Nutrient removal and biomass production: advances in microalgal biotechnology for wastewater treatment

Owing to certain drawbacks, such as energy-intensive operations in conventional modes of wastewater treatment (WWT), there has been an extensive search for alternative strategies in treatment technology. Biological modes for treating wastewaters are one of the finest technologies in terms of economy and efficiency. An integrated biological approach with chemical flocculation is being conventionally practiced in several-sewage and effluent treatment plants around the world. Overwhelming responsiveness to treat wastewaters especially by using microalgae is due to their simplest photosynthetic mechanism and ease of acclimation to various habitats. Microalgal technology, also known as phycoremediation, has been in use for WWT since 1950s. Various strategies for the cultivation of microalgae in WWT systems are evolving faster. However, the availability of innovative approaches for maximizing the treatment efficiency, coupled with biomass productivity, remains the major bottleneck for commercialization of microalgal technology. Investment costs and invasive parameters also delimit the use of microalgae in WWT. This review critically discusses the merits and demerits of microalgal cultivation strategies recently developed for maximum pollutant removal as well as biomass productivity. Also, the potential of algal biofilm technology in pollutant removal, and harvesting the microalgal biomass using different techniques have been highlighted. Finally, an economic assessment of the currently available methods has been made to validate microalgal cultivation in wastewater at the commercial level.     

Novel approach for the development of axenic microalgal cultures from environmental samples

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence‐activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.     

The potential of microalgal biotechnology: a review of production and uses of microalgae

An overview of the various aspects, promises and limitations of microalgal biotechnology is presented. The factors of importance in microalgal cultivation as well as the culture systems are briefly described. Microalgal biomasses can fulfil the nutritional requirements of aquatic larvae and organisms. The biochemical composition of algae can be improved by the manipulation of culture conditions. The nutritive value of the microalgal biomasses for human and animal consumption is also commented upon as well as some socio-economical aspects. Among the sources of required nutrients (N, P), wastewaters and manures can upgraded as culture media for microalgae the safety of which has to be evaluated. Harvesting of the biomass is one of the bottlenecks. The various techniques, physical, physico-chemical and biological are outlined and their feasibility and economic interest examined. Microalgal biomasses can be submitted to various technological transformations. Various processes are reviewed in the light of their effects on safety and nutritional value. The possible extraction of fine chemicals and the preparation of protein concentrates is also reported on. The various uses of microalgae lead to a possible competition, to be evaluated, between systems for the production of food, energy and chemicals. The review finally covers the application of genetic manipulation to microalgae.     

Dunaliella biotechnology: methods and applications

The microalga Dunaliella salina is the best commercial source of natural β-carotene. Additionally, different species of Dunaliella can accumulate significant amounts of valuable fine chemicals such as carotenoids, glycerol, lipids, vitamins, minerals and proteins. They also have a large potential for biotechnological processes such as expressing of foreign proteins and treatment of wastewater. In this review, we discussed several biotechnological aspects of the mass cultivation of D. salina like strain selection, carotenoid induction, culture conditions, culture systems and downstream processes. We also discuss several traditional and new applications of the genus.     

Phytase enzymology, applications, and biotechnology

Phytases are phosphohydrolases that initiate the step-wise removal of phosphate from phytate. These enzymes have been widely used in animal feeding to improve phosphorus nutrition and to reduce phosphorus pollution of animal waste. The potential of phytases in improving human nutrition of essential trace minerals in plant-derived foods is being explored. This review covers the basic biochemistry and application of phytases, and emphasizes the emerging biotechnology used for developing new effective phytases with improved properties.     

Improvement of tissue culture,genetic transformation,and applications of biotechnology to Brassica

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.     

Growth and biochemical characterization of microalgal biomass produced in bubble column and airlift photobioreactors: studies in fed-batch culture

Relatively large (0.19 m column diameter, 2 m tall, 0.06 m³ working volume) outdoor bubble column and airlift bioreactors (a split-cylinder and a draft-tube airlift device) were compared for monoseptic fed-batch culture of the microalga Phaeodactylum tricornutum. The three photobioreactors produced similar biomass versus time profiles and final biomass concentration (4 kg m⁻³). The maximum specific growth rate observed within a daily illuminated period in the exponential growth phase, had a value of 0.08 h⁻¹ on the third day of culture. Because of night-time losses of biomass, the specific growth rate averaged over the 4-days of exponential phase was 0.021 h⁻¹ for the three reactors.

The biomass in the vertical column reactors did not experience photoinhibition under conditions (photosynthetically active daily averaged irradiance value of 1150±52 μE m⁻² s⁻¹) that are known to cause photoinhibition in conventional thin-tube horizontal loop reactors. Because of good gas-liquid mass transfer, the dissolved oxygen concentration in the reactors at peak photosynthesis remained <120% of air saturation; thus, oxygen inhibition of photosynthesis and photo-oxidation of the biomass did not occur. Carbohydrate accumulation (up to 13% w/w) by the biomass was favored during light-limited linear growth. A declining light intensity caused a more than five-fold increase in cellular carotenoids but the chlorophylls increased only by about 2.5-fold during the course of the culture. In the stationary phase, up to 2% of the biomass was chlorophylls and carotenoids constituted up to 0.5% of the biomass dry weight.     

Bioprospecting for oil producing microalgal strains: evaluation of oil and biomass production for ten microalgal strains

Microalgae have the ability to grow rapidly, synthesize and accumulate large amounts (approximately 20-50% of dry weight) of lipids. A successful and economically viable algae based oil industry depends on the selection of appropriate algal strains. In this study ten species of microalgae were prospected to determine their suitability for oil production: Chaetoceros gracilis, Chaetoceros mulleri, Chlorella vulgaris, Dunaliella sp., Isochrysis sp., Nannochloropsis oculata, Tetraselmis sp., Tetraselmis chui, Tetraselmis tetrathele and Thalassiosira weissflogii. The study was carried out in 3 L glass flasks subjected to constant aeration and controlled artificial illumination and temperature at two different salinities. After harvesting, the extraction of oil was carried out using the Bligh and Dyer method assisted by ultrasound. Results showed that C. gracilis presented the highest oil content and that C. vulgaris presented the highest oil production.     

A model for light-limited continuous cultures: growth, shading, and maintenance

Light-limited growth in continuous cultures of phototrophic organisms is modeled. It is assumed that light energy up-take rate depends hyperbolically on light intensity and that the maintenance costs are proportional to biomass. Modeling the light distribution caused by shading within the vessel is necessary to explain the existence of steady state in light-limited chemostats. The model fits well to experimental data from literature on light-limited chemostats and turbidostats. Attention is given to the implications of the model for the estimation of the specific maintenance rate constant in light-limited continuous cultures.     

Albumin and mammalian cell culture: implications for biotechnology applications

Albumin has a long historical involvement in design of media for the successful culture of mammalian cells, in both the research and commercial fields. The potential application of albumins, bovine or human serum albumin, for cell culture is a by-product of the physico-chemical, biochemical and cell-specific properties of the molecule. In this review an analysis of these features of albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species to name a few. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival. Application of this knowledge to improve the performance in manufacturing biotechnology and in the emerging uses of cell culture for tissue engineering and stem cell derived therapies is an important prospect.     

Cyanobacteria and microalgae: a positive prospect for biofuels

Biofuel-bioenergy production has generated intensive interest due to increased concern regarding limited petroleum-based fuel supplies and their contribution to atmospheric CO2 levels. Biofuel research is not just a matter of finding the right type of biomass and converting it to fuel, but it must also be economically sustainable on large-scale. Several aspects of cyanobacteria and microalgae such as oxygenic photosynthesis, high per-acre productivity, non-food based feedstock, growth on non-productive and non-arable land, utilization of wide variety of water sources (fresh, brackish, seawater and wastewater) and production of valuable co-products along with biofuels have combined to capture the interest of researchers and entrepreneurs. Currently, worldwide biofuels mainly in focus include biohydrogen, bioethanol, biodiesel and biogas. This review focuses on cultivation and harvesting of cyanobacteria and microalgae, possible biofuels and co-products, challenges for cyanobacterial and microalgal biofuels and the approaches of genetic engineering and modifications to increase biofuel production.     

Mechanisms for hydrogen production by different bacteria during mixed-acid and photo-fermentation and perspectives of hydrogen production biotechnology

H₂ has a great potential as an ecologically-clean, renewable and capable fuel. It can be mainly produced via hydrogenases (Hyd) by different bacteria, especially Escherichia coli and Rhodobacter sphaeroides. The operation direction and activity of multiple Hyd enzymes in E. coli during mixed-acid fermentation might determine H₂ production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating the activity of different Hyd enzymes is an effective way to enhance H₂ production by E. coli in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H₂ production. Mixed carbon (sugar and glycerol) utilization studies enlarge the kind of organic wastes used in biotechnology. During photo-fermentation under limited nitrogen conditions, H₂ production by Rh. sphaeroides is observed when carbon and nitrogen sources are supplemented. The relationship of H₂ production with H⁺ transport across the membrane and membrane-associated ATPase activity is shown. On the other hand, combination of carbon sources (succinate, malate) with different nitrogen sources (yeast extract, glutamate, glycine) as well as different metal (Fe, Ni, Mg) ions might regulate H₂ production. All these can enhance H₂ production yield by Rh. sphaeroides in biotechnology Finally, two of these bacteria might be combined to develop and consequently to optimize two stages of H₂ production biotechnology with high efficiency transformation of different organic sources.     

Chitinase biotechnology: Production,purification, and application

Chitin is one of the most abundant biopolymers and is present in many organisms in different forms. Its resistance to degradation has caused many problems in industry (waste decomposition) and agriculture (as protective structures in pests); this has led to increased interest in chitin‐hydrolyzing enzymes: chitinases. Chitinases are enzymes that break down the 1→4 β‐glycoside bond of N‐acetyl d ‐glucosamine in chitin to produce mono‐ and oligomers. The inducible nature of chitinases, low activity of synthesized enzymes, and inertia of the substrate are only a few of the problems that can be solved by biotechnology to meet industry demands for green, energy‐efficient, pollution‐free, and economically profitable chitin use. This review aims to present the pitfalls and successes in research and production of chitinolytic enzymes, as well as to promote the use of chitinases in everyday practice. The focus is on the biosynthesis of chitinases: inducers, type of fermentation, and media composition. Methods for purification and future perspectives are also discussed.     

Actual,potential and speculative applications of seaweed cellular biotechnology: some specific comments on Gelidium

Cellular biotechnology is a promising application in the propagation and selection of superior strains of seaweeds. Although axenic cultures, organogenetic tissue cultures, vegetative micro-propagation, callus induction and high yields of agar from calli have been described for several species of Gelidium, a number of basic problems remain to be solved. These include standardized methods for obtaining axenic cultures, identification of requirements for organic nutrients, PGR's, cellular disorganization and reorganization, somaclonal variation and somatic incompatibilities. Future progress in seaweed biotechnology will depend on the resolution of many of these problems.     

Rosmarinic acid and its derivatives: biotechnology and applications

Rosmarinic acid (RA) is one of the first secondary metabolites produced in plant cell cultures in extremely high yields, up to 19% of the cell dry weight. More complex derivatives of RA, such as rabdosiin and lithospermic acid B, later were also obtained in cell cultures at high yields. RA and its derivatives possess promising biological activities, such as improvement of cognitive performance, prevention of the development of Alzheimer’s disease, cardioprotective effects, reduction of the severity of kidney diseases and cancer chemoprevention. The TNF-α-induced NF-κB signaling pathway has emerged as a central target for RA. Despite these impressive activities and high yields, the biotechnological production of these metabolites on an industrial scale has not progressed. We summarized data suggesting that external stimuli, the Ca²⁺-dependent NADPH oxidase pathway and processes of protein phosphorylation/dephosphorylation are involved in the regulation of biosynthesis of these substances in cultured plant cells. In spite of growing information about pathways regulating biosynthesis of RA and its derivatives in cultured plant cells, the exact mechanism of regulation remains unknown. We suggest that further progress in the biotechnology of RA and its derivatives can be achieved by using new high-throughput techniques.     

Review: Seaweed tissue culture as applied to biotechnology: Problems,achievements and prospects

Advances have been made in cell and tissue culture of seaweeds to define a unique branch of in vitro techniques; however, they are lagging far behind those of land plants and have limited applications. Explants can be cultivated axenically in enriched or artificial seawater culture media, and regeneration and even callus formation are achieved. In this state of the art technique, seaweed tissue culture may be already useful for certain biotechnological applications, such as clonal propagation of seed material for mariculture. Nevertheless, the absolute control of growth and development as it is exerted in higher plant tissue culture is lacking, and it is required for more complex biotechnological applications in seaweeds. Definitively, we need appropriate cells (competent cells) to induce growth with the most effective chemical regulators in culture medium adjusted towards the addition of carbon sources. Still, free cells and protoplast isolation and regeneration in marine seaweeds constitute the most developed topic in seaweed tissue culture. The regulation of growth and development of seaweed free cell and protoplast cultures may sustain a purposeful use of techniques in the era of genomic applications.     

Seed coats: Structure,development, composition,and biotechnology

Summary Although seeds have been the subject of extensive studies for many years, their seed coats are just beginning to be examined from the perspective of molecular genetics and control of development. The seed coat, plays a vital role in the life cycle of plants by controlling the development of the embryo and determining seed dormancy and germination. Within the seed coat are a number of unique tissues that undergo differentiation to serve specific functions in the seed. A large number of genes are known to be specifically expressed within the seed coat tissues; however, very few of them are understood functionally. The seed coat synthesizes a wide range of novel compounds that may serve the plant in diverse ways, including defense and control of development. Many of the compounds are sources of industrial products and are components of food and feeds. The use of seed coat biotechnology to enhance seed quality and yield, or to generate novel components has not been exploited, largely because of lack of knowledge of the genetic systems that govern seed coat development and composition. In this review, we will examine the recent advances in seed coat, biology from the perspective of structure, composition and molecular genetics. We will consider the diverse avenues that are possible for seed coat biotechnology in the future. This review will focus principally on the seed coats of the Brassicaceae and Fabaceae as they allow us to merge the areas of molecular biology, physiology and structure to gain a perspective on the possibilities for seed coat modifications in the future. The authors have contributed equally and are considered first authors.     

Microalgae as multi-functional options in modern agriculture: current trends,prospects and challenges

Algae are a group of ubiquitous photosynthetic organisms comprising eukaryotic green algae and Gram-negative prokaryotic cyanobacteria, which have immense potential as a bioresource for various industries related to biofuels, pharmaceuticals, nutraceuticals and feed. This fascinating group of organisms also has applications in modern agriculture through facilitating increased nutrient availability, maintaining the organic carbon and fertility of soil, and enhancing plant growth and crop yields, as a result of stimulation of soil microbial activity. Several cyanobacteria provide nitrogen fertilization through biological nitrogen fixation and through enzymatic activities related to interconversions and mobilization of different forms of nitrogen. Both green algae and cyanobacteria are involved in the production of metabolites such as growth hormones, polysaccharides, antimicrobial compounds, etc., which play an important role in the colonization of plants and proliferation of microbial and eukaryotic communities in soil. Currently, the development of consortia of cyanobacteria with bacteria or fungi or microalgae or their biofilms has widened their scope of utilization. Development of integrated wastewater treatment and biomass production systems is an emerging technology, which exploits the nutrient sequestering potential of microalgae and its valorisation. This review focuses on prospects and challenges of application of microalgae in various areas of agriculture, including crop production, protection and natural resource management. An overview of the recent advances, novel technologies developed, their commercialization status and future directions are also included.     

Progress in tissue culture,genetic transformation and applications of biotechnology to trees: an overview

Trees are an integral part of human life, and a vital component of biodiversity. Forest trees in particular are renewable sources of food, fodder, fuel wood, timber and other valuable non-timber products. Due to the rapid growth of population and the human desire to progress, there has been a tremendous reduction in forest cover from the earths surface. To maintain and sustain forest vegetation, conventional approaches have been exploited in the past for propagation and improvement. However, such efforts are confronted with several inherent bottlenecks. Biotechnological interventions for in vitro regeneration, mass micropropagation and gene transfer methods in forest tree species have been practised with success, especially in the last decade. Against the background of the limitations of long juvenile phases and life span, development of plant regeneration protocols and genetic engineering of tree species are gaining importance. Genetic engineering assumes additional significance, because of the possibility of introducing a desired gene in a single step for precision breeding of forest trees. There are no comprehensive and detailed reviews available combining research developments with major emphases on tissue culture and basic genetic transformation in tree species. The present communication attempts to overview the progress in tissue culture, genetic transformation and biotechnological applications in the last decade and future implications.     

Variations in the microalgal structure in paddy soil in Osaka, Japan: comparison between surface and subsurface soils

Seasonal variations in microalgal communities were compared between surface and subsurface paddy soils in Osaka, Japan. Soil samples were collected from depths of 0–1 (surface), 8–9, and 17–18 cm. Diatom cells were counted directly, and the numbers of other microalgae were estimated using a culture method. The microalgal community as well as the soil properties changed drastically in the surface soil as a consequence of alternate flooding and drainage. In the soil collected at a depth of 0–1 cm, the cell density of diatoms and the viable count of other microalgae markedly increased, and Chlorella spp., Nitzschia spp., and Navicula spp. were predominant during the flooding period, whereas Scenedesmus spp. and Hantzschia spp. were predominant during the drainage period. In contrast, in the soils collected at depths of 8–9 and 17–18 cm, the cell density of diatoms and the viable count of other microalgae remained constant. Despite the unavailability of light, a large number of microalgae were present in these subsurface soils throughout the annual cultivation cycle, and Scenedesmus spp. and Nitzschia spp. were always dominant. Cyanophytes were also present at all the depths but had low relative frequencies. These results suggest that the algae that are predominant in paddy soil can survive not only drastic changes in water content but also complete darkness.