a P. pinea L. provenance trial established in Portugal in 1993, the present study aimed to evaluate the genetic variability of the adaptive traits of P. pinea L. and to identify a group of provenances with high performance for growth traits to be used in future plantings. Several mixed models were fitted to survival and growth trait data to estimate provenance and provenance × site interaction variability. The empirical best linear unbiased predictors of provenance genotypic effects were used to select a superior group of provenances. The provenance genetic variability of P. pinea L. was successfully detected for survival and height of different planting ages and also for diameter at breast height at age 13 years after planting. For growth traits, the most successful methods used for evaluating provenance genetic variability were based on the linear mixed spatial models. When multi-environmental analysis was performed, provenance × site interaction variability was detected for survival, but not for height at age 6 years. The existence of provenance variability in P. pinea L. permitted the identification of a seed lot composed of a mixture of the best provenances for height and diameter.     

Characterization of exudates released by the marine diatom Skeletonema costatum exposed to copper stress: a 3D-fluorescence spectroscopy approach

In a laboratory study, metal contamination experiments were conducted to investigate the effects of two free copper concentrations (10^?9 and 10^?8 M) on cell growth and on dissolved organic matter exudation by a marine diatom Skeletonema costatum. Throughout incubation, the growth kinetics and exudation of extracellular molecules (i.e. dissolved organic carbon (DOC) and the fluorescent organic matter) were determined. Results revealed an inhibition of S. costatum growth when the free copper level increased (from 10^?9 to 10^?8). Furthermore, DOC release was more significant in cultures contaminated by 10^?9 M Cu²⁺ than in control, suggesting a coping mechanism developed by this species. In this study, samples were daily analysed by 3D-fluorescence and PARAFAC algorithm, in order to compare the fluorescent material produced during growth under different contaminations. PARAFAC treatment revealed two main contributions: one related to the biological activity (C1), the other linked to the marine organic matter (C2). The third component C3 was typically protein-like. This fluorophore was considered as a tryptophan-like fluorophore, whereas the C1 and the C2 components were associated to marine production such as humic matter.     

Glucose-induced activation of H+-ATPase in Dunaliella salina and its role in hygromycin resistance

Dunaliella salina, a eukaryotic microalga, is known for its highly halophilic nature. The high level of salts in growth medium for this alga has made its genetic transformation a comparatively difficult procedure, particularly during the selection stage. The high salt content decreases the efficiency of most antibiotics which are being used as selection markers. Studies pertaining to the interrelationship between salt concentration and antibiotic sensitivity are scarce in Dunaliella. During our previous experiment at genetic transformation of Dunaliella, an inverse relationship between the amount of antibiotic hygromycin and sodium chloride in the medium was revealed. A possible link between plasma membrane activity and the hygromycin sensitivity was investigated in the present study by modulating plasma membrane H⁺-ATPase activity using glucose. Glucose-induced activation of H⁺-ATPase, reduced the tolerance of D. salina to the antibiotic hygromycin. Hygromycin concentration required for selection during genetic transformation of Dunaliella was lowered from 100 to 25 mg L^?1 in the presence of 10 mM glucose. Conversely, the inhibitors of the plasma membrane H⁺-ATPase, orthovanadate and diethylstilbestrol were found to inhibit the glucose activation at concentrations of 10 and 15 μM, respectively. The activation of H⁺-ATPase by glucose was further confirmed through H⁺-ATPase assay and medium acidification experiments. The results indicated that the sensitivity of Dunaliella to antibiotic is related to H⁺-ATPase and the possible involvement of pH gradient, created through H⁺-ATPase activation during drug transport.