Marine ecosystems that form the marine environment are the largest habitats on Earth for a diversified group of organisms. The biotic community of marine envi­ronments is dominated by microalgae. They are among the largest primary pro­ducers of biomass in the marine environment and are common inhabitants of the tidal and intertidal areas of the marine ecosystem. These algae exhibit a character­istic geographical distribution pattern under the influence of several environmen­tal factors (Vijayaraghavan and Kaur, 1997). Nevertheless, the coastal ecosystem in the marine environment is very complex where all organisms exist in mutual dependency. Although considerable attention has been paid to distribution, abun­dance, growth, culture, biochemical constituents, by-products, and bioactivity of marine algae in different parts of the world (Faulkner, 1984), information is avail­able on the microbes, planktonic and faunal associates of marine algae, and the impact of various environmental factors on their distribution. The algal biotope with its morphological diversity is considered important in providing food, living space, and refuge, and offers a variety of potential habitats for the faunal spe­cies, including planktonic forms. A detailed investigation is therefore necessary to understand the actual nature of the association between algae and other forms to appreciate the potential importance of this interaction in the marine ecosystem. Steele (1988) described the “heuristic projection,” which illustrates the scales of importance in monitoring pelagic components of the ecological unit. The large marine ecosystem (LME) approach defines a spatial domain based on ecological principles and, thereby, provides a basis for focused temporal and spatial scientific research and monitoring efforts in support of management aimed at the long-term productivity and sustainability of marine habitats and resources. The plankton of LMEs can be studied by deploying Continuous Plankton Recorder (CPR) sys­tems (Glover, 1967) through commercial vessels. Advanced plankton recorders can be installed with sensors for intense recording of temperature, salinity, chlo­rophyll, nitrate/nitrite, petroleum hydrocarbons, light, bioluminescence, and pri­mary productivity (UNESCO, 1992; Williams, 1993), which will help monitor the changes in phytoplankton composition, dominance, and long-standing changes in the physical and nutrient characteristics of the LME. In addition, longer-term changes in relation to the biofeedback of the plankton community toward adverse climate may also be clearly understood (Hayes et al., 1993; Jossi and Goulet, 1993; Williams, 1993).

The phytoplankton community includes 5,000 marine species of unicellular algae and has a broad diversity of cell size (mostly in the range of 1 to 100 pm), morphology, physiology, and biochemical composition (Margalef, 1978). All phy­toplankton species are capable of photosynthesis, and many have the capacity for rapid cell division and population growth—up to four doublings per day. The popu­lation dynamics of the phytoplankton can be interpreted as responses to changes in the individual processes that regulate the biomass (total quantity, in measures such as carbon, nitrogen, or chlorophyll concentration), species composition, and spatial distribution of the phytoplankton population. Phytoplankton have a wide distribution in all habitats of the marine environment and play a major role in the food chain of an aquatic ecosystem. Some of the phytoplankton species also act as bio-indicators, reflecting changes in the environment. Different hydrobiological parameters, such as pH, temperature, salinity, alkalinity, nutrient concentration, solar radiation, etc., determine species composition, diversity, succession, and abundance of phytoplank­ton (Perumal et al., 1999; Redekar and Wagh, 2000a, b). Remarkable changes in the irradiance toward phytoplankton could occur due to changing seasonal, diurnal cycles and weather conditions. Diatoms are the significant and often dominant con­stituent of benthic microalgal communities in estuarine and shallow coastal regions (Sullivan, 1999).

The taxonomy of the most common species with reliable distributional informa­tion and records will allow for the design of ecological role models incorporating the effects of climatic parameters, which would be very useful in predicting shifts in distribution due to climatic changes.

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Perpetual transfer leading to long-term culturing, usually under conditions very dif­ferent from its natural environment, leads to genetic variants among the popula­tion adapted to the artificial culturing environment. To sidestep the shortcomings of serial sub-culturing, alternate methods of ex-situ conservation of algal strains are suggested. Continuous maintenance of actively growing algal strains on a long­term basis is often costly, and time and labor consuming. In contrast, cultures can be maintained alive in a retarded metabolic state that requires less attention. One approach is to maintain resting spores or other dormant stages of some algal species (such as akinetes) at ambient temperatures for many years without any attention. Leeson et al. (1984) were able to recover aplanospores of Haematococcus pluvialis Flotow from air-dried soil even after 27 years. However, it should be considered that the viability of resting stages generally declines with time, and many aquatic algae do not show any insistent dormant stage. Hence, the addition of bacteriostatic chemi­cals and agents that prevent autolysis of algal cells to help improve the cell viability during the entire storage time is generally recommended. Some of the major preser­vatives in use today are formalin, 1% Lugol solution, and 3% glutaraldehyde (Wetzel and Likens, 2000). The concentration can be altered based on the type and nature of the algae and maintenance conditions. For instance, a 3% glutaraldehyde concentra­tion is too high, causing withering or complete disintegration of cells beyond the ability for retention of normal cell shape, specifically of wall-less flagellates.

To overcome the shortcomings and inclusion of chemicals in maintenance medium, lyophilization has been accepted widely as a means of conserving viable cultures of all microorganisms in a desiccated state. However, lyophilization involves vacuum desiccation under freezing and subsequent thawing, so cell revival mandates inclusion of cryoprotective agents at high concentrations to offer protection from dam­age. The cryoprotectants extensively used for algae are methanol, dimethylsulfoxide (DMSO), and glycerol (Taylor and Fletcher, 1998). Methanol and DMSO are preferred for freshwater and terrestrial microalgal cryopreservation, while glycerol and DMSO are useful for marine phytoplanktons (Day et al., 2000). The above are penetrating cryo- protectants and passively move through the plasma membrane to equilibrate between the cell interior and the extracellular solution. Penetrating cryoprotectants are toxic at high concentrations (Adam et al., 1995; Santarius, 1996). Hence, permeating cryopro — tectants should be added prior to cryopreservation and should immediately be removed after thawing. Algal spore preservation is heavily dependent on bacterial contamination. Hence, preservation of spores of the green seaweeds Ulva fasciata and U. pertusa was improved by the addition of ampicillin in f/2 medium at 4°C (Bhattarai et al., 2007).

Cryopreservation is most suited for algae that do not require that the normal rest­ing stage be maintained indefinitely. Because microalgae are cryopreserved as large populations of algal units, the percentage of viability of identical cultures is of great concern and often varies. However, with proper physiological conditioning prior to freezing, the variability can be minimized. This is one of the key reasons that, to date, most dinoflagellates, cryptophytes, synurophytes, and raphidophytes are not successfully cryopreserved. In contrast, most marine diatoms can be effectively cryopreserved, with high viability, although freshwater diatoms fail to revive and have thus proven more problematic. Examinations of large numbers of strains have taken place at the four major protistan collections: Culture Collection of Algae and Protozoa (CCAP) (United Kingdom), The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) (United States), Sammlung von Algenku Huren Gottingen (SAG) (Germany), and The Culture Collection of Algae at UTEX (United States); examination reveals that chlorarachniophytes, eustigmatophytes, pelagophytes, phaeothamniophytes, and ulvophytes also have very high success rates, comparable with the other green algae and cyanobacteria. Algal strains that have been reestablished at NREL are being cryopreserved in an effort to reduce the workload associated with maintaining an algae collection and to prevent unintended loss or genetic drift, a risk associated with frequent transfer. The cryofreezer uses liquid nitrogen, and cultures are stored at -195°C in the vapor phase. Nevertheless, it has been distinguished that virtually all large cell sized algae, and most filamentous forms, cannot as yet be cryopreserved. Attempts to determine the fundamental rea­sons for this failure of cryopreservation on large and complex algae are not satisfy­ing. This warrants auxiliary research on the basic mechanisms of freezing damage. Furthermore, the pragmatic development of improved techniques will expand the number and diversity of algal taxa that can be successfully cryopreserved.

Subburamu Karthikeyan

Department of Agricultural Microbiology Tamil Nadu Agricultural College Coimbatore, India

CONTENTS

3.1 Bioprospecting……………………………………………………………………………………………. 17

3.2 Isolation and Characterization of Naturally Occurring Algae……………………. 21

3.3 Isolation Techniques…………………………………………………………………………………… 22

3.3.1 Media Configuration……………………………………………………………………… 23

3.3.2 Traditional Methods………………………………………………………………………. 23

3.3.3 Advanced Methods………………………………………………………………………… 27

3.4 Screening Criteria and Methods………………………………………………………………….. 28

3.5 Screening and Selection for Lipid Production…………………………………………….. 31

3.6 Preservation………………………………………………………………………………………………… 33

3.6.1 Transfer Techniques………………………………………………………………………. 33

3.6.2 Maintenance Conditions………………………………………………………………… 33

3.6.3 Cryopreservation……………………………………………………………………………. 35

3.7 Role of Repositories……………………………………………………………………………………. 37

3.8 Concluding Remarks………………………………………………………………………………….. 37

References…………………………………………………………………………………………………………… 38

3.1 BIOPROSPECTING

Bioprospecting is the collection of biological material and the exploitation of its molecular, biochemical, and/or genetic content for the development of a commer­cial product. Precisely, bioprospecting relies on the endowment of a bioresource, a stock of novel biodiversity. Bioprospecting is a time-consuming process, where new products and markets must be identified, and a compound that covers com­mercial demands and social needs must be discovered. Algae are ubiquitous and have been evolving as primary biomass producers on the Earth for billions of years. Exploring this existing, self-maintaining, and diverse life form offers a rich base for global biotechnological innovations. Indigenous species are well adapted to pre­vailing regional abiotic and biotic factors, and further local strains provide an ideal platform for additional strain improvement and process optimization. Many algal species remain unknown or unexplored in science, giving logical attention to explore

Lipid Accumulating Algal Groups in Terms of Abundance

a Seaweeds are included in the green algae (Chlorophyta); b Red algae (Rhodophyta); and c Brown algae (Ochrophyta or Heterokontophyta). d Adapted from Khan et al. (2009). e The World Conservation Union (2010).

this realm for potential application. To further illustrate this point, only fifteen of the currently known microalgal species are mass cultivated in some applied form for use in nutraceuticals, aquaculture feeds, or for wastewater treatment (Raja et al., 2008). Furthermore, the estimated unknown species for all clades of algae are projected to be two orders of magnitude greater than the currently known species (Norton et al., 1996) (Table 3.1). Of the commercialized algae, only a few species are cultivated

TABLE 3.2

Annual Biomass Potential of Microalgae in Comparison to Major Cultivated Crops

at substantial levels, which is trivial when compared to the annual global produc­tion of cultivated crops (Table 3.2). To propel algal biotechnological applications to commercially significant sustainable levels, regional species should be investigated for potential application to mass-scale cultivation. The idea of bioprospecting indig­enous microalgae for high-value or bioactive products is not innovative. The Aquatic Species Program of National Renewable Energy Laboratory (NREL) stocks more than 3,000 microalgal strains from the United States and Hawaii (Sheehan et al., 1998). Microalgae capable of producing large quantities of docosahexaenoic acid were isolated from marine environments of Western Taiwan (Yang et al., 2010).

Up to now, the key emphasis of microalgal biofuel research has focused on upstream aspects such as bioreactor designs, biomass and lipid production from microalgae, and downstream aspects such as biomass harvesting and the chemistry of oil production.

Microalgal bioprospecting includes isolation of exceptional microalgal strains from aquatic environments for potential value-added products and fine chemicals (Olaizola,
2003; Spolaore et al., 2006). A great deal of literature is accessible on the mass cultivation and sustainable use of microalgae for biofuels; however, relatively few stud­ies have focused on microalgal bioprospecting. Nevertheless, bioprospecting and the establishment of a microalgal collection exclusively for biofuel production have not been reported thus far. Algal bioprospecting or phycoprospecting of indigenous species has an advantage over other methods of sourcing algae from type culture collections and from genetically engineered organisms (Wilkie et al., 2011) (Table 3.3). Screening native algae for species with desirable traits provides a robust biological platform for bioresource production. This biological platform comes equipped with millions of years

TABLE 3.3

Comparison of Different Methods of Sourcing Algae

Source: Adapted from Wilkie et al. 2011.

FIGURE 3.1 Schematic outline of procedures in bioprospecting algae for biodiesel production.

of adaptation to the local climate and biota, meaning less energy expended on methods of environmental control and sterile techniques. Specific criteria for the production of biofuels from indigenous algae should include biomass and lipid productivity, harvesting the cells, and oil extractability. Further, the algal oil derived should contain 20%-25% C16 and C18 saturated fatty acid methyl esters and high amounts of unsaturated fatty acid chains, thus offering more cleavage sites to produce hydrocarbons (Gunstone and Harwood, 2007). Phycoprospecting may improve the efficiency of lipid extraction by yielding organisms with traits amenable to oil recovery. For specific objectives such as algal biodiesel, feedstocks for wastewater utilization or mitigation of greenhouse gases (GHGs), the chosen algal strains should satisfy requirements such as the ability to survive in wastewater, capability to grow robustly with higher cell densities, hyper­lipid content as triacylglycerol, and be capable of heterotrophic or mixotrophic growth as wastewater provides both organic and inorganic carbon sources. Until now, research on screening and acclimation of microalgae to adapt to wastewater environments is very sporadic (Zhou et al., 2011). A schematic outline and procedures in bioprospecting algae for biofuel production are outlined in Figure 3.1.

Repositories are indispensable in preserving the diversity of natural habitats, pro­tecting genetic material, and providing basic resources for research. At present, only a few major algal collection centers exist in the United States and other countries. They currently maintain thousands of different algal strains and support the research and industrial community with their expertise in algae biology. The function of a culture collection often transcends simple depository functions. They may also sup­port research on determining strain characteristics, cryopreservation, and phylogeny, either by themselves or in connection with outside collaborators. Currently, no cen­tral database exists that provides global information on the characteristics of cur­rently available algal strains. Protection of intellectual property in private industry has further exacerbated the flow of relevant strain data. Some minimal growth infor­mation is available from existing culture collections, but it is very difficult to obtain more detailed information on growth, metabolites, and the robustness of particular existing strains. The establishment of a central strain, open-access repository could accelerate research on algae-based biofuel production systems.

Above all, it is certain that many algal strains in established collections have been cultivated for several decades, and some may have lost their original properties, such as mating capability or versatility regarding nutrient requirements. To obtain ver­satile and robust strains that can be used for mass culture in biofuel applications, it would be prudent to consider the isolation of new native strains directly from unique environments. For both direct breeding and metabolic engineering approaches to improve biofuels production, it will be important to isolate a wide variety of algae for assembly into a culture collection that will serve as a bioresource for further algal biofuel research.

Despite the existence of morphologically diverse algae in a wide variety of terres­trial and aquatic habitats, work with algae has been restricted to a relatively few representatives. This seems partly the result of difficulties encountered in both
the isolation and the subsequent purification of the algae. It has been suggested that the techniques normally used to isolate algae may severely limit the number of algal species that can be readily cultured (Castenholz, 1988). The goals of algae isolation and screening efforts are to identify and maintain promising algal specimens for cul­tivation and strain development. Because it is premature to decide on the system of mass cultivation, new strains should be isolated from a wide variety of environments to provide the largest range of metabolic versatility possible. Algae can be isolated from a variety of natural aquatic habitats, ranging from freshwater to brackish water, to marine and hyper-saline environments, to soil (Round, 1984; Mutanda et al., 2011). Furthermore, large-scale sampling efforts should be coordinated to ensure the broadest coverage of environments and to avoid duplication of efforts. The selec­tion of specific locations can be determined by sophisticated site selection crite­ria through the combined use of dynamic maps, geographic information systems (GIS) data, and analysis tools. Ecosystems to be sampled could include aquatic (i. e., oceans, lakes, rivers, streams, ponds, and geothermal springs, which includes fresh, brackish, hypersaline, and acidic and alkaline environments) and terrestrial environments in a variety of geographical locations to maximize genetic diversity. Collection sites can include public lands as well as various sites within national and state park systems. In all cases, questions of ownership of isolated strains should be considered. Additionally, within an aqueous habitat, algae are typically found in planktonic (free floating) and benthic (attached) environments. Planktonic algae may be used in suspended mass cultures, whereas benthic algae may find application in biofilm-based production facilities. Sampling strategies should not only account for spatial distribution, but also for the temporal succession brought about by seasonal variations of algae in their habitats (Mutanda et al., 2011).