Diversity of algae and their biotechnological potential.

This chapter will discuss the diversity of algae and show that the diversity is much greater than just obligately oxygenic photosynthetic algae and that it includes many mixotrophic and heterotrophic organisms that are more similar to the major groups of microorganisms. The photosynthetic groups are seen as part of the plant kingdom, whereas the non-photosynthetic groups are not related to plants at all. The organisation of algal groups has become complex and confusing - The chapter will address the problems within this area of eukaryotic taxonomy. The metabolic diversity of algae and the ability to genetically engineer algae are key components in developing the biotechnology of algae. As more researchers become interested in exploiting algae for a number of industrial products, it is important to understand the relationships between different groups of algae and the relationships of algae with the rest of the living world.

Utilization and accumulation of compatible solutes in Halomonas pacifica: a species of moderately halophilic bacteria isolated from a saline lake in South Libya.

When grown in high salt concentrations, halophilic bacteria often accumulate compatible solutes, which have major applications in biotechnology because they stabilize cells and proteins. Four Gram-negative bacterial strains, belonging to the family Halomonadaceae, were isolated from Qaberoun and Um-Alma lakes in South Libya using high-salinity medium. The strains were identified using 16S rRNA gene sequencing as belonging to Halomonas pacifica (strain ABQ1), Halomonas venusta (ABQ2), Halomonas elongata (ABU1) and Halomonas salifodinae (ABU2). H. pacifica ABQ1 is a moderate halophile (salinity range 0.05 to 2.5 M NaCl), with a broad tolerance to pH (7 to 9) and temperature (25-37 °C). Addition of the compatible solutes glycine betaine (betaine) and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) to the medium had a positive effect on growth of H. pacifica at 2 M NaCl. In rich LB medium, betaine was the major compatible solute accumulated, with ectoine only being accumulated at salinities in excess of 1 M NaCl. In minimal M9 medium, betaine was not produced, but increasing amounts of ectoine were synthesized with increasing salinity, and hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was also synthesized when the cells were grown in very high salt. We have thus identified H. pacifica as a producer of ectoine and hydroxyectoine, with more being produced at higher salinities. As industrial demand for these compatible solutes continues to increase, this system has biotechnological potential.

Quantitative proteomic comparison of salt stress in Chlamydomonas reinhardtii and the snow alga Chlamydomonas nivalis reveals mechanisms for salt-triggered fatty acid accumulation via reallocation of carbon resources.

BACKGROUND: Chlamydomonas reinhardtii is a model green alga strain for molecular studies; its fully sequenced genome has enabled omic-based analyses that have been applied to better understand its metabolic responses to stress. Here, we characterised physiological and proteomic changes between a low-starch C. reinhardtii strain and the snow alga Chlamydomonas nivalis, to reveal insights into their contrasting responses to salinity stress. RESULTS: Each strain was grown in conditions tailored to their growth requirements to encourage maximal fatty acid (as a proxy measure of lipid) production, with internal controls to allow comparison points. In 0.2 M NaCl, C. nivalis accumulates carbohydrates up to 10.4% DCW at 80 h, and fatty acids up to 52.0% dry cell weight (DCW) over 12 days, however, C. reinhardtii does not show fatty acid accumulation over time, and shows limited carbohydrate accumulation up to 5.5% DCW. Analysis of the C. nivalis fatty acid profiles showed that salt stress improved the biofuel qualities over time. Photosynthesis and respiration rates are reduced in C. reinhardtii relative to C. nivalis in response to 0.2 M NaCl. De novo sequencing and homology matching was used in conjunction with iTRAQ-based quantitative analysis to identify and relatively quantify proteomic alterations in cells exposed to salt stress. There were abundance differences in proteins associated with stress, photosynthesis, carbohydrate and lipid metabolism proteins. In terms of lipid synthesis, salt stress induced an increase in dihydrolipoyl dehydrogenase in C. nivalis (1.1-fold change), whilst levels in C. reinhardtii remained unaffected; this enzyme is involved in acetyl CoA production and has been linked to TAG accumulation in microalgae. In salt-stressed C. nivalis there were decreases in the abundance of UDP-sulfoquinovose (- 1.77-fold change), which is involved in sulfoquinovosyl diacylglycerol metabolism, and in citrate synthase (- 2.7-fold change), also involved in the TCA cycle. Decreases in these enzymes have been shown to lead to increased TAG production as fatty acid biosynthesis is favoured. Data are available via ProteomeXchange with identifier PXD018148. CONCLUSIONS: These differences in protein abundance have given greater understanding of the mechanism by which salt stress promotes fatty acid accumulation in the un-sequenced microalga C. nivalis as it switches to a non-growth state, whereas C. reinhardtii does not have this response.

Microalgae for biofuel production.

Microalgae have been used commercially since the 1950s and 1960s, particularly in the Far East for human health foods and in the United States for wastewater treatment. Initial attempts to produce bulk chemicals such as biofuels from microalgae were not successful, despite commercially favorable conditions during the 1970s oil crisis. However, research initiatives at this time, many using extremophilic microalgae and cyanobacteria (e.g., Dunaliella and Spirulina), did solve many problems and clearly identified biomass productivity and harvesting as the two main constraints stopping microalgae producing bulk chemicals, such as biofuels, on a large scale. In response to the growing unease around global warming, induced by anthropogenic CO2 emissions, microalgae were again suggested as a carbon neutral process to produce biofuels. This recent phase of microalgae biofuels research can be thought to have started around 2007, when a very highly cited review by Chisti was published. Since 2007, a large body of scientific publications have appeared on all aspects of microalgae biotechnology, but with a clear emphasis on neutral lipid (triacylglycerol) synthesis and the use of neutral lipids as precursors for biodiesel production. In this review, the key research on microalgal biotechnology that took place prior to 2007 will be summarized and then the research trends post 2007 will be examined emphasizing the research into producing biodiesel from microalgae.

Effect of ammonium and high light intensity on the accumulation of lipids in Nannochloropsis oceanica (CCAP 849/10) and Phaeodactylum tricornutum (CCAP 1055/1).

BACKGROUND: Microalgae accumulate lipids when exposed to stressful conditions such as nutrient limitation that can be used to generate biofuels. Nitrogen limitation or deprivation is a strategy widely employed to elicit this response. However, this strategy is associated with a reduction in the microalgal growth, leading to overall poor lipid productivities. Here, we investigated the combined effect of a reduced source of nitrogen (ammonium) and super-saturating light intensities on the growth and induction of lipid accumulation in two model but diverse microalgal species, Phaeodactylum tricornutum and Nannochloropsis oceanica. We hypothesized that the lower energy cost of assimilating ammonium would allow the organisms to use more reductant power for lipid biosynthesis without compromising growth and that this would be further stimulated by the effect of high light (1000 µmol m-2 s-1) stress. We studied the changes in growth and physiology of both species when grown in culture media that either contained nitrate or ammonium as the nitrogen source, and an additional medium that contained ammonium with tungsten in place of molybdenum and compared this with growth in media without nitrogen. We focused our investigation on the early stages of exposure to the treatments to correspond to events relevant to induction of lipid accumulation in these two species. RESULTS: At super-saturating light intensities, lipid productivity in P. tricornutum increased twofold when grown in ammonium compared to nitrogen free medium that increased further when tungsten was present in the medium in place of molybdenum. Conversely, N. oceanica growth and physiology was not compromised by the high light intensities used, and the use of ammonium had a negative effect on the lipid productivity, which was even more marked when tungsten was present. CONCLUSIONS: Whilst the use of ammonium and super-saturating light intensities in P. tricornutum was revealed to be a good strategy for increasing lipid biosynthesis, no changes in the lipid productivity of N. oceanica were observed, under these conditions. Both results provide relevant direction for the better design of processes to produce biofuels in microalgae by manipulating growth conditions without the need to subject them to genetic engineering manipulation.

The Search for a Lipid Trigger: The Effect of Salt Stress on the Lipid Profile of the Model Microalgal Species Chlamydomonas reinhardtii for Biofuels Production.

BACKGROUND: Algal cells produce neutral lipid when stressed and this can be used to generate biodiesel. OBJECTIVE: Salt stressed cells of the model microalgal species Chlamydomonas reinhardtii were tested for their suitability to produce lipid for biodiesel. METHODS: The starchless mutant of C. reinhardtii (CC-4325) was subjected to salt stress (0.1, 0.2 and 0.3 M NaCl) and transesterification and GC analysis were used to determine fatty acid methyl ester (FAME) content and profile. RESULTS: Fatty acid profile was found to vary under salt stress conditions, with a clear distinction between 0.1 M NaCl, which the algae could tolerate, and the higher levels of NaCl (0.2 and 0.3 M), which caused cell death. Lipid content was increased under salt conditions, either through long-term exposure to 0.1 M NaCl, or short-term exposure to 0.2 and 0.3 M NaCl. Palmitic acid (C16:0) and linolenic acid (C18:3n3) were found to increase significantly at the higher salinities. CONCLUSION: Salt increase can act as a lipid trigger for C. reinhardtii.

Periodic CO2 Dosing Strategy for Dunaliella salina Batch Culture.

A periodic CO2 dosing strategy for D. salina 19/30 batch culture is proposed. A model of periodic CO2 dosing including dosing time calculation, dosing interval estimation and final chlorophyll yield prediction was established. In experiments, 5% CO2/95% N2 gas was periodically dosed into D. salina culture. Two different gas dosing flow rates were tested. The corresponding dosing time for each flow rate was estimated via the model (10 min·d-1 for 0.7 L·min-1 and 36 min·d-1 for 0.3 L·min-1). Daily pH measurements showed that the pH of these cultures dosed periodically was always kept between 7.5 and 9.5, which highlights that periodic gas supply can maintain a suitable range of pH for microalgal growth without expensive buffers. Notably the culture dosed for set daily intervals was seen to have similar growth to the culture supplied constantly, but with much higher CO2 capture efficiency (11%-18%) compared to continuous dosing (0.25%). It shows great potential for using periodic gas supply to reduce cost, wasted gas and energy use.

Analysis of protein solvent interactions in glucose dehydrogenase from the extreme halophile Haloferax mediterranei.

The structure of glucose dehydrogenase from the extreme halophile Haloferax mediterranei has been solved at 1.6-A resolution under crystallization conditions which closely mimic the "in vivo" intracellular environment. The decoration of the enzyme's surface with acidic residues is only partially neutralized by bound potassium counterions, which also appear to play a role in substrate binding. The surface shows the expected reduction in hydrophobic character, surprisingly not from changes associated with the loss of exposed hydrophobic residues but rather arising from a loss of lysines consistent with the genome wide-reduction of this residue in extreme halophiles. The structure reveals a highly ordered, multilayered solvation shell that can be seen to be organized into one dominant network covering much of the exposed surface accessible area to an extent not seen in almost any other protein structure solved. This finding is consistent with the requirement of the enzyme to form a protective shell in a dehydrating environment.

The effect of NaCl on the growth of a Halomonas species: accumulation and utilization of compatible solutes.

The effect of NaCl on growth and compatible solute utilization was investigated in a Halomonas species. Growth of Halomonas was observed in medium of low osmolarity (high water activity) when only 01 mM Na+ was present. However, lowering the water activity, by addition of KCl or sucrose, inhibited growth in this low-Na+ medium, but growth could be restored by the addition of NaCl. The bacterium could grow on glucose as the sole carbon source in up to 355 M NaCl and was shown also to metabolize glycine betaine. However NaCl concentrations greater than 2 M inhibited growth when glycine betaine was the sole carbon source. Glycine betaine was transported into the cells by a process stimulated by NaCl irrespective of whether the carbon source was glucose or glycine betaine. Cytoplasmic levels of glycine betaine were monitored throughout the growth cycle in 2 M NaCl medium with glycine betaine as sole carbon source. As the culture aged, glycine betaine was increasingly replaced by the tetrahydropyrimidine ectoine as the major cytoplasmic solute. The increased sensitivity to high NaCl concentrations when grown on glycine betaine may be due to the glycine betaine catabolic pathway enzymes being inhibited by the increasing external solute concentration.