The pH values of cultures affect the biochemical processes associated with microalgae, including the bioavailability of CO2 for photosynthesis and use of the medium nutrients.

The optimum pH is determined according to the type of microorganism. Some species have an optimum pH of around 7.0; however, some microalgae are tolerant to high pH (Spirulina, pH 11.0) or low pH (Chlorococcum, pH 4.0) (Kumar et al., 2010).

The optimum growth of the microorganism in an acidic or basic environment can be maintained if the intracellular pH is 7.5, regardless of the external pH. Living cells have the ability, within certain limits, to maintain internal pH by expelling hydrogen ions. The external pH generally has a drastic change before it affects the cell. The optimum pH of the cultures should be maintained, thereby preventing the collapse of cell cultures by the cellular process of rupture due to high pH. The control of pH must be integrated with the aeration system by the addition of alkaline solution to the culture (Wang et al., 2012).

Some microalgae have high productivity when maintained at an alkaline pH between 10 and 11. The high pH may be beneficial for outdoor cultivation because it inactivates patho­genic microorganisms and other microalgae (Kumar et al., 2010).

In the case of cultivation with addition of CO2, the concentration of this gas may be the dominant factor that will determine the pH of the culture. In this case, the CO2 demand results from the balance between the transfer of CO2 to the liquid and CO2 consumption by the cells (Wang et al., 2012). SOx and NOx, present in flue gas from burning coal, can also cause changes in pH, damaging microalgal cultivation. With high concentrations of CO2 the pH drops to 5.0, and when exposed to SOx and NOx this value is 2.6 (Westerhoff et al., 2010).

The pH also influences the removal of ammonia and phosphorus. The high pH may increase the removal of ammonia through its volatilization and phosphorus through its pre­cipitation (Craggs, 2005).

Tubular systems are widely used as close systems in commercial production. Usually tubu­lar photobioreactors are made of transparent polypropylene acrylic or polyvinylchloride

pipes with small internal diameters to increase the penetration of light. Mixing and agitation of the culture are maintained by an air pump to provide circulation (see Figure 2.4).

The most significant characteristic of this tubular system that is different from the vertical column bioreactor is the improvement of air-residence time inside the tubular bioreactor, which can provide more dissolved CO2. These systems could use artificial light, but they are also designed based on natural light (sunlight) provided from outside of the tube. The hydrodynamic stress on the algae may vary, depending on the flow characteristics of each system (e. g., turbulent flow, pump type). Likewise, the gas transfer to the culture may vary from low to high, depending on the flow characteristics and the air-supply technique adopted. The operational difficulties are similar to other systems, including growth of microalgae on the wall of the tubes, thus blocking the light penetration; high oxygen concen­tration that can inhibit photosynthesis; and limits on the length of the tube in a single run (Briassoulis et al., 2010).

Coil-type systems are often adopted to enhance the efficiency of space utilization com­pared to the other categories. Among the most important advantages of the system and com­mon to most coil-type systems are the larger ratio of surface area to culture volume to receive illumination effectively, as well as the easy control of temperature and contaminants (Briassoulis et al., 2010). The cleaning problems of tubular systems are not easy to overcome due to the small internal tube size, which has no ready mechanical way to conduct the inside cleaning for a long tube. The scale-up of these systems is relatively easy compared with other photobioreactor designs. The increase of tubular photobioreactor working volume can easily be achieved by simply extending the tube length to the designed volume if the air pump can affordably provide enough power to pump in air bubbles.

Light

source

Microalgae tank

і

Gas mixer

CO2 tank

FIGURE 2.4 Horizontal tubular photobioreactors for microalgae cultivation.

The driving force for vacuum filtration results from the application of suction on the filtrate side of the medium. Although the theoretical pressure drop for vacuum filtration is 100 kPa, it is normally limited to 70 or 80 kPa in actual operation (Shelef et al., 1984). Vacuum filtration can yield algal harvests with moisture contents comparable to those of pressure filtration at lower operating cost if the content of large algal cells in the feed is high.

Five different vacuum filters—vacuum drum filter (not precoated), vacuum drum filter precoated with potato starch, suction filter, belt filter, and filter thickener—have been tested for the harvesting of Coelastrum (Mohn, 1980). Suspended-solids content of the harvested al­gae was in the range of 5-37%. Based on energy consideration, reliability, and dewatering capability, the precoated vacuum drum filter, the suction filter, and the belt filter were recommended. The precoated filter can also be used to harvest tiny microalgae such as Scenedesmus (Shelef et al., 1984). The nonprecoated vacuum drum filter was ineffective and not reliable due to clogging problems. The filter thickeners were not recommended because of low solids content (3-7%) of the algal cake, low filtration velocity, high energy demand, and poor reliability.

Dodd and Anderson (1977) were the first to harvest microalgae by a belt filter precoated with eucalyptus and pine-crafts fibers. The use of a precoat was found to cause undesirable operational complexity and increased costs. In another study, fine-weave cloth rather than the precoated filter was investigated (Dodd, 1980). This method required a relatively low energy input and no chemicals were added. It was found to be efficient in harvesting larger species of algae such as Micractinium, but it had problems with fouling in smaller algal species such as Chlorella. Its capital costs are higher than dissolved-air floatation, but the operating expendi­tures are the lowest among all harvesting methods with the exception of natural settling (Dodd, 1980).

Vinasse is a liquid residue from the sugarcane-based ethanol industry. After sugarcane juice fermentation by yeast, ethanol concentration in the fermented broth is no more than 10% v/v (due to its toxicity). During distillation, the ethanol is recuperated and everything left is called vinasse. It is produced in high volumes (12-15 liters for each liter of ethanol) and is rich in minerals (Rego and Hernandez, 2006). Ethanol production in Brazil in 2012 is esti­mated at 27.9 billion liters (Empresa de Pesquisa Energetica, 2012), which means production of vinasse is around 365 billion liters.

The major problem related to vinasse is its high chemical and biological oxygen demand:

29,0 and 17,000 mgO2/L (Elia Neto and Nakahodo, 1995), respectively, 100 times more pollutant than average domestic wastewater. Vinasse pollutant strength is mainly due to high organic matter content and the presence of three important nutrients: nitrogen, phos­phorous, and potassium (Bittencourt et al., 1978). Due to its composition, vinasse is largely used as fertilizer in sugarcane cultivation. Theoretically, the amount of vinasse allowed per area is regulated by the Brazilian government, but inspection is difficult to be carried out, leading to indiscriminate use.

According to Manhaes et al (2003), soils irrigated with vinasse have high concentrations of nutrients at depths that can contaminate groundwater. Around 60% of the Brazilian ethanol is produced in Sao Paulo state (UNICA, 2010), which is located on the Guarani Aquifer, the second largest underground freshwater reserve in the world.

Given the clear environmental risk caused by poor allocation of vinasse, it is of great importance to apply technical and scientific knowledge for its better distribution, allowing further relocation in water bodies. When used in microalgae cultivation, biological and chem­ical oxygen demand (BOD and COD, respectively) can reach more than 90% reduction in BOD and more than 80% reduction in COD (DalmasNeto, 2012) in the first cycle of cultivation. Considering three cycles, reduction in BOD and COD can reach more than 95%.

Ultrasonic-assisted extractions can recover oils from microalgae cells through cavitation (Harun et al., 2010). During the low-pressure cycle, high-intensity small vacuum bubbles are created in the liquid. When the bubbles attain a certain size, they collapse violently during a high-pressure cycle. During the implosion very high pressures and high-speed liquid jets are produced locally, and the resulting shear forces break the cell structure mechanically. This effect supports the extraction of lipids from algae (Wei et al., 2008). The high-pressure cycles of the ultrasonic waves support the diffusion of solvents, such as hexane, into the cell structure. As ultrasound breaks the cell wall mechanically by the cavitation shear forces, it facilitates the transfer of lipids from the cell into the solvent (Cravotto et al., 2008).

Phospholipids (PLs) consist of fatty acids and a phosphate-containing moiety attached to either glycerol or (the amino alcohol) sphingosine, thus resulting in compounds with fat — soluble and water-soluble regions that are ubiquitors in cell membranes. Glycerol-containing PLs include phosphatidic acid, phosphatidylcholine (PC), phophatidylethanolamine (PE), phosphatidylinositol, and phosphatidylserine. Sphingomyelin (SPH), a major PL, consists of sphingosine and PC. Phospholipids and choline entail several benefits for human health, as depicted in Table 10.4. The level of phospholipids in various red macroalgae varies from 10-21% of total lipids; the main ones are PC (62-78%) and PG (10-23%) (Dembitsky and Rozentsvet, 1990).

Dietary phospholipids act as natural emulsifiers and as such they facilitate digestion and absorption of fatty acids, cholesterol, and other lipophilic nutrients. Algal phopholipids appear to bear a number of advantages relative to fish oils because they are more resistant to oxidation (rancidity), have higher contents of EPA and DHA and provide them with a better bioavailability, and entail a wider spectrum of health benefits for humans and animals (Holdt and Kraan, 2011).

Different seaweed species were gasified in supercritical water as biomass feedstock. The experimental conditions were 500°C of temperature and 1 h of reaction time. The coke yields were found to be significantly lower than those obtained with lignocellulosic and protein contained wastes. The gaseous species detected contained mainly hydrogen, methane, and carbon dioxide. Hydrogen yields ranging between 11.8 and 16 g H2 kg-1 seaweed have been obtained. On the other hand, the methane yields were found to be in the range of 39 and 104 g CH4 kg-1 seaweed. Dissolved organic carbon (DOC) values of aqueous phase show the extent of higher gasification (Schumacher et al., 2011).

Guan et al. reported results from a systematic study of the gasification of the alga Nannochloropsis sp. in supercritical water at 450-550°C. The gaseous products were mainly H2, CO2, and CH4, with lesser amounts of CO, C2H4, and C2H6. Higher temperatures, longer reaction times, higher water densities, and lower algae loadings provided higher gas yields. The algae loading strongly affected the H2 yield, which more than tripled when the loading was reduced from 15 wt% to 1 wt%. The water density had little effect on the gas composition. The temporal variation of intermediate products indicated that some (e. g., alkanes) reacted quickly, whereas others (aromatics) reacted more slowly (Guan et al., 2012).

Finally, biomass can be converted into biogas, either directly from the microalgal biomass or indirectly by the anaerobic digestion of the oilcakes. Various kinds of energies can thus be produced, and the perimeters of the study could be different. Methane potential strongly varies depending on the species composition and degradability (Sialve et al., 2009). In the con­sidered studies, it ranges from 0.262 (Collet et al., 2011) to 0.800 m3CH4 kgDM-1 (Brentner et al., 2011). It should be noted that this last value is higher than the theoretical maximum value for Scenedesmus (Sialve et al., 2009). Energy consumption of anaerobic digesters is usu­ally ignored. It is regrettable, since long hydraulic retention times required to digest low bio­degradable materials (from 10 to 40 days) represent a significant energy effort for mixing and heating. Heat consumption is estimated at 2.45 MJ kgDM in Collet et al. (2011), and electricity consumption is estimated at 0..47 MJ kgDM in Brentner et al. (2011) and 0.39 MJ kgDM in Collet et al. (2011).

Hybrid cultivation is a method that combines different growth stages in two types of photobioreactors, closed and open (Brennan and Owende, 2010). The hybrid culture system is designed to utilize the good qualities of both types of reactors. In the case of open and closed reactors, the first stage of the cultivation occurs in the closed photobioreactor, where the con­ditions are controlled to minimize contamination of other microorganisms and to promote continuous cell division.

In the second stage of production the cells are exposed to a nutritional stress, increasing the synthesis of a specific metabolite, such as lipids, proteins or polymers. The second stage is ideal for open ponds (Brennan and Owende, 2010). The a-shaped reactor is another type of hybrid system, developed by Lee et al. (1995). In this reactor, the culture is lifted 5 meters by air to a receiver tank, and culture flows down an inclined PVC tube (at an angle of 25° to the horizontal) to reach another set of air-riser tubes, and the process is repeated for the next set of tubes.

Photosynthesis can be defined as a redox reaction driven by light energy, in which carbon dioxide and water are converted into metabolits and oxygen. Photosynthesis is traditionally divided into two stages, the so-called light reactions and the dark reactions. The first process is the light-dependent process (light reaction), which occurs in the grana and requires the direct energy of light to make energy carrier molecules that are used in the second process. The light-independent process (or dark reaction) occurs in the stroma of the chloroplasts, where the products accumulated in the products of the light reaction are used to form C-C covalent bonds of carbohydrates. The dark reactions can usually occur if the energy carriers from the light process are present.

In the light reactions, light strikes chlorophyll a in such a way as to excite electrons to a higher energy state. In a series of reactions, the energy is converted (along an electron transport process) into ATP and NADPH. Water is split in the process, releasing oxygen as a byproduct of the reaction. The ATP and NADPH are used to make C-C bonds in the dark reactions.

In the dark reactions, carbon dioxide from the atmosphere (or water for aquatic and marine organisms) is captured and reduced by the addition of hydrogen to form carbohydrates ([CH2O]n). The incorporation of carbon dioxide into organic compounds is known as carbon fixation. The energy comes from the first phase of the photosynthetic process. Living systems
cannot directly utilize light energy, but they can, through a complicated series of reactions, convert it into C-C bond energy that can be released by glycolysis and other metabolic processes. So, the main role of the light reactions is to provide the biochemical reducing agent NADPH2 and the chemical energy carrier (ATP) for the assimilation of inorganic carbon, as presented in Equation 4.1:

2NADP + 3H2O + 2ADP + 2Pi $ 2NADPH2 + 3ATP + O2 (4.1)

The fixation of carbon dioxide happens in the dark (in the stroma of chloroplasts) using the NADPH2 and ATP produced in the light reaction of photosynthesis (Equation 4.2):

CO2 + 4H + 2NADPH + 3ATP $ (CH2O) (4.2)

Carbon dioxide is available in water in three different forms: CO2, bicarbonate (HCOg), or carbonate (HCO32~) (Figure 4.1), the relative amounts of which are pH dependent. Although plants and algae are known to be dependent exclusively on the Calvin-Benson-Bassham cycle (also known as the Calvin cycle) (Atomi, 2002), six autotrophic carbon-fixation pathways are known. These are (1) the Calvin cycle, (2) the acetyl-CoA pathway, (3) the 3-hydroxypropionate cycle, (4) the reverse tricarboxylic acid cycle, (5) 3-Hydroxypropionate/4-hydroxybutyrate cycle, and (6) Dicarboxylate/4-hydroxybutyrate cycle (GeorgeFuchs, 2011). This section discusses the Calvin cycle, which is the most important in microalgae.

In the Calvin cycle there is only one enzyme responsible for CO2 fixation: ribulose 1,5-biphosphate carboxylase/oxygenase, also known as Rubisco. Figure 4.2 shows the Calvin

CO2(g) ^—► CO2(aq) ^—► H2C03 ч—► HCO — ч—► C032- FIGURE 4.1 Different forms in which carbon dioxide is

available in water.

cycle, where one molecule of ribulose 1,5-biphosphate and a CO2 are converted into two glycerate phosphate. CO2 diffuses through the cell and is captured by the enzyme ribulose biphosphate (Rubisco).

CO2(g) $ CO2 (aq) $ H2CO3 $ HCO3 $ CO32- The fixation of CO2 occurs in four distinct phases (Masojidek et al., 2004):

1. Carboxylation. A reaction whereby CO2 is added to the five carbon sugar ribulose bisphosphate (Ribulose-bis-P) to form two molecules of phosphoglycerate (Glycerate-P). This reaction is catalyzed by the enzyme ribulose biphosphate carboxylase/oxygenase (Rubisco).

2. Reduction. To convert Glycerate-P into 3-carbon sugars (Triose-P), energy must be added in the form of ATP and NADPH2 in two steps, which are the phosphorylation of Glycerate-P to form diphosphoglycerate (Glycerate-bis-P) and the reduction of Glycerate-bis-P to phosphoglyceraldehyde (Glyceraldehyde-P) by NADPH2.

3. Regeneration. Ribulose-P is regenerated for further CO2 fixation in a complex series of reactions combining 3-, 4-, 5-, 6-, and 7-carbon sugar phosphates, which are not explicitly shown in the diagram.

4. Production. The primary end products of photosynthesis are considered to be carbohydrates, fatty acids, amino acids, and organic acids.

Besides the carboxylase activity described here, all Rubiscos (there is more than one type) are known to display an additional oxygenase activity in which an oxygen molecule, com­peting with CO2 for the enzyme-bound eno-diolate of RuBP, reacts with RuBP to form

3- phosphoglycerate and phosphoglycolate (Atomi, 2002). The latter product is subse­quently oxidatively metabolized via photorespiration, leading to a net loss in carbon dioxide fixation. Photorespiration thus represents a competing process to carbon fixation, where the organic carbon is converted into CO2 without any metabolic gain. Photorespiration depends on the relative concentrations of oxygen and CO2 where a high O2/CO2 ratio stimulates this process, whereas a low O2/CO2 ratio favors carboxylation. Rubisco has low affinity by CO2; its Km (half saturation) is approximately equal to the level of CO2 in air. Thus, under high irradiance, high oxygen level, and reduced CO2, the reaction equilibrium is shifted toward photorespiration. For optimal yields in microalgal mass cultures, it is necessary to minimize the effects of photorespiration, achieved by an effective stripping of oxygen and by CO2 enrichment. For this reason, microalgal mass cultures are typically grown at a much higher CO2/O2 ratio than that found in air, which is in turn an opportunity to reuse industrial gas emissions.

The source of nitrogen in cultivation of microalgae seems to cause changes in oxygen production during photosynthesis. The ratio between O2 evolution rate and CO2 uptake rate (the photosynthetic quotient, PQ) depends on the composition of the produced biomass and the substrates that are used. Especially oxidized nitrogen sources, which must be reduced before they are incorporated into the biomass, affect the PQ. When nitrate is used, it is expected at an evolution of 1.3 mol O2 per mol of CO2 assimilated, whereas nitrite promotes a release of 1.2 mol O2 and ammonia 1.0 mol O2 (Eriksen et al., 2007). Approximately 20% of O2 evolution equivalents can be accounted for by NO33 uptake and assimilation under N-replete conditions (Turpin, 1991).