Systematic, continental scale temporal monitoring of marine pelagic microbiota by the Australian Marine Microbial Biodiversity Initiative.

Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. The Australian Marine Microbial Biodiversity Initiative (AMMBI) provides methodologically standardized, continental scale, temporal phylogenetic amplicon sequencing data describing Bacteria, Archaea and microbial Eukarya assemblages. Sequence data is linked to extensive physical, biological and chemical oceanographic contextual information. Samples are collected monthly to seasonally from multiple depths at seven sites: Darwin Harbour (Northern Territory), Yongala (Queensland), North Stradbroke Island (Queensland), Port Hacking (New South Wales), Maria Island (Tasmania), Kangaroo Island (South Australia), Rottnest Island (Western Australia). These sites span ~30° of latitude and ~38° longitude, range from tropical to cold temperate zones, and are influenced by both local and globally significant oceanographic and climatic features. All sequence datasets are provided in both raw and processed fashion. Currently 952 samples are publically available for bacteria and archaea which include 88,951,761 bacterial (72,435 unique) and 70,463,079 archaeal (24,205 unique) 16 S rRNA v1-3 gene sequences, and 388 samples are available for eukaryotes which include 39,801,050 (78,463 unique) 18 S rRNA v4 gene sequences.

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis.

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.

Evaluation of growth, nutrient utilization and production of bioproducts by a wastewater-isolated microalga.

Treatment of wastewater while producing microalgal biomass is receiving ever-increasing attention, particularly in the biofuels arena. In this study, a wastewater chlorophyte isolate, Kirchneriella sp., was tested for its ability to be mass cultivated, utilize nutrients from defined media and wastewater, and produce bioproducts of commercial interest. Growth studies were carried out in various systems at scales up to 60L, with Kirchneriella sp. showing an excellent amenability to being cultured. Biomass concentrations of greater than 1gL(-1) were consistently achieved, nitrogen and phosphorus uptake was rapid, and stable medium-term cultures were maintained. Nitrogen limitation affected biomass yield, fatty acid methyl ester (FAME) yield, and cetane index. In contrast, a low phosphorus condition had no effect. Kirchneriella sp. showed an ability to produce several products of commercial value, including carbohydrate-rich biomass, FAME/biodiesel and the pigments ß,ß-carotene and lutein.

Archaeal dominated ammonia-oxidizing communities in Icelandic grassland soils are moderately affected by long-term N fertilization and geothermal heating.

The contribution of ammonia-oxidizing bacteria and archaea (AOB and AOA, respectively) to the net oxidation of ammonia varies greatly between terrestrial environments. To better understand, predict and possibly manage terrestrial nitrogen turnover, we need to develop a conceptual understanding of ammonia oxidation as a function of environmental conditions including the ecophysiology of associated organisms. We examined the discrete and combined effects of mineral nitrogen deposition and geothermal heating on ammonia-oxidizing communities by sampling soils from a long-term fertilization site along a temperature gradient in Icelandic grasslands. Microarray, clone library and quantitative PCR analyses of the ammonia monooxygenase subunit A (amoA) gene accompanied by physico-chemical measurements of the soil properties were conducted. In contrast to most other terrestrial environments, the ammonia-oxidizing communities consisted almost exclusively of archaea. Their bacterial counterparts proved to be undetectable by quantitative polymerase chain reaction suggesting AOB are only of minor relevance for ammonia oxidation in these soils. Our results show that fertilization and local, geothermal warming affected detectable ammonia-oxidizing communities, but not soil chemistry: only a subset of the detected AOA phylotypes was present in higher temperature soils and AOA abundance was increased in the fertilized soils, while soil physio-chemical properties remained unchanged. Differences in distribution and structure of AOA communities were best explained by soil pH and clay content irrespective of temperature or fertilizer treatment in these grassland soils, suggesting that these factors have a greater potential for ecological niche-differentiation of AOA in soil than temperature and N fertilization.

High-throughput analysis of ammonia oxidiser community composition via a novel, amoA-based functional gene array.

Advances in microbial ecology research are more often than not limited by the capabilities of available methodologies. Aerobic autotrophic nitrification is one of the most important and well studied microbiological processes in terrestrial and aquatic ecosystems. We have developed and validated a microbial diagnostic microarray based on the ammonia-monooxygenase subunit A (amoA) gene, enabling the in-depth analysis of the community structure of bacterial and archaeal ammonia oxidisers. The amoA microarray has been successfully applied to analyse nitrifier diversity in marine, estuarine, soil and wastewater treatment plant environments. The microarray has moderate costs for labour and consumables and enables the analysis of hundreds of environmental DNA or RNA samples per week per person. The array has been thoroughly validated with a range of individual and complex targets (amoA clones and environmental samples, respectively), combined with parallel analysis using traditional sequencing methods. The moderate cost and high throughput of the microarray makes it possible to adequately address broader questions of the ecology of microbial ammonia oxidation requiring high sample numbers and high resolution of the community composition.

Isolation and characterisation of a high-efficiency desaturase and elongases from microalgae for transgenic LC-PUFA production.

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2omega6) and alpha-linolenic acid (ALA; 18:3omega3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Delta6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Delta6-elongase and a Delta5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39-75% conversion for omega3 substrate fatty acids), and the Delta5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5omega3) to docosapentaenoic acid (22:5omega3)) than previously reported genes. In addition, the Delta6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the omega3 substrate ALA.