**9. The physical and chemical properties of biodiesel produced from algal cell**

Analysis of the produced biodiesel from the promising alga *Dictyochloropsis splendida* (Table 5). showed that the unsaturated fatty acids percentage was increased in alga cultivated in nitrogen free media (0.0g/l N) two times more than normal conditions (13.67, 4.81% respectively). However, the composition of fatty acids was different in these algae depending on its growth condition as showed in table 3. These results were in agreements with those reported by Wood (1974) relative to *Chlorophycean* species. Furthermore Ramos *et al*. (2009) reported that monounsaturated, polyunsaturated and saturated methyl esters were built in order to predict the critical parameters of European standard for any biodiesel, composition. The extent of unsaturation of microalgae oil and its content of fatty acids with more than four double bonds can be reduced easily by partial catalytic hydrogenation of the oil (Jang *et al*., 2005, Dijkstra, 2006). Concerning the fatty acids contents of the produced biodiesel from microalgae, Chisti (2007) reported in his review that, microalgal oils differ from vegetable oils in being quite rich in polyunsaturated fatty acids with four or more double bands (Belarbi *et al*., 2000) as eicosapentanoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) which occurred commonly in algal oils. The author added that, fatty acids and fatty acid methyl esters with four and more double bands are susceptible to oxidation during storage and this reduces their acceptability for use in biodiesel especially for vehicle use (European standard EN 14214 limits to 12%) while no such limitation exists for biodiesel intended for use as healing oil. In addition to the content of unsaturated fatty acids in the biodiesel also its iodine value (represented total unsaturation) must be taken in consideration (not exceeded 120 g iodine/100g biodiesel according to the European standard.

410 2.7 21c

49 0.3 25c

Table 4. Comparison of estimated biodiesel production efficiencies from vascular plants and

**9. The physical and chemical properties of biodiesel produced from algal cell**  Analysis of the produced biodiesel from the promising alga *Dictyochloropsis splendida* (Table 5). showed that the unsaturated fatty acids percentage was increased in alga cultivated in nitrogen free media (0.0g/l N) two times more than normal conditions (13.67, 4.81% respectively). However, the composition of fatty acids was different in these algae depending on its growth condition as showed in table 3. These results were in agreements with those reported by Wood (1974) relative to *Chlorophycean* species. Furthermore Ramos *et al*. (2009) reported that monounsaturated, polyunsaturated and saturated methyl esters were built in order to predict the critical parameters of European standard for any biodiesel, composition. The extent of unsaturation of microalgae oil and its content of fatty acids with more than four double bonds can be reduced easily by partial catalytic hydrogenation of the oil (Jang *et al*., 2005, Dijkstra, 2006). Concerning the fatty acids contents of the produced biodiesel from microalgae, Chisti (2007) reported in his review that, microalgal oils differ from vegetable oils in being quite rich in polyunsaturated fatty acids with four or more double bands (Belarbi *et al*., 2000) as eicosapentanoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) which occurred commonly in algal oils. The author added that, fatty acids and fatty acid methyl esters with four and more double bands are susceptible to oxidation during storage and this reduces their acceptability for use in biodiesel especially for vehicle use (European standard EN 14214 limits to 12%) while no such limitation exists for biodiesel intended for use as healing oil. In addition to the content of unsaturated fatty acids in the biodiesel also its iodine value (represented total unsaturation) must be taken in consideration (not exceeded 120 g iodine/100g biodiesel according to the European

**Area required as a percent of total global land** 

**Area required as a percent of total arable global land** 

**Area needed to meet global oil demand (106 hectans)** 

cAssuring that microalgal ponds and bioreactors are located on non-arable land

**Cotton** 15000 101 757 **Soybean** 10900 73 552 **Mastard seed** 8500 57 430 **Sunflower** 5100 34 258 **Rapeseed/Canola** 4100 27 207 **Jatropha** 2600 17 130b **Oil palm** 820 5.5 41

**Biodiesel feedstock** 

**Microalgae (10 g/m3/day, 30%TAG)** 

**Microalgae (50 g/m3/day, 50%TAG)** 

microalgae

standard.

b Jatropha is mainly grown on marginal land


Table 5. Analysis of fatty acids of the obtained biodiesel from the promising green microalgae *Dictyochloropsis* sp

#### **10. Enhancement the biodiesel production from algae**

Lipid productivity, the mass of lipid that can be produced per day, is dependent upon plant biomass production as well as the lipid content of this biomass. Algal biodiesel production will therefore be limited not only by the standing crop of microalgae, but also by its lipid content, which can vary from <1% to >50% dry weight [Shifrin, N.S. and Chisholm, 1980]. Given that a strong and predictable response of microalgal biomass to phosphorus enrichment has consistently been exhibited by freshwater ecosystems worldwide (Box 2), it can be expected that the volumetric lipid content (in mg L\_1) of water contained in algal bioreactors should also in general increase with an increase in the total phosphorus content of the system, as has been reported for lakes by Berglund *et al*. [Berglund, 2001]. However, both the quantity and the quality of lipids produced will vary with the identity of the algal species that are present in the water, as well as with site-specific growth conditions. This variability probably reflects modifications in the properties of cellular membranes, and alterations in the relative rates of production and utilization of storage lipids [Roessler, 1990]. In the presence of moderate temperatures and sufficient light, many dozens of studies during the past several decades have revealed that algal lipid content is particularly sensitive to conditions of nutrient limitation . For example, silicon-starved diatoms can contain almost 90% more lipids than silicon-sufficient cells [Shifrin, N.S. and Chisholm, 1980]. However, silicon will be a growth-limiting nutrient only for the limited subset of microalgal species that have an absolute requirement of this element for their cellular growth. A stronger stimulation of lipid production occurs in response to conditions of nitrogen limitation, which potentially can occur in all known microalgae. Nitrogen-starved cells can contain as much as four times the lipid content of Nsufficient cells [Shifrin, N.S. and Chisholm, 1980], and maximizing the lipid production of pond bioreactors should

Algal Biomass and Biodiesel Production 125

biodiesel and sediment (glycerin and pigments) percentages. Hexane/ ether (1:1, v/v) extraction solvent system resulted in low lipid recoveries (2.3-3.5% dry weight) while; chloroform/methanol (2: 1, v/v) extraction solvent system was proved to be more efficient for lipid and biodiesel extraction (2.5 – 12.5% dry weight) depending on algae species (Table 7). The green microalga *Dictyochloropsis splendida* extract produced the highest lipid and biodiesel yield (12.5 and 8.75% respectively) followed by the cyanobacterium *Spirulina maxima* (9.2 and 7.5 % respectively). On the other hand, the macroalga (red, brown and green) produced the lowest biodieselyield. The fatty acids of *Dictyochloropsis splendida*  Geitler biodiesel were determined using gas liquid chromatography. Lipids, biodiesel and glycerol production of *Dictyochloropsis splendida* Geitler (the promising alga) were markedly enhanced by either increasing salt concentration or by nitrogen deficiency (Table 8) with maximum production of (26.8, 18.9 and 7.9 % respectively) at nitrogen starvation condition.

Table 7. Total lipid, biodiesel, sediment percentage and biodiesel color of eight algal species Natural biotic communities in outdoor bioreactors require the external provision of potentially growth-limiting resources (e.g. light, carbon dioxide and the essential mineral nutrients N and P). These resources act as ''bottom-up'' regulators of the potential microalgal biomass that can be produced. Once harvested, the cellular lipids in this microalgal biomass can be extracted and processed to create biodiesel fuels. The lipid content of microalgal biomass is not constant, however, and can be influenced by many factors, including nitrogen:phosphorus supply ratios, light, CO2 and the hydraulic residence time of the bioreactor. Moreover, natural assemblages of microalgae are taxonomically diverse: some species are small and can easily be consumed by herbivorous zooplankton. Undesirable grazing losses of edible microalgae (and their cellular lipids) to large-bodied zooplankton can be reduced by adding zooplanktivorous fish, which can greatly restrict large-bodied zooplankton growth via sizeselective predation (''top-down'' regulation).

(Afify *et al*., 2010)

therefore depend on their operators' ability to reliably and consistently induce N-limitation in the resident algal cells. Resource-ratio theory and the principles of ecological stoichiometry, provide additional new insights into the control of algal biomass and lipid production in pond bioreactors. the nutrient limitation status of microalgae can be directly controlled by regulating the ratio of nitrogen and phosphorus (N:P) supplied in the incoming nutrient feed: nitrogen limitation occurs at N:P supply ratios that lie below the optimal N:P ratio for microalgal growth, whereas phosphorus limitation occurs at ratios that exceed this ratio. A transition between N- and P-limitation of phytoplankton growth typically occurs in the range of N:P supply ratios between ca. 20:1 to ca. 50:1 by moles . Such shifts between N- and P-limitation have extremely important implications for algal biofuel production because diverse species of microalgae grown under nitrogen-limited conditions (i.e. low N:P supply ratios) can exhibit as much as three times the lipid content of cells grown under conditions of phosphorus limitation (high N:P supply ratios) . Both the total phosphorus concentration as well as the total nitrogen concentration in the nutrient feeds to pond bioreactors should therefore impact algal biodiesel production, because the N:P ratio of incoming nutrients will strongly influence algal biomass production as well as the cellular lipid content. Given the inverse relationship observed between N:P and cellular lipids , and the positive, hyperbolic relationship observed between N:P and microalgal biomass , we conclude that optimal lipid yields (in terms of mass of lipid produced per unit bioreactor volume per day) should occur at intermediate values of the N:P supply ratio. From the strong apparent interactions between the effects of nitrogen and carbon dioxide availability on microalgal lipids, we also conclude that the effects of N:P supply ratios on volumetric lipid production might be even greater if the bioreactors are simultaneously provided with supplemental CO2 (cf. Figure 2).


Table 6. Comparison between lipid percentage (%) produced by eight algal species using two different extraction system.

Eight algal species (4 *Rhodo*, 1 *chloro* and 1 *phaeophycean* macroalgae, 1 *cyanobacterium* and 1 green microalga) were used for the production of biodiesel using two extraction solvent systems (Hexane/ether (1:1, v/v)) and (Chloroform/ methanol (2:1, v/v)) Table 6. Biochemical evaluations of algal species were carried out by estimating biomass, lipid,

therefore depend on their operators' ability to reliably and consistently induce N-limitation in the resident algal cells. Resource-ratio theory and the principles of ecological stoichiometry, provide additional new insights into the control of algal biomass and lipid production in pond bioreactors. the nutrient limitation status of microalgae can be directly controlled by regulating the ratio of nitrogen and phosphorus (N:P) supplied in the incoming nutrient feed: nitrogen limitation occurs at N:P supply ratios that lie below the optimal N:P ratio for microalgal growth, whereas phosphorus limitation occurs at ratios that exceed this ratio. A transition between N- and P-limitation of phytoplankton growth typically occurs in the range of N:P supply ratios between ca. 20:1 to ca. 50:1 by moles . Such shifts between N- and P-limitation have extremely important implications for algal biofuel production because diverse species of microalgae grown under nitrogen-limited conditions (i.e. low N:P supply ratios) can exhibit as much as three times the lipid content of cells grown under conditions of phosphorus limitation (high N:P supply ratios) . Both the total phosphorus concentration as well as the total nitrogen concentration in the nutrient feeds to pond bioreactors should therefore impact algal biodiesel production, because the N:P ratio of incoming nutrients will strongly influence algal biomass production as well as the cellular lipid content. Given the inverse relationship observed between N:P and cellular lipids , and the positive, hyperbolic relationship observed between N:P and microalgal biomass , we conclude that optimal lipid yields (in terms of mass of lipid produced per unit bioreactor volume per day) should occur at intermediate values of the N:P supply ratio. From the strong apparent interactions between the effects of nitrogen and carbon dioxide availability on microalgal lipids, we also conclude that the effects of N:P supply ratios on volumetric lipid production might be even greater if the bioreactors are simultaneously

Table 6. Comparison between lipid percentage (%) produced by eight algal species using

Eight algal species (4 *Rhodo*, 1 *chloro* and 1 *phaeophycean* macroalgae, 1 *cyanobacterium* and 1 green microalga) were used for the production of biodiesel using two extraction solvent systems (Hexane/ether (1:1, v/v)) and (Chloroform/ methanol (2:1, v/v)) Table 6. Biochemical evaluations of algal species were carried out by estimating biomass, lipid,

provided with supplemental CO2 (cf. Figure 2).

two different extraction system.

biodiesel and sediment (glycerin and pigments) percentages. Hexane/ ether (1:1, v/v) extraction solvent system resulted in low lipid recoveries (2.3-3.5% dry weight) while; chloroform/methanol (2: 1, v/v) extraction solvent system was proved to be more efficient for lipid and biodiesel extraction (2.5 – 12.5% dry weight) depending on algae species (Table 7). The green microalga *Dictyochloropsis splendida* extract produced the highest lipid and biodiesel yield (12.5 and 8.75% respectively) followed by the cyanobacterium *Spirulina maxima* (9.2 and 7.5 % respectively). On the other hand, the macroalga (red, brown and green) produced the lowest biodieselyield. The fatty acids of *Dictyochloropsis splendida*  Geitler biodiesel were determined using gas liquid chromatography. Lipids, biodiesel and glycerol production of *Dictyochloropsis splendida* Geitler (the promising alga) were markedly enhanced by either increasing salt concentration or by nitrogen deficiency (Table 8) with maximum production of (26.8, 18.9 and 7.9 % respectively) at nitrogen starvation condition. (Afify *et al*., 2010)


Table 7. Total lipid, biodiesel, sediment percentage and biodiesel color of eight algal species

Natural biotic communities in outdoor bioreactors require the external provision of potentially growth-limiting resources (e.g. light, carbon dioxide and the essential mineral nutrients N and P). These resources act as ''bottom-up'' regulators of the potential microalgal biomass that can be produced. Once harvested, the cellular lipids in this microalgal biomass can be extracted and processed to create biodiesel fuels. The lipid content of microalgal biomass is not constant, however, and can be influenced by many factors, including nitrogen:phosphorus supply ratios, light, CO2 and the hydraulic residence time of the bioreactor. Moreover, natural assemblages of microalgae are taxonomically diverse: some species are small and can easily be consumed by herbivorous zooplankton. Undesirable grazing losses of edible microalgae (and their cellular lipids) to large-bodied zooplankton can be reduced by adding zooplanktivorous fish, which can greatly restrict large-bodied zooplankton growth via sizeselective predation (''top-down'' regulation).

Algal Biomass and Biodiesel Production 127

Governorate, Egypt was used after filtered using glass microfiber filter to remove large particles and indigenous bacteria for the experiment and the chemical and physical parameters were analysis as reported by APHA (1998) Table (2). The supplementation of NaNO3, K2HPO4 and FeSO4.7H2O in amounts equal to those of the standard BG11, Bold and Zarrouk were used as basal media. The algal strains were grown in 500 ml Erlenmeyer flasks containing 200 ml of 100% effluent supplemented with basal nutrients and 100% effluent without basal nutrients with/without sterilization and the synthetic media (BG11, Bold and Zarrouk) were used as control. Two per cent algal inoculums were added to each flask. The experiment was conducted in triplicates and cultures were incubated at 25 ºC ±1ºC, under continuous shaking (150 rpm) and illumination (2000 lux) for 15 days. This work aimed to evaluate the laboratory cultivation of nine algal strains belonging to Nostocales and Chlorellales in secondary treated municipal domestic wastewater for

**Color pH Glycerin +** 

7.5±0.50 Yellowish

**pigments**

biomass and biodiesel production as shown in Table (9 and 10).

**content Algal species Total lipids**

*muscorum* 

*aquae*

*vulgaris*

*platensis*

*oryzae* 

*humifusum* 

according to Duncan's multiple range tests.

sterilization T4: waste water+ nutrients without sterilization

from different microalgae species cultivated in different waste water

**Biodiesel** 

16.80±3.62 12.52±1.74 4.28±1.74 Brown 7.4±0.33 *Nostoc* 

5.50±0.58 4.00±0.41 1.50±0.41 Red 6.9±0.95 *Anabaena flous* 

12.50±1.20 8.8±0.16 3.70±0.16 Green 8.1±1.0 *Chlorella* 

*Oscillatoria sp* 8.00±0.58 4.30±0.32 3.70±0.32 Yellow 7.5±0.85

10.0±0.11 7.80±0.17 2.20±0.17 Light green 8.0±0.32 *Spirulina* 

7.40±0.90 4.50±0.10 2.90±0.10 Orange 7.3±0.96 *Anabaena* 

*Wollea sp* 6.30±1.31 3.90±0.60 2.40±0.60 Yellow 7.8±0.35

*Phormedium sp* 12.20±1.66 10.10±1.50 2.10±1.50 Dark brown 7.1±0.0

Each value is presented as mean of triplet treatments, LSD: Least different significantly at P ≤ 0.05

T1: waste water without treatment; T2: waste water after sterilization; T3: waste water+ nutrients with

Table 9. Total lipids, biodiesel, glycerine+pigments percentage and color, pH of biodiesel

LSD 0.159 0.151 0.151 1.659

brown 14.80±2.40 10.20±1.30 4.6±1.30 *Nostoc* 


Table 8. Total lipid, biodiesel, sediment percentage and biodiesel color of Dictyochloropsis sp cultivated under stress
