The Rhodophyta is a relatively well-defined group of about 6,000 algal species with several features that differentiate them from other algal divisions, such as the pres­ence of accessory phycobilin pigments, the absence of flagella and centrioles [36]. The vast majority of red algae are marine multicellular, macroscopic species, which account for the majority of the so-called seaweeds [37]. The main habitats are near­shore and offshore zones (down to 40-60 m) in tropical and temperate climate regions while the presence of accessory pigments allow algae to grow at depths down to 200-250 m. Species with calcified cell walls are important for the estab­lishment and support of coral reef formation. Red algae are also found in brackish and fresh water, as well as in soil [38, 39].

Porphyra species are an important food source for humans in the Asia region [40]. Several Rhodophyta species (Gelidium, Gracilaria) are an important source of agar and agarose [41] . These polysaccharides are used in many laboratories for preparing culture media and separating nucleic acids [42]. Carrageenan is widely used in the food industry as a gel forming substance and stabilizer [43] (Tables 5-7). Structural, biochemical characteristics and productivity of selected red algae species are presented in Tables 5-7.

The simultaneous application of two pretreatment methods can potentially enhance COD solubilization, VS reduction, and methane yield. The following combination of treatment methods have been studied with WAS:

— Thermochemical (alkali-, acid-thermal pretreatment)

— Ultrasonic plus thermal

— Irradiation-assisted methods

Thermal treatment of WAS after adjustment of the pH to 12 (121°C for 30 min) led to an increase in the COD solubilization. With NaOH, KOH, Mg(OH)2 , and Ca(OH)2, solubilization reached 51.8%, 47.8%, 18.3%, 17.1% after treatment at 121°C for 30 min [201] or 71.6%, 83.7%, 55.6%, 51.5% at 140°C for 30 min [225], respectively. Other authors reported a COD solubilization of 55% at pH 12 (NaOH) and 140°C for 30 min compared to 48% without alkali reagent [236].

Thermochemical pretreatment enhanced methane yield by 34 and 19% compared to untreated and chemically pretreated samples, respectively [201]. Combined ther­mochemical pretreatment of A. maxima had a stronger impact on increasing the amount of sCOD (Fig. 12a) [233]. A maximum solubilization value of 78% was achieved at pH 13 and temperature 150°C. In most cases, methanogenesis was inhibited compared to untreated fresh algae (Fig. 12b). At pH 11, the methane yield increased by 5%, 10%, and 20% at temperatures 50, 100, and 150°C, respectively. Overall, alkali-thermal pretreatment led to higher levels of solubilization and to larger methane yields com­pared to acid-thermal pretreatment. Strong inhibition of methanogens can be caused by ammonia, toxic chemicals, and/or fatty acids formed during pretreatment.

Wet oxidation is a pretreatment process when organic materials are treated by gaseous oxygen at high temperatures. It is able to convert poorly biodegradable lignocellulose to carbon dioxide, water, and carboxylic acid [253] . Newspaper bio­mass pretreated by wet oxidation (190°C) and fermented in a batch anaerobic reac­tor showed 59% lignin removal, 74-88% cellulose removal, and 59% of the total COD converted to methane [223]. A doubling of methane yield from raw yard waste after wet oxidation pretreatment was reported [254] .

Microwave irradiation can be used as a volumetrically distributed heat source, and it can be applied with acid or alkali pretreatment. Microwave-assisted acid pretreatment of herbal-extraction process residue enhanced biogas production by 65, 29, and 14% compared to nontreated, acid, or microwave pretreated samples [255, 256]. Microwave-assisted alkali pretreatment of switchgrass increased the cellulase hydrolysis yield by 53% [257]. Ultrasonic treatment (42 kHz for 120 min) after ther­mal treatment (121°C for 30 min) had no effect on COD solubilization. Solubilization enhancement was not statistically significant—19.4% vs. 18.4% (ultrasonic only) — and was comparable with the value for single thermal pretreatment of 17.6% [201].

5.1.1 Algal Metabolic Manipulations Through Environmental Factors

The algal biochemical composition has a dramatic impact on algal biomass digest­ibility and methane yield (Figs. 5 and 7). Improving the extent of algal biomass biodegradability in anaerobic digesters is a critical research need.

Metabolic manipulation is an effective tool for control and influence of algal growth rate and biochemical composition. Several environmental factors, such as availability of carbon dioxide, nutrients (nitrogen, phosphorus), trace metals, silicon for diatoms, level of irradiation, salinity, and temperature affect the enzyme activity and algal biochemical profile significantly.

Generally, stress conditions (nitrogen, phosphorus, or trace metals depletion, photo-oxidative stress, and high salinity) lead to increasing cellular lipid content. Exposure to stress conditions resulted in increasing lipid content on average from

25.5 to 45.7% in green microalgae, from 22.7 to 44.6% in diatoms, and from 27.1 to 44.6% in other oleaginous algae identified as chrysophytes, haptophytes, eustig — matophytes, dinophytes, xanthophytes, or rhodophytes [258]. A major disadvantage of using metabolic methods to modify the lipid content is a decrease in the algal growth/division rate and productivity [259, 260] . This can be countered by an increase in the overall calorific value and theoretical methane potential of the algal biomass due to accumulation of more reduced lipid compounds [261-263]. Li observed the highest lipid productivity at 5 mM nitrate while the highest lipid con­tent was observed at 3 mM nitrate [264]. Optimization of all environmental param­eters is necessary to achieve the highest biomass and lipid productivity [265] .

The NER values for coupled production of biodiesel and biogas (residues) from

H. pluvialis and Nannochloropsis are 0.4 and 0.09, respectively, with approximately 58 and 76% of the output energy coming from biogas [488]. The sensitivity analysis showed that it is not possible to achieve an NER value larger than 1 even at most optimistic algal yield and oil content. Pumping, drying, and cell-disruption are the most energy consuming steps. Lardon et al. emphasize that integration of ADP into biodiesel production is a promising solution for external energy demand reduction and partial recycling of essential nutrients [489].

Energy output from T. suecica was reported for several conversion techniques (Fig. 22) [435]. Anaerobic digestion as a sole conversion technology has the largest energy output compared to biodiesel and bioethanol. Coupling of biogas production with biodiesel production gave slightly larger total energy output, and decreased the estimated biodiesel production cost from $72 to $47 per liter. But this cost is significantly larger than the current cost of petroleum-based biodiesel.

The life-cycle assessment of microalgal cultivation and biogas production shows that the NER value is equal to 1.51 for the following conditions: C. vulgaris is culti­vated in ponds with area of 100 ha, productivity of 25 g/m2-day, carbon dioxide sup­plied from biogas purification and methane combustion, and the supernatant liquid from the digester provides a portion of the fertilizers necessary for algal growth [490]. Technical parameters of the ADP include: CSTR digester, methane yield 0.292 L/ gVS, OLR 1 gVS/L-day, HRT 46 days, and algal biodegradability of 56%.

Zamalloa and colleagues reviewed three scenarios of growing algae in wastewa­ter effluent with productivity 20, 25, and 30 g/m2-day [491]. The algal production facility utilized carbon dioxide produced during electricity production. For all sce­narios, the NER values are larger than 1 and equal to 2.48, 2.67, and 3.34, respec­tively. This means that the energy output is larger than energy demand for algal production and anaerobic digestion. The major energy consuming processes are digester heating, mixing, algal pre-concentration, and pumping. The cost of bio­mass for three scenarios were $169, $138, and $117 per kg of dry weight and the levelized cost of energy were $0.232, $0.154, and $0.119 per kWh (given €1/$1 equal to 1.3652). A minimum algal productivity of 25 g/m2-day is required in order to achieve profitability with an OLR of 18 gVS/L-day and methane yield of 0.5 L/gVA. The fermentation of at least 75% of the VS is crucial for an economically feasible process. These assumptions are relatively optimistic but can be achieved in advanced anaerobic reactors.

6 Conclusions

Cyanobacteria and algae are feasible feedstocks for biogas production through ADP. Moreover, with current understanding and technology, the anaerobic digestion of algae has the promise to be the technology that can be applied for biofuel produc­tion in the nearest future. Despite more than 60 years of research and several advan­tages, the technology of methane production from algae is still far from wide application at a large scale. One of the reasons for that is the vast diversity of algae and cyanobacteria. They have different cell morphology, ecology, photosynthesis biochemistry, cell structure, and biochemical composition.

Further research directions that are critical for making ADP with algae

economically feasible for successful commercialization include:

• Engineering of efficient systems for algal cultivation and anaerobic digestion:

— Design of algal production units for better light illumination, penetration, car­bon dioxide dissolution, and oxygen off-take

— Design algal harvesting and dewatering units

— Develop methods of biomass pretreatment for greater VS reduction and higher methane yield

— Design of anaerobic reactors with lower HRT and high SRT

— Design of biogas upgrading systems

• Isolation and characterization of prospective natural organisms:

— Isolation of algal strains with high production potential, biochemical compo­sition with high colorific value, lower recalcitrance, robustness for cultivation in waste streams and to aeration by exhausted gases

— Isolation of anaerobic bacteria responsible for digesting algal biomass in nature

• Developing and application molecular biology and genetic tools, and “omics”

technologies for cyanobacteria, algae, and anaerobic organisms to target goals

such as:

— Decreasing algal photoinhibition, increasing photosynthesis efficiency and carbon dioxide uptake

— Algal simultaneous lipid accumulation and high growth rate

— High digestibility of algal biomass

— Algal persistence to bacterial and viral infections

— Development of stronger hydrolytic apparatus of anaerobic bacteria

— Robustness of methanogens for fluctuations in environmental and operational parameters

• Integration of algal production and AD with other technologies:

— Co-digestion with domestic, industrial, and agricultural wastes can improve the C:N:P balance

— Algal cultivation in wastewaters containing organic carbon and nutrients

— Co-location of algal production with carbon power plants that can be a source of carbon dioxide and waste heat

— Application of algal anaerobic digestion with other biofuel production pro­cesses or production of high-value products from algae

• Development of information technologies:

— For techno-economical analysis

— Dynamic modeling of systems

— Bioinformatics technologies

— Metabolic networks modeling

Acknowledgments This research was supported by The Bureau ofEducation and Cultural Affairs of United States Department of State though an International Fulbright Science and Technology Award to Pavlo Bohutskyi.

Sediment sampling and the interpretation of properties measured using samples are among the most challenging tasks in geo-engineering. A sample is expected (1) to represent the sediment constitution (grain size, mineralogy, and fluids), (2) to cap­ture the statistics of the sediment characteristics, and (3) to preserve its physical properties. Unfortunately, most of these characteristics are seriously compromised in the sampling of HBS because of (a) unavoidable changes in effective stress and ensuing strains, (b) SH changes during sample recovery, handling, and storage, and

(c) discrepancies between GH feature scale and scale of sampling [127].

There are several initiatives to overcome or circumvent these difficulties. New technology developed at the Georgia Institute of Technology makes possible pres­sure core testing to characterize HBSs without ever exposing them outside the PT-stability field [222]. A significant effort is in progress to relate index properties to the bounds of HBS properties [31,45, 221], and to further complement the analy­sis with geomechanical and geophysical testing [31, 70, 101, 113]. We can antici­pate that further developments in sediment characterization will include more extensive developments in in situ testing. Finally, proper formation characterization will combine information gathered using index-property-based bounds, reconsti­tuted specimens, pressure cores, and in situ testing data.

Christopher J. Brigham, Claudia S. Gai, Jingnan Lu, Daan R. Speth, R. Mark Worden, and Anthony J. Sinskey

Abstract Isobutanol (IBT) can be used as a 100% replacement for gasoline in existing automobile engines, has >90% of the energy density of gasoline and is compatible with established fuel distribution infrastructure. The facultatively auto­trophic bacterium Ralstonia eutropha can utilize H2 for energy and CO2 for carbon and is also employed in industrial processes that produce biodegradable plastics. Using a carefully designed production pathway, R. eutropha, a genetically tractable organism, can be modified to produce biofuels from autotrophic growth. Microbial

C. J. Brigham • C. S. Gai

Department of Biology, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

J. Lu

Department of Chemistry, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

D. R. Speth

Department of Biology, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

Department of Microbiology, IWWR, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands

R. M. Worden

Department of Chemical Engineering and Materials Science,

Michigan State University, East Lansing, MI 48824, USA

A. J. Sinskey (H)

Department of Biology, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

Engineering Systems Division, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

Health Sciences Technology Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA e-mail: asinskey@mit. edu

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_39, 1065 © Springer Science+Business Media New York 2013

production of IBT can be achieved by directing the flow of carbon through a synthetic production pathway involving the branched-chain amino acid biosynthe­sis pathway, a heterologously expressed ketoisovalerate decarboxylase, and a broad substrate specificity alcohol dehydrogenase. We discuss the motivations and the methods used to engineer R. eutropha to produce the liquid transportation fuel IBT from CO2, H2, and O2.

1 Introduction

Increased demand for fossil fuels along with dwindling reserve supplies reveals an immediate need for alternative fuel sources. Bio-based fuels, or biofuels, are pro­duced from many sources of biomass. Microbially produced biofuels offer a sus­tainable approach to fuel production using inexpensive carbon sources, such as agricultural waste or CO2 [1] . The availability of H2 derived from solar-powered electrolysis will eventually increase dramatically, creating demand for microbes that use this energy source to convert CO2 into value-added chemical compounds, including liquid transportation fuels. Ethanol has been long discussed as a biofuel since Beall et al. [2, 3] developed a method for producing ethanol from sugars using a recombinant Escherichia coli strain. However, ethanol is not the most suitable alcohol for biofuel use as its hygroscopicity is higher and its energy density is lower than for longer chain alcohols [4]. Isobutanol (IBT), on the other hand, can be used without gasoline-blending in existing internal combustion engines and is compati­ble with the existing fuel infrastructure [5] . Recently, an automobile competed in the American Le Mans racing Series running on 100% IBT [6]. Although the source of IBT was not disclosed, the American Le Mans racing Series had recently approved IBT from corn, sugarcane, and cellulosic feedstocks for use as a fuel [7].

Microalgal biomass can be pre-treated via hydrolysis using the cellulosic enzymes obtained from fungi, protozoa or bacteria. Widely used cellulosic enzymes are cellu — lase from Thrichoderma reseei and cellulase from Aspergillus niger. The cellulase enzyme consists of three main components [63]; (1) 1,4-b-D-glucan glucanohydro — lases (endoglucanases); break down the cellulose crystallinity, (2) 1,4-b-D-glucan cellobiohydrolases and 1,4-b-D-glucan glucanohydrolases (exoglucanases); the exog- lucanases hydrolyse the individual cellulose fibres into simple sugars and cellobiohy — drolases attack the chain ends producing cellobiose, (3) b-D-glucoside glucohydrolases (b-glucosidases); release glucose monomers by hydrolysing the disaccharides and tetrasaccarides of cellulose and form glucose that is ready to be used in the fermenta­tion process. Even though it is a common practice for biomass to be pre-treated prior to enzymatic hydrolysis, microalgal biomass can undergo the hydrolysis process directly without any pre-treatment due to its non-lignin composition. This makes production of bioethanol from microalgal biomass more economical.

The use of co-solvents such as dimethyl ether (DME), diethyl ether (DEE), methyl tert-butyl ether (MTBE) and tetra-hydro-furan (THF) has attracted much attention since they allow one to increase the reaction rate, under milder conditions, by dimin­ishing the mixture polarity [15, 43].

For example, the use of co-solvents and high mixing intensity for the reaction reduces the need to use higher temperatures to enhance the solubility among reac­tants and the mass transfer between both phases [11]. Moreover, transesterification in supercritical methanol, employing propane and CO2 as co-solvents, was also developed [23,48,84,98]. Nevertheless, the selection of the appropriate co-solvent and the mixing intensity are critical factors contributing to the correct operation and performance of the reaction system.

Sabudak and Yildiz [83] studied biodiesel production from waste frying oils by applying three different processes: a one-step alkali-catalyzed transesterification, a two-step alkali-catalyzed transesterification, and a two-step acid-catalyzed transesterification followed by alkali-catalyzed transesterification. For each reac­tion, these authors added THF as a co-solvent concluding that the effect of THF on reaction yield is not significant, and instead of using the co-solvent, it is more eco­nomical to improve the mixing ability of the reactor.

Unit operations such as centrifugation and microfiltration may be preceded by flocculation to improve the efficiency of recovery [21]. Flocculants can improve dewatering characteristics during centrifugation and fil tration because of their binding capabilities. Flocculants can help to maintain cellular properties when the culture experiences high shear forces during processes such as centrifugation [3]. Multi-stage dewatering processes have the potential to significantly reduce the energy consumption involved with large-volume cultures. It is estimated that bio­mass recovery contributes up to 20-30% of the total biomass production cost [12]. Therefore, multi-stage dewatering processes have the potential to reduce the eco­nomics involved with biomass production.

Note that only Scope 1 and Scope 2 emissions are considered in this section. Scope

1 emissions refer to the release of GHG as a direct result of an activity or series of activities (including ancillary activities) that constitute the facility. Scope 2 emissions refer to emissions caused by the activity of the facility but in this case the emissions are not directly released from the facility [37]. Even though Scope 2 emissions are not direct emissions from within the system boundary, the activity within the system boundary causes these emissions to occur at another facility; hence, Scope 2 emissions are considered as well. An example of a Scope 2 emission is the emissions from electricity usage. Even though the use of electricity does not directly increase GHG emissions from within the system boundary, it creates GHG emissions at another facility which is the power station. As per the NGER Act, both Scope 1 and Scope

2 emission have to be reported; however, the financial liability of a corporation only rests with Scope 1 emissions as per CPRS. Scope 3 emissions (process unrelated emissions such as administrative and transportation emissions) are not be consid­ered in this study, as there is insufficient information to undertake an accurate analy­sis. All GHGs and energy usage will be converted to tonnes of carbon dioxide equivalent (tonne CO2-e) to enable ease of comparison. It is assumed that the plant is operating at normal conditions when the audit takes place, and all equipments utilise electricity from the grid, unless otherwise stated.

1.1.1 Carbon

Since S. platensis is composed of approximately 50% carbon [30], the use of carbon dioxide in the cyanobacterium cultivation may contribute significantly to reducing the cost of production and, at the same time, to reducing the emission of this green­house gas. Photosynthetic microorganisms can efficiently assimilate carbon dioxide from different sources, including the atmosphere, industrial exhaust gases, and soluble carbonate salts [112] . In fact, carbon dioxide taken up by photosynthetic

microorganisms is among the most productive biological methods of treating industrial waste emissions, and the yield of biomass per acre is three — to fivefold greater than in typical crops [2, 55]. The most important trace gases, which intensify the greenhouse effect, are carbon dioxide (CO2) , methane (CH4) , nitrous oxide (N2O), and ozone (O3). Among all these emissions, carbon dioxide makes the great­est contribution to global warming. For this reason, most of the measures for miti­gating climate change target the reduction of carbon dioxide emissions to the atmosphere [77]. In this sense, there are several studies that point out the use of carbon dioxide in the cultivation of Arthrospira (Spirulina) platensis [8,15,41,63, 79, 96, 114].

Traditionally, Arthrospira spp. grow autotrophically in open tanks (Fig. 2) in the presence of high sodium carbonate and bicarbonate levels as carbon sources because they are relatively inexpensive and provide a high pH in the culture medium. Bicarbonate, the main carbon source, is actively transported into the cell, where the enzyme carbonic anhydrase, present intracellularly and/or in the periplasmic mem­brane, promotes the release of carbon dioxide. This is incorporated in the Calvin cycle to produce organic molecules such as carbohydrates, proteins, and lipids [52] .

At low extracellular concentration of bicarbonate, cyanobacteria have the ability to accumulate bicarbonate intracellularly [30] and use carbon dioxide as a carbon source for its metabolism. In medium containing only carbonate, there is no increase in cell concentration and the pH remains almost unchanged, emphasizing the impor­tance of bicarbonate in cyanobacterial metabolism [8].

Taking into account that cultivations in tubular photobioreactors (Fig. 3) lead to a high cell concentration, it is essential that CO2 is added during cell growth to sus­tain it [63], thus justifying the fed-batch process for this carbon source. Soletto et al. [96] evaluated the performance of a bench-scale helical photobioreactor in fed-batch

cultures of S. platensis under different conditions of light intensity and CO2 feeding rate. The optimum feeding rate of carbon dioxide for the microalgal growth was cor­related with the light intensity to which it was exposed. In general, the behavior of S. platensis was more influenced by the CO2 feeding pattern at low PPFD. Irrespective of the light intensity studied, at a CO2 feeding rate of 1.03 g L-1 d-1, the excessive amount of CO2 caused an inhibition of biomass growth due to excess carbon levels and likely due to pH reduction.

Matsudo et al. [63] studied the use of CO2 released from alcoholic fermentation, without any prior treatment, for carbon source replacement and pH control, in the continuous cultivation of A. platensis, using urea as nitrogen source, in a bench scale tubular photobioreactor. Irrespective of the carbon source used in the cultiva­tion of this cyanobacterium (pure CO2 or from alcoholic fermentation), it was obtained similar behavior in cell growth. In both cases, the maximum cell concen­tration in steady state condition (X) occurred at dilution rate (D) of 0.2 d-1, being
obtained Xs = 2,446 ± 75 and 2,261 ± 71 mg L-1 in cultivations carried out using pure CO2 and CO2 from alcoholic fermentation, respectively. It was not observed any difference in the protein content of the dry biomass when these two kinds of carbon sources were used. The higher the D, the higher the protein contents of the dry bio­mass, which were as high as about 50% when the cultivations were carried with D=0.8 d-1. These results indicated that the possibility of using such cost-free carbon source and a cheap nitrogen source like urea may contribute to reduce the cost of culture medium. Besides, this work suggests that the biofixation of CO2 released from alcoholic fermentation of sugar raw materials, which represents about 33% of the whole CO2 involved in the use of ethanol as fuel, may help to mitigate the green­house effect.

Carvalho et al. [15] have proposed methods for the recovery and purification of CO2 from alcoholic fermentation and/or burning of lignocellulosic materials and feed it into the cultivation of photosynthetic microorganisms. Application of the fed-batch process would be particularly important for the fixation of CO2 from industrial plants. Such statement can be evidenced by the fact that the ethanol pro­duction in Brazil was as high as 27.5 billion liters in 2008/2009 [107], and it can be estimated that the release of CO2 associated only with this fermentation process was about 20.8 billion kg. Moreover, considering that the all correspondent sugar cane bagasse was burned, an additional CO2 production of about 83 billion kg would occur [108].

Regardless of the importance of the fed-batch process when using CO2 as a car­bon source, organic carbon sources are the most cited cases in which the fed-batch process is employed in the cultivation of photosynthetic microorganisms. Despite the increase in the risk of contamination, which requires running the process in closed reactors under aseptic conditions, the use of an organic carbon source can provide readily usable energy and make it possible to reach a high final biomass concentration. It can be done under dark (heterotrophic) or light (mixotrophic) con­ditions. Taking into account that the organic carbon source can lead to inhibition of the growth beyond a limit concentration [103] or even repress the formation of a desired metabolite, several fed-batch processes have been used to improve cell or metabolite production by different microalgae and cyanobacteria [20, 103] or even to remove organic pollutants from wastewater [58].

Marquez et al. [61] showed that S. platensis can grow heterotrophically in a medium containing glucose in aerobic and dark conditions but also mixotrophically under illuminated conditions. Chojnacka and Noworyta [20] also observed Spirulina sp. growth under heterotrophic conditions using glucose as carbon and energy sources. Chen et al. [19] showed that acetate may be used as a carbon source, in mixotrophic S. platensis cultivations, for the production of several photosynthetic pigments.

S. platensis can also use glycerol as the sole carbon source. Nevertheless, when this microorganism takes up glycerol for the first time, it forms aggregates of cells and a lag phase takes place. After the lag phase, however, it was possible to achieve a cell concentration higher than with cultivation using bicarbonate as the sole car­bon source [68] .

Three organic carbon sources differing in molecular complexity (glucose, acetate, and propionate) were tested by Lodi et al. [58] in a fed-batch mixotrophic process with minimum volume variation. To avoid carbon source accumulation in the medium, acetate and proprionate were added by pulse feeding equimolar amounts about 12 h after their complete depletion, namely acetate every 3.4 days and propi­onate every 4.0 days, whereas glucose was added once a day. The results of cultiva­tions performed under continuous illumination show that glucose was metabolized for algal growth faster than acetate and proprionate. Besides, the values of nitrate and phosphate removals are near those observed with traditional biological treat­ment plants [29]. This suggests that mixotrophic metabolism of S. platensis could be exploited in a tertiary treatment system for simultaneous removals of mixtures of organic pollutants, nitrate, and phosphate [58] .

The chlorophyll content in the biomass did not appreciably vary during the course of cultivations (2-5%), thereby indicating that most of the dry weight increase was the result of microbial growth. Besides, it was shown to be almost independent on the type of organic carbon source and about 17% lower than that determined by Danesi et al. [33] under autotrophic conditions, thus confirming the underutilization of the photosynthetic apparatus when cyanobacteria are grown with an organic car­bon source [58] .

Chen and Zhang [18] reported that S. platensis cell concentration in a fed-batch culture with intermittent glucose feeding with a peristaltic pump was 4.25-fold higher than in the mixotrophic batch culture and 5.1-fold higher than in the photoau­totrophic batch culture. The biomass output rate of the fed-batch culture was 3.1-fold higher than in the mixotrophic batch culture and 3.8-fold that of the photoautotrophic culture. Additionally, these authors demonstrated that a mixotrophic fed-batch cul­ture of S. platensis with glucose is suitable for the production of high-value products, particularly the light-induced pigments such as phycocyanin.