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Microalgae are unicellular photoautotrophs (ranging from 0.2 μm up to 100 μm) and are classified in various taxonomic groups. Microalgae can be found in most environments but are mostly found in aquatic environments. Phytoplankton are responsible for over 45% of world's primary production as well as generating over 50% of atmospheric Osub2/sub. In general, there is no major difference in photosynthesis of microalgae and higher plants (Deppeler et al. 2018). However, due to their smaller size and the reduction in a number of internally competitive physiological organelles, microalgae can grow much faster than higher plants (Moheimani et al. 2015). Microalgae can also grow under limited nutrient conditions and have the ability to adapt to a wider range of environmental conditions (Gordon and Polle 2007). Most importantly, microalgal culture does not compete with food crop production regarding arable land and freshwater (Moheimani et al. 2015). Furthermore, microalgae can efficiently utilize inorganic nutrients from waste effluents (Ayre et al. 2017). In general, microalgal biomass contains up to 50% carbon making them a perfect candidate for bioremediating atmospheric COsub2/sub (Moheimani et al. 2012).
The increase in extensive worldwide agriculture and animal farming has resulted in significant increases in biologically available nitrogen and phosphorus entering the terrestrial biosphere (Galloway et al. 2004). Crop and animal farming and sewage systems contribute significant amounts to these nutrient loads (Schoumans et al. 2014). The infiltration of these nutrients into water streams can cause massive environmental issues such as harmful algal blooms and mass fish mortality. For instance, in the USA, nutrient pollution from agriculture is acknowledged as one of the major sources of eutrophication (Sharpley et al. 2008). Controlling the flow of nutrients from farming operations into the surrounding environment results in both technical and economic challenges that must be overcome to reduce such effects. There have been various successful processes developed to treat waste effluent with high organic loads. However, almost all of these methods are not very effective in removing inorganic elements from water. Furthermore, some of these methods are rather expensive to operate. One simple method for treating organic waste is anaerobic digestion (AD). The AD process is well understood and when operated efficiently, it can convert over 90% of the wastewater organic matters to bio-methane and COsub2/sub (Parkin and Owen 1986). The methane can be used to generate electricity and the generated heat can be used for various additional purposes. However, the AD process results in creating an anaerobic digestion effluent (ADE) which is very rich in inorganic phosphate and nitrogen as well as high COD (carbon oxygen demand). In certain locations, this effluent can be treated using microalgae and macroalgae (Ayre et al. 2017).
Since the United Nations committee recommended that conventional agricultural crops be supplemented with high-protein foods of unconventional origin, microalgae have become natural candidates (Richmond and Becker 1986). The first microalgal cultivation was achieved though in 1890 by culturing *Chlorella vulgaris* (Borowitzka 1999). Due to the fact that microalgae normally divide at a certain time of the day, the term cyclostat was developed in order to introduce a light/dark (circadian) cycle to the culture (Chisholm and Brand 1981). The largescale culturing of microalgae and the partial use of its biomass especially as a base for certain products such as lipids was probably started seriously as early as 1953 with the aim of producing food from a large-scale culture of *Chlorella* (Borowitzka 1999). Typically, algae can be cultured in liquid using open ponds (Borowitzka and Moheimani 2013), closed photobioreactors (Moheimani et al. 2011), or a combination of these systems. Alga can also be cultured as biofilms (Wijihastuti et al. 2017).
Closed Photobioreactors (after Moheimani et al. 2011): Closed algal cultures (photobioreactors) are not exposed to the atmosphere but are covered with a transparent material or contained within transparent tubing. Photobioreactors have the distinct advantage of preventing evaporation. Closed and semi-closed photobioreactors are mainly used for producing high-value algal products. Due to the overall cost of operating expenditure (OPEX) and capital expenditure (CAPEX), closed photobioreactors are less economical than open systems. On the other hand, there is less contamination and less COsub2/sub losses, and by creating reproducible cultivation conditions and flexibility in technical design, this makes them a good substitute for open ponds. Some of the closed systems' weaknesses can be overcome by (a) reducing the light path, (b) solving shear (turbulence) complexity, reducing oxygen concentration, and (c) a temperature control system. Closed photobioreactors are mainly divided into (a) carboys, (b) tubular, (c) airlift and (d) plate photobioreactors.
Open Ponds (after Borowitzka and Moheimani 2013): Open ponds are most commonly used for large-scale outdoor microalgal cultivation. Major algal commercial production is based in open channels (raceways) which are less expensive, easier to build and operate when compared to closed photobioreactors. In addition, the growth of microalgae meets less difficulties in open than closed cultivation systems. However just a few species of microalgae (e.g. Dunaliella salina, Spirulina sp., Chlorella sp.) have been grown successfully in open ponds. Commercial microalgal production costs are high, approximated to be between 4 and 20 \$US/gsup-1/sup. Largescale outdoor open pond commercial microalgal culture has developed over the last 70 years, and both still (unstirred) and agitated ponds have been developed and have been used on a commercial basis. The very large unstirred open ponds are simply constructed from natural water ponds with open beds that are usually less than 0.5 m in depth. In some smaller ponds the surface may be lined with plastic lining sheets. Unstirred open ponds represent the most economical and least technical of all commercial culture methods and have been commercially used for Dunaliella salina β-carotene production in Australia. Such ponds are mainly limited to growing microalgae which are capable of surviving in poor conditions or have a competitive advantage that allows them to outgrow contaminants such as protozoa, unwanted microalgae, viruses, and bacteria. Agitated ponds on the other hand have the advantage of a mixing regime. Most agitated ponds are either (a) circular ponds with rotating agitators or (b) single or joined raceway ponds.
Circular cultivation ponds have primarily been used for the large-scale cultivation of microalgae especially in South East Asia. The circular ponds up to 45 m in diameter and usually 0.3—0.7 m in depth are uncovered, but there are some examples which are covered by glass domes. The low shear stresses that are required for microalgae production are produced in these systems particularly in the centre of the pond, and this is a distinct advantage of these kinds of systems. Some disadvantages include expensive concrete structures, inefficient land use with large footprints, difficulties in controlling the movement of the agitating device and the added cost in supplying COsub2/sub.
Paddlewheel-driven raceways are the most common commercial microalgal cultivation system. Raceways are usually constructed in either a single channel or as linked channels. Raceways are usually shallow (0.15 to 0.25 m deep), are constructed in a loop and normally cover an area of approximately 0.5 to 1.5 ha. Raceways are mostly used and recommended for the major commercial culturing of three species of microalgae including Chlorella, Spirulina and Dunaliella. A high risk of contamination and low productivity, resulting mainly from poor mixing regimes and light penetration, are the main disadvantages of these open systems. In raceways, biomass concentrations of up to 1000 mg dry weight.Lsup-1/sup and productivities of 20 g dry weight.msup2/sup.dsup-1/sup have been shown to be possible.
The price of microalgal production makes economic achievement highly dependent on the marketing of expensive and exclusive products, for which demand is naturally restricted. Raceways are also the most used cultivation system used for treating wastewater (Parks and Craggs 2010).
Solid Cultivation (after Wijihastuti et al. 2017): An alternative microalgal cultivation method is immobilizing the cells in a polymer matrix or attaching them to the surface of a solid support (biofilm). In general, the biomass yield of such biomass cultures are at least 99% more concentrated than liquid-based cultures. Dewatering is one of the most expensive and energy-intensive parts of any mass algal production. The main advantage of biofilm growth is the potential of reducing the dewatering process and the related energy consumption and thus costs. Biofilm cultivation can also increase cellular light capture, reduce environmental stress (e.g. pH, salinity, metal toxicity, very high irradiance), reduce the cost of production and reduce nutrient consumption. Solid-based cultivation methods can be used for treating wastewater (nutrient and metal removal). There are three main methods for biofilm cultivation: (a) 100% directly submerged in medium, (b) partially submerged in medium and (c) using a porous substrate to deliver the nutrients and moisture from the medium to the cells.
A number of inhibitory physical, chemical and biological factors can inhibit high microalgal production. These are described in Table 12.1.
A basic knowledge of the critical growth limitations is probably the most essential factor before applying any microalgae to any process. Light is by far the most important limiting factor affecting the growth of any alga. Temperature is also a critical factor for mass algal production (Moheimani and Parlevliet 2013). However, these variables are difficult to control (Moheimani and Parlevliet 2013). Next to light and temperature, nutrients are the most important limiting factor affecting the growth of any alga (Moheimani and Borowitzka 2007) and each microalgal species tends to have its own optimum nutrient requirements. The most important nutrients are nitrogen, phosphorus and carbon (Oswald 1988). Most
table tbody tr thAbiotic factors/th td Light (quality, quantity) /td /tr tr class="odd" td/td td Temperature /td /tr tr class="even" td/td td Nutrient concentration /td /tr tr class="odd" td/td td Osub2/sub /td /tr tr class="even" td/td td COsub2/sub and pH /td /tr tr class="odd" td/td td Salinity /td /tr tr class="even" td/td td Toxic chemicals /td /tr tr class="odd" thBiotic factors/th td Pathogens (bacteria, fungi, viruses) /td /tr tr class="even" td/td td Competition by other algae /td /tr tr class="odd" thOperational factors/th td Shear produced by mixing /td /tr tr class="even" td/td td Dilution rate /td /tr tr class="odd" td/td td Depth /td /tr tr class="even" td/td td Harvest frequency /td /tr tr class="odd" td/td td Addition of bicarbonate /td /tr /tbody /table
Table 12.1 Limits to growth and productivity of microalgae (Moheimani and Borowitzka 2007)
algae respond to N-limitation by increasing their lipid content (Moheimani 2016). For example, Shifrin and Chisholm (1981) reported that in 20 to 30 species of microalgae that they examined, the algae increased their lipid content under N-deprivation. Phosphorus is also an important nutrient required for microalgal growth as it plays an essential role in cell metabolism and regulation, being involved in the production of enzymes, phospholipids and energy-supplying compounds (Smith 1983). Brown and Button's (1979) studies on green alga Selenastrum capricornutum showed an apparent growth limitation when the phosphate concentration of the medium was lower than 10 nM. COsub2/sub is also a critical nutrient for achieving high algal productivity (Moheimani 2016). For example, if additional COsub2/sub is not added to the algal culture, the average productivity can be reduced by up to 80% (Moheimani 2016). However, the addition of COsub2/sub to algal ponds is rather costly (Moheimani 2016). The most economical way for introducing COsub2/sub to a culture media is the direct transfer of the gas into the media by bubbling through sintered porous stones or using pipes under submerged plastic sheets as COsub2/sub injectors (Moheimani 2016). Unfortunately, in all of these methods there is still high loss of COsub2/sub to the atmosphere because of the short retention time of the gas bubbles in the algal suspension.
Although adding N, P and C is critical, other nutrients also affect microalgal growth and metabolism. A lack of other nutrients, such as manganese (Mn) and various other cations (Mgsup2+/sup, Ksup+/sup and Casup2+/sup), is also known to reduce algal growth (Droop 1973). Trace elements are also critical for microalgal growth and some microalga also require vitamins for their growth (Croft et al. 2005). One effective and inexpensive way of supplying nutrients is by combining algal culture and wastewater treatment which is discussed immediately below.
With an increase in environmental deterioration and a greater necessity to generate alternative food and energy sources, there is the impetus to explore the feasibility of biological wastewater treatments coupled with resource recovery. Microalgal wastewater treatments have been particularly attractive, due to algal photosynthetic activities, where light is transferred into profitable biomass. Under certain conditions, wastewater-grown microalgal biomass can be equivalent or superior in biomass production to higher plant species. Thus, the process can transform a waste product into useful products (e.g. animal feed, aquaculture feed, bio-fertilizer and bioenergy). Thus, the waste effluent is no longer a negative waste product, but it becomes a valuable substrate for producing important substances and successful microalgal wastewater bioremediation has been reported for over half a century (Oswald and Gotass 1957; Delrue et al. 2016). Algal phytoremediation indeed provides an environmentally favourable solution for the treatment of wastewater as it can utilize organic and inorganic nutrients efficiently (Nwoba et al. 2017). Microalgal cultures hold an enormous potential for the later steps of wastewater treatment, especially for reducing 'N', 'P' and 'COD' (Nwoba et al. 2016). Moreover, the added ability of microalgae to grow via different nutritional conditions such as photoautotrophic, mixotrophic and heterotrophic conditions also enhances its capabilities in removing various different types of pollutants and chemicals from aqueous matrices. The ability of microalgae in sequestrating carbon (COsub2/sub) allows COsub2/sub bioremediation. The synchronized algal-bacteria relationship established is also ideally synergetic for the bioremediation of wastewater (Munoz and Guieysse 2006). Through photosynthesis, microalgae provide oxygen required by aerobic bacteria for the mineralization of organic matter as well as the oxidation of NHsub4/subsup+/sup (Munoz and Guieysse 2006). In return, the bacteria supply carbon dioxide for the growth of microalgae, significantly reducing the amount of oxygen required for the overall wastewater treatment process (Delrue et al. 2016). In general, waste effluents with low carbon to nitrogen ratios are fundamentally suited to the growth of photosynthetic organisms. Most importantly, the microalgal domestic and agricultural wastewater treatments is an attractive option since the technology is relatively easy and they require very low energy compared to the standard of effluent treatment. Optimization of microalgal wastewater treatment in large-scale raceway ponds is appealing since it combines the effective treatment of a harmful waste product and the production of potentially valuable protein-rich algal biomass. Figure 12.1 summarizes a closed loop system for treating any organic waste by combination of anaerobic digestion and algal cultivation.
Microalgae in aquaculture and in aquaponic systems is most often seen to be a nuisance as they can restrict water flows by clogging up pipes, consume oxygen,
Fig. 12.1 Integrated process system to use algal culture for treating organic waste and potential end users. (The process is designed based on information from Ayre et al. 2017 and Moheimani et al. 2018)
may attract insects, reduce water quality and when decomposing can deplete oxygen. However, an experiment by Addy et al. (2017) shows that algae can improve water quality in an aquaponic system, help control pH drops related to the nitrification process, generate dissolved oxygen in the system, 'produce polyunsaturated fatty acids as a value-added fish feed and add diversity and improve resilience to the system'. One of the 'holy grails' of aquaponics is to produce at least part of the food that is fed to the fish as part of the system and it is here that research is required in producing algae that could be grown with part of the aquaponics water, most probably in a separate loop, which can then be fed as part of the diet to the fish.