Nitrogen budgets in microalgae are strongly affected by growth conditions and physiological state of the cultures. As a consequence, protein N (PN) to total N (TN) ratio may be variable in microalgae grown in batch cultures, and this may limit the usefulness of the nitrogen-to-protein conversion factors (N-Prot factors), the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of specific N-Prot factors, and experimental data are needed to fill this gap. Complementing a previous study, the present work was designed to quantify the fluctuations of the main nitrogenous compounds during the growth of 12 species of marine microalgae, as well as to determine N-Prot factors for them. The microalgae were cultured in two experimental conditions: (a) using a N-replete culture medium (initial N concentration, 1.18 mM) and aeration, and (b) with a N-depleted culture medium (initial N concentration, 235 mM) and no aeration. The distribution of intracellular nitrogen was studied by constructing budgets of different nitrogen pools in different growth phases of the cultures. In all species, large variations occurred in the distribution of PN and non-protein N (NPN) in the treatments tested and in different growth phases. Intracellular inorganic nitrogen (NO 3 7 , NO 2 7 and NH 3 + NH 4 +) was the most important NPN component (0.4-30.4% of TN) in all species, followed by nucleic acids (0.3-12.2% of TN), and chlorophylls (0.1-1.8% of TN). The relative importance of NPN was greater in the exponential phase, decreasing during growth. PN ranged from 59.3 to 96.8% of TN. N-Prot factors are proposed for each of the species studied, based on the ratio of amino acid residues to TN, with values ranging from 2.53 to 5.77. Based on current results and on the previous study, we establish an overall average N-Prot factor for all species, treatments and growth phases of 4.78 + 0.62 (n = 354). This study confirms that the use of the traditional factor 6.25 is unsuitable for marine microalgae, and the use of the N-Prot factors proposed here is recommended.

The eastern tropical Pacific Ocean holds two of the main oceanic oxygen minimum zones of the global ocean. The presence of an oxygen-depleted layer at intermediate depths, which also impinges on the seafloor and in some cases the euphotic zone, plays a significant role in structuring both pelagic and benthic communities, and also in the vertical partitioning of microbial assemblages. Here, we assessed the genetic diversity and distribution of natural populations of the cyanobacteria Prochlorococcus and Synechococcus within oxic and suboxic waters of the eastern tropical Pacific using cloning and sequencing, and terminal restriction fragment length polymorphism (T-RFLP) analyses applied to the 16S-23S rRNA internal transcribed spacer region. With the T-RFLP approach we could discriminate 19 cyanobacterial clades, of which 18 were present in the study region. Synechococcus was more abundant in the surface oxic waters of the eastern South Pacific, while Prochlorococcus dominated the subsurface low-oxygen waters. Two of the dominant clades in the oxygen-deficient waters belong to novel and yet uncultivated lineages of low-light adapted Prochlorococcus.

Most climate and environmental change models predict significant increases in temperature and precipitation by the end of the 21st Century, for which the current functional output of certain symbioses may also be altered. In this context we address the following questions: 1) How the expected changes in abiotic factors (temperature, and water) differentially affect the ecophysiological performance of the plant Colobanthus quitensis? and 2) Will this environmental change indirectly affect C. quitensis photochemical performance and biomass accumulation by modifying its association with fungal endophytes? Plants of C. quitensis from King George Island in the South Shetland archipelago (62°09′ S), and Lagotellerie Island in the Antarctic Peninsula (65°53′ S) were put under simulated abiotic conditions in growth chambers following predictive models of global climate change (GCC). The indirect effect of GCC on the interaction between C. quitensis and fungal endophytes was assessed in a field experiment carried out in the Antarctica, in which we eliminated endophytes under contemporary conditions and applied experimental watering to simulate increased precipitation input. We measured four proxies of plant performance. First, we found that warming (+W) significantly increased plant performance, however its effect tended to be less than watering (+W) and combined warming and watering (+T°+W). Second, the presence of fungal endophytes improved plant performance, and its effect was significantly decreased under experimental watering. Our results indicate that both biotic and abiotic factors affect ecophysiological performance, and the directions of these influences will change with climate change. Our findings provide valuable information that will help to predict future population spread and evolution through using ecological niche models under different climatic scenarios.

Our understanding of the icy-habitat microbiome is likely limited by a lack of reliable data on microorganisms inhabiting underground ice that has accumulated inside caves. To characterize how environmental variation impacts cave ice microbial community structure, we determined the composition of total and potentially active bacterial communities along a 13,000-year-old ice core from Scarisoara cave (Romania) through 16S rRNA gene Illumina sequencing. An average of 2,546 prokaryotic gDNA operational taxonomic units (OTUs) and 585 cDNA OTUs were identified across the perennial cave ice block and analyzed in relation to the geochemical composition of ice layers. The total microbial community and the putative active fraction displayed dissimilar taxa profiles. The ice-contained microbiome was dominated by Actinobacteria with a variable representation of Proteobacteria, while the putative active microbial community was equally shared between Proteobacteria and Firmicutes. Accordingly, a major presence of Cryobacterium, Lysinomonas, Pedobacter , and Aeromicrobium phylotypes homologous to psychrotrophic and psychrophilic bacteria from various cold environments were noted in the total community, while the prevalent putative active bacteria belonged to Clostridium, Pseudomonas, Janthinobacterium, Stenotrophomonas , and Massilia genera. Variation in the microbial cell density of ice strata with the dissolved organic carbon (DOC) content and the strong correlation of DOC and silicon concentrations revealed a major impact of depositional processes on microbial abundance throughout the ice block. Post-depositional processes appeared to occur mostly during the 4,000–7,000 years BP interval. A major bacterial composition shift was observed in 4,500–5,000-year-old ice, leading to a high representation of Beta- and Deltaproteobacteria in the potentially active community in response to the increased concentrations of DOC and major chemical elements. Estimated metabolic rates suggested the presence of a viable microbial community within the cave ice block, characterized by a maintenance metabolism in most strata and growth capacity in those ice deposits with high microbial abundance and DOC content. This first survey of microbial distribution in perennial cave ice formed since the Last Glacial period revealed a complex potentially active community, highlighting major shifts in community composition associated with geochemical changes that took place during climatic events that occurred about 5,000 years ago, with putative formation of photosynthetic biofilms.

Ice entrenched microcosm represents a vast reservoir of novel species and a proxy for past climate reconstitution. Among glacial ecosystems, ice caves represent one of the scarcely investigated frozen habitats. To characterize the microbial diversity of perennial ice from karst ecosystems, Roche 454 sequencing of 16S rRNA gene amplicons from the underground ice block of Scarisoara Ice Cave (Romania) was applied. The temporal distribution of bacterial and archaeal community structures from newly formed, 400, and 900 years old ice layers was surveyed and analyzed in relation with the age and geochemical composition of the ice substrate. The microbial content of cave ice layers varied from 3.3 104 up to 7.5 105 cells mL−1, with 59–78% viability. Pyrosequencing generated 273,102 reads for the five triplicate ice samples, which corresponded to 3,464 operational taxonomic units (OTUs). The distribution of the bacterial phyla in the perennial cave ice varied with age, organic content, and light exposure. Proteobacteria dominated the 1 and 900 years old organic rich ice deposits, while Actinobacteria was mostly found in 900 years old ice strata, and Firmicutes was best represented in 400 years old ice. Cyanobacteria and Chlorobi representatives were identified mainly from the ice block surface samples exposed to sunlight. Archaea was observed only in older ice strata, with a high incidence of Crenarchaeota and Thaumarchaeaota in the 400 years old ice, while Euryarchaeota dominated the 900 years old ice layers, with Methanomicrobia representing the predominant taxa. A large percentage (55.7%) of 16S rRNA gene amplicons corresponded to unidentified OTUs at genus or higher taxa levels, suggesting a greater undiscovered bacterial diversity in this glacial underground habitat. The prokaryotes distribution across the cave ice block revealed the presence of 99 phylotypes specific for different ice layers, in addition to the shared microbial community. Ice geochemistry represented an important factor that explained the microbial taxa distribution in the cave ice block, while the total organic carbon content had a direct impact on the cell density of the ice microcosm. Both bacterial and archaeal community structures appeared to be affected by climate variations during the ice formation, highlighting the cave ice microbiome as a source of putative paleoclimatic biomarkers. This report constitutes the first high-throughput sequencing study of the cave ice microbiome and its distribution across the perennial underground glacier of an alpine ice cave.

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A B S T R A C TMethods of extraction, changes in concentrations with growth, and effects of culture conditions on intracellular inorganic nitrogen pools (IIN -ammonia, nitrite, and nitrate) were studied in nine species of marine microalgae in batch cultures. The microalgae were analysed to compare three methods of extraction of IIN, one of them developed in this study. The extraction of IIN occurs efficient by with all three methods for four out of the nine species tested. However, for five species significant differences were found among the methods, the best results being obtained with the new method. Microalgae accumulate inorganic forms of nitrogen in different proportions. The species show higher concentrations of either ammonia or nitrate, and always lower concentrations of nitrite. Microalgae of smaller cellular volumes tend to attain higher values of IIN per cubic micrometer (the converse in large-volume species), with some exceptions (Amphidinium carterae and Nannochloropsis oculata). The use of aeration in the cultures determines a decrease in the concentrations of IIN, favours nitrogen assimilation, and generates an increase in growth rates and C:N ratio. High concentrations of IIN are characteristic of the exponential growth phase, but in some cases their occurrence may result from carbon deficiency. R E S U M OMétodos de extração, mudanças na concentração durante o crescimento e efeitos de condições de cultivo sobre conteúdos de nitrogênio inorgânico intracelular (NII -amônia, nitrito e nitrato) foram estudados em nove espécies de microalgas marinhas em cultivos estanques. As microalgas foram analisadas para comparar três métodos de extração de NII, um dos quais desenvolvido neste estudo. A extração de NII ocorre de forma eficiente com os três métodos para quatro espécies. Contudo, para cinco espécies diferenças significativas foram encontradas e os melhores resultados foram obtidos com o método novo. As microalgas acumulam formas inorgânicas de nitrogênio em proporções diferentes. As espécies apresentam concentrações de amônia ou nitrato como as mais altas e sempre menores concentrações de nitrito. Microalgas de menores volumes celulares tendem a atingir valores mais altos de NII por micrômetro cúbico (contrariamente para espécies de volumes celulares maiores), com algumas exceções (Amphidinium carterae e Nannochloropsis oculata). A adição de aeração nos cultivos determina um decréscimo na concentração de NII, favorece a assimilação de nitrogênio e gera um aumento na taxa de crescimento e na razão C:N. Concentrações altas de NII são características da fase de crescimento exponencial, mas em alguns casos sua ocorrência pode resultar de deficiência por carbono.