The Nutritional and Pharmacological Potential of New Australian Thraustochytrids Isolated from Mangrove Sediments.

Mangrove sediments represent unique microbial ecosystems that act as a buffer zone, biogeochemically recycling marine waste into nutrient-rich depositions for marine and terrestrial species. Marine unicellular protists, thraustochytrids, colonizing mangrove sediments have received attention due to their ability to produce large amounts of long-chain ω3-polyunsaturated fatty acids. This paper represents a comprehensive study of two new thraustochytrids for their production of valuable biomolecules in biomass, de-oiled cakes, supernatants, extracellular polysaccharide matrixes, and recovered oil bodies. Extracted lipids (up to 40% of DW) rich in polyunsaturated fatty acids (up to 80% of total fatty acids) were mainly represented by docosahexaenoic acid (75% of polyunsaturated fatty acids). Cells also showed accumulation of squalene (up to 13 mg/g DW) and carotenoids (up to 72 µg/g DW represented by astaxanthin, canthaxanthin, echinenone, and ß-carotene). Both strains showed a high concentration of protein in biomass (29% DW) and supernatants (2.7 g/L) as part of extracellular polysaccharide matrixes. Alkalinization of collected biomass represents a new and easy way to recover lipid-rich oil bodies in the form of an aqueous emulsion. The ability to produce added-value molecules makes thraustochytrids an important alternative to microalgae and plants dominating in the food, pharmacological, nutraceutical, and cosmetics industries.

Reduced lignin content and altered lignin composition in the warm season forage grass Paspalum dilatatum by down-regulation of a Cinnamoyl CoA reductase gene.

C4 grasses are favoured as forage crops in warm, humid climates. The use of C4 grasses in pastures is expected to increase because the tropical belt is widening due to global climate change. While the forage quality of Paspalum dilatatum (dallisgrass) is higher than that of other C4 forage grass species, digestibility of warm-season grasses is, in general, poor compared with most temperate grasses. The presence of thick-walled parenchyma bundle-sheath cells around the vascular bundles found in the C4 forage grasses are associated with the deposition of lignin polymers in cell walls. High lignin content correlates negatively with digestibility, which is further reduced by a high ratio of syringyl (S) to guaiacyl (G) lignin subunits. Cinnamoyl-CoA reductase (CCR) catalyses the conversion of cinnamoyl CoA to cinnemaldehyde in the monolignol biosynthetic pathway and is considered to be the first step in the lignin-specific branch of the phenylpropanoid pathway. We have isolated three putative CCR1 cDNAs from P. dilatatum and demonstrated that their spatio-temporal expression pattern correlates with the developmental profile of lignin deposition. Further, transgenic P. dilatatum plants were produced in which a sense-suppression gene cassette, delivered free of vector backbone and integrated separately to the selectable marker, reduced CCR1 transcript levels. This resulted in the reduction of lignin, largely attributable to a decrease in G lignin.

eDNA metabarcoding reveals biodiversity and depth stratification patterns of dinoflagellate assemblages within the epipelagic zone of the western Coral Sea.

BACKGROUND: Dinoflagellates play critical roles in the functioning of marine ecosystems but also may pose a hazard to human and ecosystem health by causing harmful algal blooms (HABs). The Coral Sea is a biodiversity hotspot, but its dinoflagellate assemblages in pelagic waters have not been studied by modern sequencing methods. We used metabarcoding of the 18 S rRNA V4 amplicon to assess the diversity and structure of dinoflagellate assemblages throughout the water column to a depth of 150 m at three stations in the Western Coral Sea. Additionally, at one station we compared metabarcoding with morphological methods to optimise identification and detection of dinoflagellates. RESULTS: Stratification of dinoflagellate assemblages was evident in depth-specific relative abundances of taxonomic groups; the greatest difference was between the 5-30 m assemblages and the 130-150 m assemblages. The relative abundance of Dinophyceae (photosynthetic and heterotrophic) decreased with increasing depth, whereas that of Syndiniales (parasitic) increased with increasing depth. The composition of major taxonomic groups was similar among stations. Taxonomic richness and diversity of amplicon sequence variants (ASVs) were similar among depths and stations; however, the abundance of dominant taxa was highest within 0-30 m, and the abundance of rare taxa was highest within 130-150 m, indicating adaptations to specific depth strata. The number of unclassified ASVs at the family and species levels was very high, particularly for Syndinian representatives. CONCLUSIONS: Dinoflagellate assemblages in open water of the Coral Sea are highly diverse and taxonomically stratified by depth; patterns of relative abundance along the depth gradient reflect environmental factors and ecological processes. Metabarcoding detects more species richness than does traditional microscopical methods of sample analysis, yet the methods are complementary, with morphological analysis revealing additional richness. The large number of unclassified dinoflagellate-ASVs indicates a need for improved taxonomic reference databases and suggests presence of dinoflagellate-crypto and-morphospecies.

Biosynthesis of proanthocyanidins in white clover flowers: cross talk within the flavonoid pathway.

Proanthocyanidins and anthocyanins are produced by closely related branches of the flavonoid pathway and utilize the same metabolic intermediates. Previous studies have shown a flexible mechanism of flux diversion at the branch-point between the anthocyanin and proanthocyanidin pathways, but the molecular basis for this mechanism is poorly understood. Floral tissues in white clover plants (Trifolium repens) produce both proanthocyanidins and anthocyanins. This makes white clover amenable to studies of proanthocyanidin and anthocyanin biosynthesis and possible interactions within the flavonoid pathway. Results of this study show that the anthocyanin and proanthocyanidin pathways are spatially colocalized within epidermal cells of petals and temporally overlap in partially open flowers. A correlation between spatiotemporal patterns of anthocyanin and proanthocyanidin biosynthesis with expression profiles of putative flavonoid-related genes indicates that these pathways may recruit different isoforms of flavonoid biosynthetic enzymes. Furthermore, in transgenic white clover plants with down-regulated expression of the anthocyanidin reductase gene, levels of flavan 3-ols, anthocyanins, and flavonol glycosides and the expression levels of a range of genes encoding putative flavonoid biosynthetic enzymes and transcription factors were altered. This is consistent with the hypothesis that flux through the flavonoid pathway may be at least partially regulated by the availability of intermediates.

Structure-function analyses of a caffeic acid O-methyltransferase from perennial ryegrass reveal the molecular basis for substrate preference.

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde.

Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne).

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts.

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.

An improved method for quantitative analysis of total fructans in plant tissues.

Current methods for measuring fructan levels in plant tissues are time-consuming and costly. They often involve multiple or sequential extractions, enzymatic or acid hydrolysis of fructan polymers, and multiple HPLC runs to quantify fructan-derived hexoses. Here we describe a new method that requires a single extraction step, followed by selective precipitation of fructans by acetone, acid hydrolysis of the precipitate, and a short (10 min) HPLC run to complete the procedure. We used perennial ryegrass samples to show that the new method has similar sensitivity, but better reproducibility, than a more complex method that is widely used. We have used the new method to study developmentally related changes in fructan levels in glasshouse-grown perennial ryegrass plants.

Efficient Bioflocculation of Chlorella vulgaris with a Chitosan and Walnut Protein Extract.

Bioflocculation represents an attractive technology for harvesting microalgae with the potential additive effect of flocculants on the production of added-value chemicals. Chitosan, as a cationic polyelectrolyte, is widely used as a non-toxic, biodegradable bioflocculant for many algal species. The high cost of chitosan makes its large-scale application economically challenging, which triggered research on reducing its amount using co-flocculation with other components. In our study, chitosan alone at a concentration 10 mg/L showed up to an 89% flocculation efficiency for Chlorella vulgaris. Walnut protein extract (WPE) alone showed a modest level (up to 40%) of flocculation efficiency. The presence of WPE increased chitosan's flocculation efficiency up to 98% at a reduced concentration of chitosan (6 mg/L). Assessment of co-flocculation efficiency at a broad region of pH showed the maximum harvesting efficiency at a neutral pH. Fourier transform infrared spectroscopy, floc size analysis, and microscopy suggested that the dual flocculation with chitosan and walnut protein is a result of the chemical interaction between the components that form a web-like structure, enhancing the bridging and sweeping ability of chitosan. Co-flocculation of chitosan with walnut protein extract, a low-value leftover from walnut oil production, represents an efficient and relatively cheap system for microalgal harvesting.

Diversity of dinoflagellate assemblages in coastal temperate and offshore tropical waters of Australia.

BACKGROUND: Dinoflagellates are a ubiquitous and ecologically important component of marine phytoplankton communities, with particularly notable species including those associated with harmful algal blooms (HABs) and those that bioluminesce. High-throughput sequencing offers a novel approach compared to traditional microscopy for determining species assemblages and distributions of dinoflagellates, which are poorly known especially in Australian waters. RESULTS: We assessed the composition of dinoflagellate assemblages in two Australian locations: coastal temperate Port Phillip Bay and offshore tropical waters of Davies Reef (Great Barrier Reef). These locations differ in certain environmental parameters reflecting latitude as well as possible anthropogenic influences. Molecular taxonomic assessment revealed more species than traditional microscopy, and it showed statistically significant differences in dinoflagellate assemblages between locations. Bioluminescent species and known associates of HABs were present at both sites. Dinoflagellates in both areas were mainly represented by the order Gymnodiniales (66%-82% of total sequence reads). In the warm waters of Davies Reef, Gymnodiniales were equally represented by the two superclades, Gymnodiniales sensu stricto (33%) and Gyrodinium (34%). In contrast, in cooler waters of Port Phillip Bay, Gymnodiniales was mainly represented by Gyrodinium (82%). In both locations, bioluminescent dinoflagellates represented up to 0.24% of the total sequence reads, with Protoperidinium the most abundant genus. HAB-related species, mainly represented by Gyrodinium, were more abundant in Port Phillip Bay (up to 47%) than at Davies Reef (28%), potentially reflecting anthropogenic influence from highly populated and industrial areas surrounding the bay. The entire assemblage of dinoflagellates, as well as the subsets of HAB and bioluminescent species, were strongly correlated with water quality parameters (R2 = 0.56-0.92). Significant predictors differed between the subsets: HAB assemblages were explained by salinity, temperature, dissolved oxygen, and total dissolved solids; whereas, bioluminescent assemblages were explained only by salinity and dissolved oxygen, and had greater variability. CONCLUSION: High-throughput sequencing and genotyping revealed greater diversity of dinoflagellate assemblages than previously known in both subtropical and temperate Australian waters. Significant correlations of assemblage structure with environmental variables suggest the potential for explaining the distribution and composition of both HAB species and bioluminescent species.

Differential Production of Phenolics, Lipids, Carbohydrates and Proteins in Stressed and Unstressed Aquatic Plants, Azolla filiculoides and Azolla pinnata.

The metabolic plasticity of shikimate and phenylpropanoid pathways redirects carbon flow to different sink products in order to protect sessile plants from environmental stresses. This study assessed the biochemical responses of two Azolla species, A. filiculoides and A. pinnata, to the combined effects of environmental and nutritional stresses experienced while growing outdoors under Australian summer conditions. These stresses triggered a more than 2-fold increase in the production of total phenols and their representatives, anthocyanins (up to 18-fold), flavonoids (up to 4.7-fold), and condensed tannins (up to 2.7-fold), which led to intense red coloration of the leaves. These changes were also associated with an increase in the concentration of carbohydrates and a decrease in concentrations of lipids and total proteins. Changes in lipid biosynthesis did not cause significant changes in concentrations of palmitoleic acid (C16:0), linolenic acid (C18:3), and linoleic acid (C18:2), the fatty acid signatures of Azolla species. However, a reduction in protein production triggered changes in biosynthesis of alanine, arginine, leucine, tyrosine, threonine, valine, and methionine amino acids. Stress-triggered changes in key nutritional components, phenolics, lipids, proteins, and carbohydrates could have a significant impact on the nutritional value of both Azolla species, which are widely used as a sustainable food supplement for livestock, poultry, and fish industries.

Aquatic Plants, Landoltia punctata, and Azolla filiculoides as Bio-Converters of Wastewater to Biofuel.

The aquatic plants, Azolla filiculoides, and Landoltia punctate, were used as complementing phytoremediators of wastewater containing high levels of phosphate, which simulates the effluents from textile, dyeing, and laundry detergent industries. Their complementarities are based on differences in capacities to uptake nitrogen and phosphate components from wastewater. Sequential treatment by L. punctata followed by A. filiculoides led to complete removal of NH4, NO3, and up to 93% reduction of PO4. In experiments where L. punctata treatment was followed by fresh L. punctata, PO4 concentration was reduced by 65%. The toxicity of wastewater assessed by shrimps, Paratya australiensis, showed a four-fold reduction of their mortality (LC50 value) after treatment. Collected dry biomass was used as an alternative carbon source for heterotrophic marine protists, thraustochytrids, which produced up to 35% dry weight of lipids rich in palmitic acid (50% of total fatty acids), the key fatty acid for biodiesel production. The fermentation of treated L. punctata biomass by Enterobacter cloacae yielded up to 2.14 mol H2/mole of reduced sugar, which is comparable with leading terrestrial feedstocks. A. filiculoides and L. punctata can be used as a new generation of feedstock, which can treat different types of wastewater and represent renewable and sustainable feedstock for bioenergy production.

Physicochemical Characteristics of Protein Isolated from Thraustochytrid Oilcake.

The oil from thraustochytrids, unicellular heterotrophic marine protists, is increasingly used in the food and biotechnological industries as it is rich in omega-3 fatty acids, squalene and a broad spectrum of carotenoids. This study showed that the oilcake, a by-product of oil extraction, is equally valuable as it contained 38% protein/dry mass, and thraustochytrid protein isolate can be obtained with 92% protein content and recovered with 70% efficiency. The highest and lowest solubilities of proteins were observed at pH 12.0 and 4.0, respectively, the latter being its isoelectric point. Aspartic acid, glutamic acid, histidine, and arginine were the most abundant amino acids in proteins. The arginine-to-lysine ratio was higher than one, which is desired in heart-healthy foods. The denaturation temperature of proteins ranged from 167.8-174.5 °C, indicating its high thermal stability. Proteins also showed high emulsion activity (784.1 m2/g) and emulsion stability (209.9 min) indices. The extracted omega-3-rich oil melted in the range of 30-34.6 °C and remained stable up to 163-213 °C. This study shows that thraustochytrids are not only a valuable source of omega 3-, squalene- and carotenoid-containing oils, but are also rich in high-value protein with characteristics similar to those from oilseeds.

Marine Protists and Rhodotorula Yeast as Bio-Convertors of Marine Waste into Nutrient-Rich Deposits for Mangrove Ecosystems.

This paper represents a comprehensive study of two new thraustochytrids and a marine Rhodotorula red yeast isolated from Australian coastal waters for their abilities to be a potential renewable feedstock for the nutraceutical, food, fishery and bioenergy industries. Mixotrophic growth of these species was assessed in the presence of different carbon sources: glycerol, glucose, fructose, galactose, xylose, and sucrose, starch, cellulose, malt extract, and potato peels. Up to 14g DW/L (4.6gDW/L-day and 2.8gDW/L-day) of biomass were produced by Aurantiochytrium and Thraustochytrium species, respectively. Thraustochytrids biomass contained up to 33% DW of lipids, rich in omega-3 polyunsaturated docosahexaenoic acid (C22:6, 124mg/g DW); up to 10.2mg/gDW of squalene and up to 61µg/gDW of total carotenoids, composed of astaxanthin, canthaxanthin, echinenone, and ß-carotene. Along with the accumulation of these added-value chemicals in biomass, thraustochytrid representatives showed the ability to secrete extracellular polysaccharide matrixes containing lipids and proteins. Rhodotorula sp lipids (26% DW) were enriched in palmitic acid (C16:0, 18mg/gDW) and oleic acid (C18:1, 41mg/gDW). Carotenoids (87µg/gDW) were mainly represented by ß-carotene (up to 54µg/gDW). Efficient growth on organic and inorganic sources of carbon and nitrogen from natural and anthropogenic wastewater pollutants along with intracellular and extracellular production of valuable nutrients makes the production of valuable chemicals from isolated species economical and sustainable.

Lipid production in aquatic plant Azolla at vegetative and reproductive stages and in response to abiotic stress.

The aquatic plant Azolla became increasingly popular as bioenergy feedstock because of its high growth rate, production of biomass with high levels of biofuel-producing molecules and ability to grow on marginal lands. In this study, we analysed the contribution of all organs of Azolla to the total yield of lipids at vegetative and reproductive stages and in response to stress. Triacylglycerol-containing lipid droplets were detected in all (vegetative and reproductive) organs with the highest level in the male microsporocarps and microspores. As a result, significantly higher total yields of lipids were detected in Azolla filiculoides and Azolla pinnata at the reproductive stage. Starving changed the yield and composition of the fatty acid as a result of re-direction of carbon flow from fatty acid to anthocyanin pathways. The composition of lipids, in regard the length and degree of unsaturation of fatty acids, in Azolla meets most of the important requirements for biodiesel standards. The ability of Azolla to grow on wastewaters, along with their high productivity rate, makes it an attractive feedstock for the production of biofuels.

Applications of microalgal biofilms for wastewater treatment and bioenergy production.

BACKGROUND: Microalgae have shown clear advantages for the production of biofuels compared with energy crops. Apart from their high growth rates and substantial lipid/triacylglycerol yields, microalgae can grow in wastewaters (animal, municipal and mining wastewaters) efficiently removing their primary nutrients (C, N, and P), heavy metals and micropollutants, and they do not compete with crops for arable lands. However, fundamental barriers to the industrial application of microalgae for biofuel production still include high costs of removing the algae from the water and the water from the algae which can account for up to 30-40% of the total cost of biodiesel production. Algal biofilms are becoming increasingly popular as a strategy for the concentration of microalgae, making harvesting/dewatering easier and cheaper. RESULTS: We have isolated and characterized a number of natural microalgal biofilms from freshwater, saline lakes and marine habitats. Structurally, these biofilms represent complex consortia of unicellular and multicellular, photosynthetic and heterotrophic inhabitants, such as cyanobacteria, microalgae, diatoms, bacteria, and fungi. Biofilm #52 was used as feedstock for bioenergy production. Dark fermentation of its biomass by Enterobacter cloacae DT-1 led to the production of 2.4 mol of H2/mol of reduced sugar. The levels and compositions of saturated, monosaturated and polyunsaturated fatty acids in Biofilm #52 were target-wise modified through the promotion of the growth of selected individual photosynthetic inhabitants. Photosynthetic components isolated from different biofilms were used for tailoring of novel biofilms designed for (i) treatment of specific types of wastewaters, such as reverse osmosis concentrate, (ii) compositions of total fatty acids with a new degree of unsaturation and (iii) bio-flocculation and concentration of commercial microalgal cells. Treatment of different types of wastewaters with biofilms showed a reduction in the concentrations of key nutrients, such as phosphates, ammonia, nitrates, selenium and heavy metals. CONCLUSIONS: This multidisciplinary study showed the new potential of natural biofilms, their individual photosynthetic inhabitants and assembled new algal/cyanobacterial biofilms as the next generation of bioenergy feedstocks which can grow using wastewaters as a cheap source of key nutrients.

Clovers (Trifolium spp.).

Legumes constitute one of the most important global groups of agricultural species, providing a major source of protein and oil for humans and animals as well as fixing nitrogen and improving the fertility of soils. Gene technology can assist plant improvement efforts in clovers (Trifolium spp.), aiming to improve forage quality, yield, and adaptation to biotic and abiotic stresses. An efficient and reproducible protocol for Agrobacterium-mediated transformation of a range of Trifolium species, using cotyledonary explants and different selectable marker genes, is described. The protocol is robust and allows for genotype-independent transformation of clovers. Stable meiotic transmission of transgenes has been demonstrated for selected transgenic clovers carrying single T-DNA inserts recovered from Agrobacterium-mediated transformation. This methodology can also be successfully used for 'isogenic transformation' in clovers: the generation of otherwise identical plants with and without the transgene from the two cotyledons of a single seed.

Aquatic plant Azolla as the universal feedstock for biofuel production.

BACKGROUND: The quest for sustainable production of renewable and cheap biofuels has triggered an intensive search for domestication of the next generation of bioenergy crops. Aquatic plants which can rapidly colonize wetlands are attracting attention because of their ability to grow in wastewaters and produce large amounts of biomass. Representatives of Azolla species are some of the fastest growing plants, producing substantial biomass when growing in contaminated water and natural ecosystems. Together with their evolutional symbiont, the cyanobacterium Anabaena azollae, Azolla biomass has a unique chemical composition accumulating in each leaf including three major types of bioenergy molecules: cellulose/hemicellulose, starch and lipids, resembling combinations of terrestrial bioenergy crops and microalgae. RESULTS: The growth of Azolla filiculoides in synthetic wastewater led up to 25, 69, 24 and 40 % reduction of NH4-N, NO3-N, PO4-P and selenium, respectively, after 5 days of treatment. This led to a 2.6-fold reduction in toxicity of the treated wastewater to shrimps, common inhabitants of wetlands. Two Azolla species, Azolla filiculoides and Azolla pinnata, were used as feedstock for the production of a range of functional hydrocarbons through hydrothermal liquefaction, bio-hydrogen and bio-ethanol. Given the high annual productivity of Azolla, hydrothermal liquefaction can lead to the theoretical production of 20.2 t/ha-year of bio-oil and 48 t/ha-year of bio-char. The ethanol production from Azolla filiculoides, 11.7 × 103 L/ha-year, is close to that from corn stover (13.3 × 103 L/ha-year), but higher than from miscanthus (2.3 × 103 L/ha-year) and woody plants, such as willow (0.3 × 103 L/ha-year) and poplar (1.3 × 103 L/ha-year). With a high C/N ratio, fermentation of Azolla biomass generates 2.2 mol/mol glucose/xylose of hydrogen, making this species a competitive feedstock for hydrogen production compared with other bioenergy crops. CONCLUSIONS: The high productivity, the ability to grow on wastewaters and unique chemical composition make Azolla species the most attractive, sustainable and universal feedstock for low cost, low energy demanding, near zero maintenance system for the production of a wide spectrum of renewable biofuels.

Lipid production in association of filamentous fungi with genetically modified cyanobacterial cells.

BACKGROUND: Numerous strategies have evolved recently for the generation of genetically modified or synthetic microalgae and cyanobacteria designed for production of ethanol, biodiesel and other fuels. In spite of their obvious attractiveness there are still a number of challenges that can affect their economic viability: the high costs associated with (1) harvesting, which can account for up to 50 % of the total biofuel's cost, (2) nutrients supply and (3) oil extraction. Fungal-assisted bio-flocculation of microalgae is gaining increasing attention due to its high efficiency, no need for added chemicals and low energy inputs. The implementation of renewable alternative carbon, nitrogen and phosphorus sources from agricultural wastes and wastewaters for growing algae and fungi makes this strategy economically attractive. RESULTS: This work demonstrates that the filamentous fungi, Aspergillus fumigatus can efficiently flocculate the unicellular cyanobacteria Synechocystis PCC 6803 and its genetically modified derivatives that have been altered to enable secretion of free fatty acids into growth media. Secreted free fatty acids are potentially used by fungal cells as a carbon source for growth and ex-novo production of lipids. For most of genetically modified strains the total lipid yields extracted from the fungal-cyanobacterial pellets were found to be higher than additive yields of lipids and total free fatty acids produced by fungal and Synechocystis components when grown in mono-cultures. The synergistic effect observed in fungal-Synechocystis associations was also found in bioremediation rates when animal husbandry wastewater was used an alternative source of nitrogen and phosphorus. CONCLUSION: Fungal assisted flocculation can complement and assist in large scale biofuel production from wild-type and genetically modified Synechocystis PCC 6803 strains by (1) efficient harvesting of cyanobacterial cells and (2) producing of high yields of lipids accumulated in fungal-cyanobacterial pellets.

Fungal-assisted algal flocculation: application in wastewater treatment and biofuel production.

BACKGROUND: The microalgal-based industries are facing a number of important challenges that in turn affect their economic viability. Arguably the most important of these are associated with the high costs of harvesting and dewatering of the microalgal cells, the costs and sustainability of nutrient supplies and costly methods for large scale oil extraction. Existing harvesting technologies, which can account for up to 50% of the total cost, are not economically feasible because of either requiring too much energy or the addition of chemicals. Fungal-assisted flocculation is currently receiving increased attention because of its high harvesting efficiency. Moreover, some of fungal and microalgal strains are well known for their ability to treat wastewater, generating biomass which represents a renewable and sustainable feedstock for bioenergy production. RESULTS: We screened 33 fungal strains, isolated from compost, straws and soil for their lipid content and flocculation efficiencies against representatives of microalgae commercially used for biodiesel production, namely the heterotrophic freshwater microalgae Chlorella protothecoides and the marine microalgae Tetraselmis suecica. Lipid levels and composition were analyzed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources from wheat straw and swine wastewater, respectively. The biomass of fungal-algal pellets grown on swine wastewater was used as feedstock for the production of value-added chemicals, biogas, bio-solids and liquid petrochemicals through pyrolysis. Co-cultivation of microalgae and filamentous fungus increased total biomass production, lipid yield and wastewater bioremediation efficiency. CONCLUSION: Fungal-assisted microalgal flocculation shows significant potential for solving the major challenges facing the commercialization of microalgal biotechnology, namely (i) the efficient and cost-effective harvesting of freshwater and seawater algal strains; (ii) enhancement of total oil production and optimization of its composition; (iii) nutrient supply through recovering of the primary nutrients, nitrogen and phosphates and microelements from wastewater. The biomass generated was thermochemically converted into biogas, bio-solids and a range of liquid petrochemicals including straight-chain C12 to C21 alkanes which can be directly used as a glycerine-free component of biodiesel. Pyrolysis represents an efficient alternative strategy for biofuel production from species with tough cell walls such as fungi and fungal-algal pellets.