Microalgae aquaculture feeds

Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the production of larvae and juvenile shell- and finfish, as well as for raising the zooplankton required for feeding of juvenile animals. The blue-green algaSpirulina is used in substantial amounts (over 100 t y^–1) as a fish and shrimp feed, and even larger markets can be projected if production costs could be reduced. Another potential large-scale application of microalgae is the cultivation ofHaematococcus for the production of the carotenoid astaxanthin, which gives salmon flesh its reddish color. In the long-term microalgae biomass high in lipids (omega-3 fatty acids) may be developed as substitutes for fish oil-based aquaculture feeds. In shrimp ponds the indigenous algal blooms supply a part of the dietary requirements of the animals, but it is difficult to maximize algal productivities. A separate algal production system could feed the shrimps and minimize the need for added feed. Bivalves feed essentially exclusively on marine microalgae throughout their life cycle. The development of cultivation technologies for such microalgae would allow the onshore production of these animals, with greatly improved product quality and safety.This paper was presented at the Symposium on Applied Phycology at the Fourth International Phycological Congress, Duke University.     

Nitrogen fixation by Anabaena cylindrica

Hydrogen-supported nitrogenase activity was demonstrated in Anabaena cylindrica cultures limited for reductant. Nitrogen-fixing Anabaena cylindrica cultures sparged in the light with anaerobic gases in the presence of the photosynthesis inhibitor DCMU slowly lost their ability to reduce acetylene in the light under argon but exhibited near normal activities in the presence of 11% H₂ (balance argon). The hydrogen-supported nitrogenase activity was half-saturated between 2 and 3% H₂ and was strongly inhibited by oxygen (50% inhibition at about 5–6% O₂). Batch cultures of Anabaena cylindrica approaching stationary growth phase (“old” cultures) lost nitrogenase-dependent hydrogen evolution almost completely. In these old cultures hydrogen relieved the inhibitory effects of DCMU and O₂ on acetylene reduction. Our results suggest that heterocysts contain an uptake hydrogenase which supplies an electron transport chain to nitrogenase but which couples only poorly with the respiratory chain in heterocysts and does not function in CO₂ fixation by vegetative cells.     

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica

The mechanism of O₂ protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O₂ inhibition of nitrogenase activity correlated with the O₂ tension of the medium in which heterocyst formation was induced. O₂ resistance also correlated with the apparent K_m for acetylene, indicating that O₂ tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O₂ that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O₂ sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O₂ but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O₂ diffusion barrier and active respiration by the heterocyst.     

Hydrogen Production by the Thermophilic Alga Mastigocladus laminosus: Effects of Nitrogen, Temperature, and Inhibition of Photosynthesis

Hydrogen production by nitrogen-limited cultures of a thermophilic blue-green alga (cyanobacterium), Mastigocladus laminosus, was studied to develop the concept of a high-temperature biophotolysis system. Biophotolytic production of hydrogen by solar radiation was also demonstrated. Hydrogen consumption activity in these cultures was relatively high and is the present limiting factor on both the net rate and duration of hydrogen production.     

Cultivation of algae and nutrient removal in a waste heat utilization process

A process providing a beneficial use for waste heat and excess nutrients in the cooling waters of nuclear reactors and fossil-fueled power generating plants has been developed. The process involves the cultivation of selected strains of thermotolerant microalgae in heated discharge waters and the subsequent harvesting of the algal biomass for nutrient removal, recovery of energy and fertilizer, and extraction of high value products. The design of such a process is presented for a large cooling reservoir receiving a discharge of 10⁹1^?1 d^?1 of secondary cooling water containing 100 μg 1^?1 of available P and 400 μg 1^?1 of available N. Based on this nutrient load, with a 1% P content in the algal biomass and a productivity of 10 g m^?2 d ^?1, a 100 ha region would be needed for the process. Hydraulic barriers (submerged plastic curtains) would isolate the 100 ha algal production area “cultivation zone” in the influent end of the reservoir to create a hydraulic and thermal environment conductive to the selective growth of filamentous, thermotolerant, nitrogen-fixing, blue-green algae. The algal culture would be inoculated into the thermal plume and harvested near the distal barrier of the cultivation zone with rotating, backwashed, fine mesh screens (“microstrainers”). A portion of the harvested biomass would be recycled to the inoculation site to maintain a dense culture. This process could mitigate both thermal and nutrient loadings on receiving bodies of water.     

Microalgal industry in China: challenges and prospects

Over the past 15 years, China has become the major producer of microalgal biomass in the world. Spirulina (Arthrospira) is the largest microalgal product by tonnage and value, followed by Chlorella, Dunaliella, and Haematococcus, the four main microalgae grown commercially. China’s production is estimated at about two-thirds of global microalgae biomass of which roughly 90 % is sold for human consumption as human nutritional products (‘nutraceuticals’), with smaller markets in animal feeds mainly for marine aquaculture. Research is also ongoing in China, as in the rest of the world, for other high-value as well as commodity microalgal products, from pharmaceuticals to biofuels and CO₂ capture and utilization. This paper briefly reviews the main challenges and potential solutions for expanding commercial microalgae production in China and the markets for microalgae products. The Chinese Microalgae Industry Alliance (CMIA), a network founded by Chinese microalgae researchers and commercial enterprises, supports this industry by promoting improved safety and quality standards, and advancement of technologies that can innovate and increase the markets for microalgal products. Microalgae are a growing source of human nutritional products and could become a future source of sustainable commodities, from foods and feeds, to, possibly, fuels and fertilizers.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Hydrogen production by microalgae

The production of H₂ gas from water and sunlightusing microalgae, `biophotolysis', has been a subjectof applied research since the early 1970s. A numberof approaches have been investigated, but most provedto have fundamental limitations or requireunpredictable research breakthroughs. Examples areprocesses based on nitrogen-fixing microalgae andthose producing H<sub>2</sub> and O<sub>2</sub> simultaneously fromwater (`direct biophotolysis'). The most plausibleprocesses for future applied R & D are those whichcouple separate stages of microalgal photosynthesisand fermentations (`indirect biophotolysis'). Theseinvolve fixation of CO₂ into storagecarbohydrates followed by their conversion to H₂by the reversible hydrogenase, both in dark andpossibly light-driven anaerobic metabolic processes. Based on a preliminary engineering and economicanalysis, biophotolysis processes must achieve closeto an overall 10% solar energy conversion efficiencyto be competitive with alternatives sources ofrenewable H₂, such as photovoltaic-electrolysisprocesses. Such high solar conversion efficiencies inphotosynthetic CO₂ fixation could be reached bygenetically reducing the number of light harvesting(antenna) chlorophylls and other pigments inmicroalgae. Similarly, greatly increased yields ofH₂ from dark fermentation by microalgae could beobtained through application of the techniques ofmetabolic engineering. Another challenge is toscale-up biohydrogen processes with economicallyviable bioreactors.Solar energy driven microalgae processes forbiohydrogen production are potentially large-scale,but also involve long-term and economically high-riskR&D. In the nearer-term, it may be possible tocombine microalgal H₂ production with wastewatertreatment.     

Bioengineering aspects of biophotolysis

The practical aspects of producing hydrogen by photosynthetic microorganisms are reviewed. Various alternative concepts for hydrogen production are discussed, both single and two-stage systems. The best developed process currently is based on nitrogen-fixing heterocystous blue-green algae which can produce hydrogen and oxygen simultaneously. Solar energy conversion to hydrogen efficiencies of 0.2% averaged over several weeks have been obtained with outdoor systems. Practical systems would require a ten-fold increase in conversion efficiencies. Also, systems which produce pure hydrogen are preferred. Photosynthetic bacteria are of near-term applications. A general design for a biophotolysis system is proposed consisting of vertically arranged, thin-walled glass tubes with an inert gas recirculated through the cultures for mixing and removal of hydrogen. Gas mass transfer considerations, energy utilization, and economics favour such a system.