The mass production of microalgae can be achieved through raceway ponds or pho­tobioreactors. The feasibility of biodiesel production from algae is totally depen­dent on the technologies used in the downstream processing of algae. Downstream processing of algae involves dewatering, drying, oil extraction, biofuel production, and by-product utilization. Dewatering of algae is an energy-consuming process that requires a high capital investment for equipment. There is a greater possibility of quick spoilage of the harvested biomass slurry (5% to 15% dry solids) under hot climatic conditions due to its high moisture content (Brennan and Owende, 2010). The harvested slurry must be dewatered and dried quickly after harvesting. Drying is an important process in the downstream process that enhances biomass shelf life and lipid recovery efficiency. The process employed for oil extraction from dried algae is similar to that of oil extraction from oil seeds. In order to achieve economically viable algal oil-based biodiesel production, the technical feasibility must be thoroughly studied; that is, downstream technologies/processes must be optimized.

The oil content of dried algal biomass varies from 20% to 50% oil by weight and can be increased by optimizing growth parameters (Hu et al., 2008). Before com­mencing oil extraction from microalgae, it must be dewatered and dried to remove the moisture. The present oil extraction techniques applicable for wet/dry algae bio­mass have limitations due to technical barriers, difficulty in scaling-up, high cost investment, and extraction efficiency (Cooney et al., 2009). The choice of oil extrac­tion technology depends on the moisture content, quantity to be treated, quality of the end-product, extraction efficiency, safety aspects, and cost economics. The main outputs from the oil extraction process are oil and oil cake. The extracted oil can be used for biodiesel production with/without pretreatment, depending on the qual­ity of the oil. The actions involved in the oil extraction process are (1) breaking the algae cell walls, (2) freeing the oil, and (3) separating out the oil and oil cake. The most common methods used for the oil extracted from algae biomass are mechanical press, solvent extraction, and supercritical fluid extraction. The methods employed for oil extraction from microalgae are depicted in Figure 7.1.

The two most significant essential fatty acids found in considerable levels in meat and coldwater fish are the omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (see Table 10.7). New evidence suggests beneficial effects of omega-3 on diseases such as cardiovascular disease (CVD), inflammatory disease, and brain function. Recent studies have also shown the positive impact of omega-3 in curing mental health disorders (Simopoulos, 1999; Arterburn et al., 2000; Nemets et al., 2002; Kris-Etherton et al., 2003, Wen and Chen, 2003; Ruxton et al., 2004; Freeman et al., 2006; Von Schacky and Harris, 2007; Mischoulon et al., 2008; The Ocean Nutrition Canada website, 2010). Aside from human health, omega-3 has significant advantages for growth and development (Ruxton et al., 2004). As a result, more than 14,000 studies have been conducted over the past 35 years, promoting the benefits of omega-3 fatty acids in the human diet at every stage of life (The Ocean Nutrition Canada website, 2010).

Currently, algal EPA and DHA are the only alternative to fish oils. Apart from being a complete vegetarian alternative, microalgae are considered sustainable feed­stocks for the production of EPA and DHA compared to other sources such as sar­dines, krill, and genetically engineered oilseed crops. Consumers are well aware of the choices they make, and they prefer an omega-3 source that is “naturally bio­diverse and not genetically engineered” (Watson, 2011a). In general, algal cultures are pure and eliminate concerns about high levels of toxins, pollutants, and heavy metals. Algal oils have a high unsaturation index and with the aid of novel process­ing techniques to improve stabilization by reducing oxidation potential, algal oils provide fish-free odor and taste. New and improved technologies, such as microen­capsulation, allow these oils to be employed in a broader food and beverage applica­tion profile for both vegetarians and nonvegetarians (Pulz and Gross, 2004; Ward and Singh, 2005; Whelan and Rust, 2006). Unlike the highly competitive marine

TABLE 10.7

Life Stage Benefits of Omega-3 EPA and DHA)

Source: Adapted from Life stages benefits (2011), Ward and Singh (2005), Holub (2011).

fish oil market, the vegetarian omega-3 EPA and DHA source remains moderately competitive, with the dominant sources of omega-3 being algal and flaxseed oil. DHA market estimations alone are valued at US$15 million, and are considered one of the fast-growing microalgal products.

Martek Biosciences has been the dominant player in sustainable algal omega-3 technology and, as a result, this platform has given rise to products such as algae-derived omega-3 DHA in infant formula, dietary supplements, functional foods and beverages, as well as animal feed products. Martek Biosciences was recently (December 21, 2010) acquired by DSM (a global life sciences and material sciences company). The company’s “flagship” product is life’s DHA™, a completely vegetarian source of algal DHA. Martek’s omega-3 technologies are secured by a robust intellectual property portfolio and supported by a strong R&D platform, with particular emphasis on the infant formula and infant nutrition area. This provides new opportunities for DSM in the infant nutrition segment, as well as in the food, beverage, and dietary supplement industries. Formulas incorporating Martek’s DHA oil are available in more than sixty countries, including the United Kingdom, Mexico, China, the United States, and Canada (Spolaore et al., 2006).

Chemical manipulations of algae are reflected in their biochemical constituents. Nitrogen starvation increased lipid production from 117 to 204 mg L-1d-1 (Rodolfi et al., 2008). By manipulating the nutrients in Scenedesmus obliquus, up to 58.3% lipid was attained, which was five — to tenfold higher than controls (Mandal and Mallick, 2009). It is of interest to note that carotenoids increased only in Dunaliella salina as the salinity increased (Gomez et al., 2003; Coesel et al., 2008). The carotenoid levels (mg L-1) corresponded to 6.9, 10.8, and 12.9 mg L-1 in Provosoli medium of 1 M, 2 M, and 3 M sodium chloride (NaCl), respectively; in an arti­ficial medium, they were more pronounced and were 8, 12.9, and 29.5 mg L-1 in 1 M, 2 M, and 3 M NaCl, respectively. Takagi et al. (2006) showed that the salt content of the medium could also be a stressor in Dunaliella. In the initial stages of cultures, when the NaCl was increased from 0.5 M (equivalent to seawater) to 1.0 M, lipid increased by 67%; when mid- or late-log phase cultures were subjected to a similar stress, cellular lipid increased to 70%. So while harvesting cells for biotechnological applications, the strain and physiological state of the algae play critical roles in determining output.

In addition to production rates, variations in biochemical profiles must be con­sidered for optimization of harvesting. Carbohydrates, proteins, lipids, and fats are known to vary with the medium used among seven species of marine microalgae (Fernandez-Reiriz et al., 1989) and in sixteen species of microalgae commonly used in aquaculture (Brown, 1991). The medium used (Walne, ES, f/2, and Algal-1) for cultivation also influenced the biochemical profiles of four species (Fernandez — Reiriz et al., 1989).

Through biochemical manipulation, lipid synthesis can be regulated; this involves imposing a physiological stress such as nutrient starvation to channel metabolic pro­cesses toward lipid accumulation. In experiments by Li et al. (2008), cultures of Neochloris oleoabundans were supplied with sodium nitrate, urea, and ammonium bicarbonate as the nitrogen source; only at lower levels of sodium nitrate did cel­lular lipid increase. Co-limitation for inorganic phosphorus and carbon dioxide in Chlamydomonas acidophila Negoro resulted in high photosynthetic rates and also in a mismatch between photosynthesis and growth rates in phosphorus-limited cul­tures (Spijkerman, 2010). In Monodus subterraneus when phosphate was decreased from 175 to 52.5, 17.5, or 0 pM, cellular lipid increased (Khozin-Goldberg, and Cohen, 2006). Limitation of nitrogen in cultures of the green alga Scenedesmus obliquus resulted in an increase of lipid from 12.7% to 43% of cell dry weight (DW) (Mandal and Mallick, 2009); a deficiency of phosphate increased lipid to 29.5% (DW). Lipids in nitrogen-limited Chaetoceros mulleri increased five — to sevenfold compared to nitrogen-replete cultures (McGinnis et al., 1997). Results obtained with Nannochloropsis oculata and Chlorella vulgaris (Converti et al., 2009) con­firmed such an impact of nitrogen limitation. Lipid production is enhanced to 90 kg ha-1d-1 by a two-stage culture system that involves raising high-density cultures under optimal conditions initially and then transferring them to a nitrogen-deficient medium (Rodolfi et al., 2008).

In Dunaliella salina cultures, an increase in CO2 from 2% to 10% increased lipid production by 170% in 7 days (Muradyan et al., 2004). In Chlamydomonas vulgaris, the addition of 1.2 x 10-5 M Fe3+ not only suppressed cell growth initially, but also enhanced the accumulation of lipids up to 56.6% DW. Furthermore, the accu­mulation of lipids occurred earlier during the stationary phase (Liu et al., 2008). To enhance the yield of microalgal biomass, rigorous experiments should be carried out to establish the impact of several micronutrients, such as selenium and boron. Optimized growth of commercial algae should account for the effects of manipula­tions in nutrients, temperature, and chemical composition of media.

A wide variety of open and closed reactor systems have been proposed for microal­gal cultivation, possibly reflecting the diversity in the physiology and requirements of different algal species. Ultimately, the overall goal is the continuous maintenance of a desired algal culture under conditions for optimal productivity. High volumet­ric and areal yields reduce cost by minimizing the reactor volume and land area required, respectively. Important factors in achieving this include (Richmond, 2000):

• Provision of sufficient light, despite daily and seasonal variations and dense algal culture

• Optimal mixing and mass transfer, while avoiding damage to cells by shear stress

• Minimization of deviation from optimal temperature (requires cooling in summer and heating in winter)

• Minimization of dissolved oxygen tension

• Simple cleaning and maintenance

• Minimization of energy input requirements

• Minimization of water use (e. g., evaporation from ponds, evaporative cooling use)

• Low capital and operating costs per unit of harvested product

Over the past two decades, much work has focused on methodology to assess the environmental impact of processes and products. A number of these approaches are summarized in Table 9.2, indicating the methodology and nature of the assessment. It must be noted that while initially bioprocesses and energy processes from renewable resources were assumed to be preferential with respect to lower

TABLE 9.2

Approaches to the Quantification of Environmental Sustainability of Process Options

environmental impact, it has been demonstrated clearly that this does not necessarily hold; hence, objective assessment of the environmental burden of each process is essential in product and process selection, in a similar manner to that used to ensure economic feasibility.

Life cycle assessment (LCA) systematically identifies environmental impact and opportunities to minimize it, and evaluates these (Curran 2000). It is supported by a strong literature database and a well-defined methodology. A track record exists for its use in the environmental assessment of biofuels (Kaltschmitt et al., 1997; Kim and Dale, 2005; von Blottnitz and Curran, 2007; Harding et al., 2008; Evans et al., 2009). In conducting the LCA, setting the goal and scope of the study allows for selecting a functional unit for comparison and setting the system boundaries. A full inventory of the process flowsheet is required, including all raw materials and energy, and all emissions and products generated. Data are preferably obtained from operating plants; where this is not feasible in new process development, data are obtained experimentally, from the literature or through modeling, and validated through material and energy balancing. Typically, a cradle-to-gate approach is used where the products formed are the same. Where the products formed differ from the existing product and result in different emissions and by-products on use, a cradle — to-grave approach is needed to consider product use and disposal. In both cases, the raw material and energy requirements are expanded to include their pre-processing, taking into account extraction from abiotic reserves, cultivation, agricultural pro­cesses, etc. Typically, the impact of construction of the process plant and equipment is negligible with respect to the impact of the operating plant. In new technology environments, this should be verified. This has been demonstrated for algal bio­diesel in all categories except land use (Lardon et al., 2009). Where reactors having a short life span are used (e. g., polyethylene bags or PVC linings), these need to be included in the analysis. For multiple products or by-products, as in the biorefinery, environmental burden allocation or substitution is required to allocate the overall burden representatively across the products formed. Burden allocation may be done based on the mass or volume ratio of useful products or, in some cases, based on cost. According to ISO (International Organization for Standardization) guidelines, substitution is preferred where possible; that is, the additional product or by-product is accounted for through the inventory typical of its conventional process route. This handling of multiple products is important as typically the production of multiple biofuels has been shown to increase the material and energy efficiency and process economics of biomass utilization (Kaparaju et al., 2009).

Life cycle inventory (LCI) data are used in life cycle impact assessments (LCIAs), typically using appropriate software to group the impacts into a manageable set of impact categories (mid-point categories), such as abiotic depletion, global warming, eutrophication, acidification, toxicity, etc. These may be further grouped into end­point categories, such as human health, climate change, and ecosystem quality, where appropriate.

The importance of the holistic study, considering all aspects of resource utilization and emission generation, is demonstrated through early-stage biofuel analyses where the carbon benefits of land use were counted for first-generation biofuels; however, the emissions caused by clearing of the land to grow new feedstock (land-use change) were not estimated (Searchinger, 2008). Fargione (2008) determined that the greenhouse gases (GHGs) released from changing natural habitats to biofuel cropland were several-fold greater than the offset from displacing fossil fuels, and hence a “carbon payback time” was defined to determine the time required before a true reduction in GHG resulted. This example drives home the need for an integrated assessment of environmental impacts.

A freshwater green microalga, Haematococcus pluvialis, has been cultivated as a source of natural astaxanthin, a ketocarotenoid. Astaxanthin in microalgal cells is located in the cytoplasmic lipid globules. The biflagellate and motile cells of

H. pluvialis transform into resting cyst cells, the aplanospores, and develop a distinct red color due to astaxanthin accumulation. After maturation, the cysts germinate, releasing flagellated cells (Margalith, 1999). The transformation of vegetative micro­algal cells to astaxanthin-accumulating resting cells could be achieved by subjecting the microalgal culture to environmental and nutritional stress, for example, nitrogen and phosphorus limitation, increases in culture temperature, increases in the salinity of the culture medium, and exposure of the culture to high irradiance (Del Campo et al., 2007). The astaxanthin content of Haematococcus cells can go up to 3%, mak­ing them an attractive source of the carotenoid pigment.

The accumulation of astaxanthin in Haematococcus cells in the resting phase necessitates a two-phase cultivation protocol where in the first phase the microalga is grown under optimal growth conditions to achieve high biomass yields, and then the green biomass is subjected to nutritional and environmental stress in a second phase to induce cyst (aplanospore) formation and the accumulation of astaxanthin (Del Campo et al., 2007). Complete outdoor cultivation of Haematococcus has not been feasible due to its high sensitivity to contamination and extreme environmen­tal conditions during the growth phase. Commercial production of Haematococcus biomass is generally carried out in closed photobioreactors, or it combines closed photobioreactors and open ponds where the first stage of biomass generation is car­ried out in closed photobioreactors, followed by a short residence period of culture in open ponds for the second phase of induction of astaxanthin accumulation (Olaizola and Huntley, 2003; Cysewski and Lorenz, 2004; Del Campo, 2007).

Astaxanthin has major commercial application in aquaculture as a source of pigmentation for salmon, trout, and red sea bream (Lorenz and Cysewski, 2000; Guerin et al., 2003; Cysewski and Lorenz, 2004), and the market is dominated by synthetic astaxanthin. Natural astaxanthin from Haematococcus is not competitive with synthetic astaxanthin for aquaculture applications due to high production costs (Guerin et al., 2003; Olaizola, 2003). Therefore, the economic viability of large — scale cultivation of Haematococcus to produce natural astaxanthin for aquaculture applications alone may not be feasible, but finding high-value markets is important. Human nutraceuticals have emerged as the high-value market for natural astaxan — thin from Haematococcus. Several in vitro and in vivo studies have demonstrated the beneficial health effects of Haematococcus-derived natural astaxanthin (Guerin et al, 2003; Olaizola, 2003; Kamath et al., 2008; Yuan et al., 2011). Haematococcus has been cleared by the U. S. FDA for application as an ingredient in dietary supple­ments for humans and has also been approved for human consumption in several European countries (Lorenz and Cysewski, 2000). This has paved the way for mar­keting Haematococcus biomass for application as a food supplement.

The sedimentation rates of algae are influenced by the settling velocity of micro­algae, which can be increased by increasing cell dimensions (i. e., by aggregation of cells into large bodies) (Schenk et al., 2008). This principle is being applied to algal harvesting, wherein chemicals are added to enhance flocculation, causing the large algal flocs to settle more readily to the bottom of the container. The floccula­tion of algal biomass is generally followed by gravity sedimentation for settling of algal flocs, thus enhancing the efficiency of this process. Gravity sedimentation preceded by flocculation is one of the most commonly used techniques for first — stage (1% to 5% solids) algae biomass harvesting (Girma et al., 2003; Pittman et al., 2011). However, the gravity settling rate for very small sized microalgae is too low for routine high rate algae harvesting, and holding algal biomass for a long time under dark and static conditions can result in significant biomass loss via respiration and bacterial decomposition. Moreover, during flocculation, flocs may float due to adsorption of tiny air bubbles and do not settle by gravitational forces. The classical approach to gravity settling may therefore not be very efficient for rapid biomass recovery from high-rate algal ponds. The sedimentation rate can be increased by increasing the gravitational force via centrifugation. The latter has very high biomass recovery (>95%) and can be applied to a wide range of microal­gae, although it cannot be used for an algae farm producing an energy feedstock, owing to cost constraints.

The motivation to overcome the challenges with respect to environmental and economic sustainability of microalgal culture for biodiesel production and other renewable products stems from the significant advantages of the microalgal sys­tem as a biomass source. These include the potential to use nonarable land for microalgal cultivation, the homogeneity of the biomass formed and the ability to process all components, the much-improved oil production per unit area (15 to 300 times greater), the higher growth rate, and the photosynthetic efficiency of microalgae compared with terrestrial plants (up to tenfold increase) (Chisti, 2007; Schenk, 2008; Rodolfi et al., 2009). Freshwater, seawater, brines, and wastewater are all potential water sources for algal growth (Vasudevan and Briggs, 2008). CO2 uptake by the autotrophic algae enables CO2 cycling through uptake for bio­mass generation and release on fuel combustion. Furthermore, the multiple energy forms attainable from microalgae span liquid fuels, and heat and electricity genera­tion, enabling ongoing support of existing technologies while developing a reduced carbon economy. On attaining an energy economy in which dependence on carbon combustion is reduced, the technology lessons learned through achieving environ­mental and economically sustainable algal biomass will readily be transferred to the production of carbon-based commodities with simultaneous carbon sequestra­tion or cycling.

The NER and LCA studies conducted to date have highlighted the great sensi­tivity of the GWP, fossil fuel requirements, and NER on the productivity of algal biomass and of algal oil attainable in the algal cultivation process. While maxi­mum specific growth rates and lipid content are partly defined through the algal species selected, culture conditions may be used to enhance these through improved light supply, mass transfer, and mixing. The energy requirement of the bioreactor to achieve mixing and mass transfer is a major contributor to the energy requirement of the integrated algal process, as is the CO2 provision to the reactor. Based on the volumetric concentrations attainable, pumping energies can be defined.

Supply of nutrients, yield of products from these nutrients, and recycle of unused nutrients impact GWP and fossil fuel requirements significantly. Opportunities exist in selecting algal species able to scavenge low nutrient concentrations, having reduced nitrogen content, as well as to utilize nitrogen and phosphorous resources efficiently, either by their provision from wastewater or through their recycle.

The typically dilute biomass concentrations required to minimize light limitation result in the need to process large culture volumes; hence, natural flocculation to facilitate settling is beneficial. In downstream processing, the most important factors pertain to the ability to process wet biomass, thereby eliminating the drying process, as well as the ability to recover product from the algal cell readily, thus minimizing the requirement for conventional, energy-intensive cell disruption.

As reported by Harding (2009), the production phase typically has the greatest impact on the overall LCA; hence, its optimization is required in the first instance.

Opportunities to improve the economics correlate well with the environmental analysis with respect to operating costs. These highlight mass transfer and mixing, provision of CO2, provision of nutrients, biomass recovery, and the avoidance of rigorous drying. More importantly, the economic studies suggest that algal biofuel technology requires further enhancement prior to its economic feasibility as a stand­alone technology. However, opportunity exists to establish a cost-effective algal biorefinery delivering a combination of products such as biodiesel, biogas, animal feed, and protein extracts. To this, high-value products may be added. Further, the predicted cost of algal biomass positions it attractively as a raw material source for bulk products, including fuels, chemicals, materials feeds, and food supplements.

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Yang, J., Xu, M., Zhang, X., Hu, Q., Sommerfeld, M., and Chen, Y. (2011). Life-cycle analysis on biodiesel production from microalgae: Water footprint and nutrients balance. Bioresource Technology, 102(1): 159-165.

The removal of nitrogen and phosphorus from wastewater is essential in prevent­ing ecological damage to receiving water bodies. Phosphorus is particularly dif­ficult to remove (Pittman et al., 2011). Chemical precipitation is currently the main commercial process for removing phosphorus from wastewater. Biological removal efficiencies vary from 20% to 30% for most organisms (de-Bashan et al., 2004). The phosphorus is then converted into activated sludge that cannot be fully recycled and is buried in landfills or treated to render sludge fertilizer. Microalgae are effec­tive in removing nitrogen, phosphorus, and toxic metals from wastewater, thus mak­ing them ideal candidates for nutrient removal and recovery (Pittman et al., 2011). Microalgal uptake of phosphorus has been shown to be as efficient as chemical treat­ment (Pittman et al., 2011).

Carbon, nitrogen, phosphorus, and sulfur are essential growth requirements for most microalgae (Chisti, 2007; Tsai et al., 2011; Zeng et al., 2011). These elements are commonly found in domestic wastewater in concentrations that support microal­gal cultivation. Minimal nutritional requirements can be estimated using the approx­imate molecular formula of the microalgal biomass, that is, CO048H183N0.nP0.01 (Chisti, 2007; Putt et al., 2011). Nitrogen is the critical factor for the growth and lipid content regulation of microalgae. Phosphorus, although required in smaller amounts, must be supplied in excess as it complexes with metal ions and is thus not fully bio­available for cell uptake (Chisti, 2007). Microalgae naturally utilize suitable nutri­ents and energy sources from their environment, thereby optimizing the efficiency of utilization for growth and survival. They are resilient organisms, in that a single species may be able to undergo various types of metabolism, depending on the avail­able nutrients for growth as well as other environmental factors (Amaro et al., 2011). Nitrogen is utilized in the form of nitrate and ammonia, with ammonia being used preferentially in the presence of both chemical species (Feng et al., 2011).

Phototrophic cultivation uses sunlight and CO2 as an inorganic carbon source for energy production and growth (Mata et al., 2010). Phototrophic cultivation is less prone to contamination than other types of cultivation. Heterotrophic growth occurs in the absence of light using organic carbon sources such as glucose, acetate, glycerol, fructose, sucrose, lactose, galactose, and mannose (Amaro et al., 2011). Organisms that are able to undergo mixotrophic growth have the ability to photosyn — thesize or use organic substrates as a carbon source. Mixotrophic production reduces photo-inhibition and decreases the loss of biomass due to dark-phase respiration (Brennan and Owende, 2010; Pittman et al., 2011). Organic carbon sources in waste­water allow microalgae to undergo mixotrophic growth followed by phototrophic growth. This effectively removes nutrients while improving biomass and potential lipid productivity (Feng et al., 2011).

The efficiency of nutrient removal depends on the species of algae cultivated and has been shown to be influenced positively by the cultivation of algal strains that are tolerant to certain extremes, such as extreme temperatures, quick sedimentation, or the ability to grow mixotrophically (Olguin, 2003). Choosing a strain for cultivation in HRAPs should preferentially (1) have a high growth rate, (2) have a high protein concentration when grown under nutrient-limited conditions, (3) be used for animal/ fish feed, (4) have the ability to tolerate high nutrient levels, (5) produce a value-added product, (6) be able to grow mixotrophically, and (7) be easily harvested (Sheehan et al., 1998; Olguin, 2003; Rawat et al., 2011). Chlorella vulgaris, Haematococcus pluvialis, and Arthrospira (Spirulina) platensis, among others, are examples of spe­cies that can grow under photo-autotrophic, heterotrophic, and mixotrophic condi­tions (Amaro et al., 2011).

Microalgal wastewater treatment has the potential to significantly reduce the costs of treatment when compared to conventional chemical methods; this is par­tially achieved by negation of the requirement for mechanical aeration as microalgae produce oxygen via the process of photosynthesis (Pittman et al., 2011). The simul­taneous treatment of wastewater and production of biomass reduces the cost of both processes (Brennan and Owende, 2010; Christenson and Sims, 2011). Furthermore, the production of biofuel in conjunction with wastewater treatment has been put for­ward as the most viable method for biofuel production from microalgae in the near future (Brennan and Owende, 2010).

Several studies have proven the potential for nutrient removal from synthetic waste­water by microalgal biomass production. Phosphorus removal of 98% and total ammo­nia removal has been achieved by (Martinez et al., 2000) using Scenedesmus obliquus. Boelee et al. (2011) demonstrated simultaneous removal of nitrate and phosphate to

2.2 mg L-1 and 0.15 mg L-1, respectively, using microalgal biofilms. Su et al. (2012) reported phosphorus removal efficiency of algae to be 89%. Certain photosynthetic bacteria and green microalgae such as Rhodobacter sphaeroides and Chlorella soro — kiniana can, under heterotrophic conditions, remove high concentrations of organic acids (>1,000 mg L-1) and ammonia (400 mg L-1) (Olguin, 2003). The bacterial removal of substances such as polycyclic aromatic hydrocarbons, organic solvents, and phenolic compounds may be assisted by the use of microalgae that produce the oxygen required for bacterial action. Heavy-metal biosorption may be achieved by microalgae grown under phototrophic conditions (Brennan and Owende, 2010).

There are several methods available for the enumeration of microalgal cells. However, due to the small size of the microalgal cells, most methods are not very accurate. In addition, some of the methods described here require sophisticated and expensive equipment, which is beyond the reach of some research laboratories. The choice of counting device depends on culture density, the size and shape of the cells or colonies being counted, and the presence and amount of extracellular threads, sheaths, or dissolved mucilage, which can influence the filling of the counting cham­ber (Guillard and Sieracki, 2005). Moreover, the method to be adopted for a par­ticular sample will therefore depend on other factors such as detection range, costs, sample throughput, health and safety considerations, inter alia.