Disposal of wastewaters from human activities and animal production is both an environmental and a financial issue. These nitrogen and phospho­rus rich wastewaters have been proven suitable for microalgae growth. The feasibility of growing microalgae to remove inorganic nitrogen, phosphorus, metal elements and other pollutants in the wastewaters has long been recognized [56,57]. Therefore, a wastewater based microal­gae production process has the due benefits of wastewater treatment and production of algal biomass with minimum external input of fresh water and nutrients. It has been argued that microalgae biodiesel production in conjunction with wastewater treatment is the area with the most plausible commercial potential in the short term [58,59]. Several applications in wastewater treatment have been reported in the literature. Chinnasamy et al. [60] found that both fresh water and marine algae showed good growth in carpet industry effluents and municipal sewage. Kim et al. [61] added fermented swine urine (3%) (v/v) to the medium to culture Scenedesmus sp, and received the growth rate (3-fold), dry weight (2.6-fold). Wang et al. [62] investigated the effectiveness of using digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp, and found that the total fatty acid content of dry weight was increased from 9.00% to 13.7%, along with removal of ammonia, total nitrogen, total phosphorus, and COD by 100%, 75.7%-82.5%, 62.5%-74.7%, and 27.4%-38.4%, respectively. Li et al. [63] cultivated Scenedesmus sp. LX1 in secondary effluent, achieving high biomass yield (0.11 g L-1, dry weight) and lipid content (31%-33%, dry weight).

Because biomass can provide energy supply in a multitude of different energy carrier types, often in the same conversion route, the biomass use was channelled through a multitude of bioenergy routes, taking into con­sideration residues resulting from some of these routes. This approach is illustrated in a simplified diagram in Fig. 2.

In order to keep the projection robust, the key principle in selecting the supply and technology options was to only use options that are cur­rently available or for which only incremental technological development is needed. One exception, where technological change of a more radical character is needed, is the inclusion of oil from algae as a supply option. To allow for development still needed in algae growing and harvesting, we included the use of significant amounts of algae oil from 2030 onwards only.

Another important assumption is made on the traditional use of bio­mass. Currently, about 35 EJ of primary biomass is used in traditional applications. This consists primarily of woody biomass and agricultural

l IX
ALGAE
Transport: Aviation
Oll/FAT TO FUEL
OIL ♦ FAT CROPS
Transport: Shipping
SUGAR♦ STARCH CROPS
{HGNO)CELLUlO$IC CROPS
Transport: Road & Rail
FERMENTATION
Electricity
TRADITIONAL BIOMASS
COMBUSTION
COMPLEMENTARY FELLINGS
Building h«at
DIGESTION ♦ GRID UPGRADE
AGRICULTURAL RESIDUES	Industry Ga>
FOREST RESIDUES ANO WOOO WASTE
Industry: Wood tor paper
DIGESTION ♦ COMBUSTION
WET WASTES
Industry: Oeircnt fuel
OIL ANO FAT RESIDUES ANO WASTE
CHARCOAL PRODUCTION
Industry Steel fuel
DRY WASTES
FIGURE 2: Bioenergy approach. Shown are the different biomass input types and conversion routes which result in various energy carrier types that meet demand.

residues harvested for home heating and cooking in developing countries. Towards 2050, the Ecofys Energy Scenario will supply energy for these demands through a route alternative to traditional biomass use. The tradi­tional use of biomass is therefore phased out over time. A proportion of this phased out biomass is used within this work in a sustainable manner in other bioenergy routes, see also Section 3.3.2.

Within this study, the different bioenergy routes displayed in Fig. 2 are prioritised according to their feedstock as follows:

1. Traditional biomass: As this biomass is currently in use, it is used first in the Ecofys Energy Scenario to reflect the current situation. Over time, the contribution of this category becomes less as it is completely phased out towards 2050.

2. Sustainable residues and waste: Sustainable residues and waste, originating from agriculture, forestry and the food processing in­dustry for example, are used to meet as much demand as possible.

3. Sustainable complementary fellings: This category consists of woody biomass gained from sustainable harvesting of additional forest growth and of the sustainable share of the biomass currently used in traditional uses. It is used to fill remaining demand in lig — nocellulosic routes, as much as possible.

4. Sustainable energy crops: Energy crops are used to fill as much of the remaining energy demand as possible while staying within their sustainable potential. Energy crops include oil crops, starch and sugar crops and (ligno) cellulosic crops.

5. Sustainable algae: Algae are used to yield oil to fill the remaining demand in the oil routes. Algae are used last because their growing and harvesting is currently not a proven technology on a commer­cial scale. However, although they are prioritised below sustain­able energy crops, their use can be necessary before the entire po­tential for cropland for energy cropping is used. This is caused by the fact that we based our land availability on suitability for grow­ing different crop types and only a share of the cropland potential is suitable for growing oil crops. Section 3.1 provides more detail on this methodology.

We assumed conversion efficiencies for each route as displayed in Table 1.

Based on the methane productivity presented above, 15.9 kg of methane would be produced per L of bio-oil in the Experimental Case, which yields

16.6 EJ/yr of methane energy produced for 19 GL/yr of bio-oil (5 Bgal/ yr). This methane yield would displace 60% of the total U. S. natural gas consumption (~28.1 EJ/yr in 2009 [39]) (although this result is not a re­alistic expectation, as the EROI for this scenario is several orders of magnitude less than 1). In the Highly Productive Case a smaller portion of the biomass is used to produce methane (70% rather than ~95%-99% in the Experimental Case) because of the much higher lipid fraction. As a result, only 0.6 kg of methane would be co-produced for each L of bio-oil, yielding 0.60 EJ/yr of methane co-product for 19 GL/yr of algal bio-oil (5 Bgal/yr). This methane production could replace ~2.2% of the total U. S. natural gas consumption [39].

5.4 CONCLUSIONS

JESSICA JONES, CHENG-HAN LEE, JAMES WANG, and MARTIN POENIE

8.1 INTRODUCTION

Some strains of algae show promise as a sustainable source of biofuel due to their rapid growth, ability to grow on non-arable land, and high triacyl — glycerol (TAG) content. TAGs can be easily converted to biodiesel, which is compatible with current fuel infrastructure [1]. Biodiesel has a higher energy density than ethanol, yet it is relatively non-toxic, biodegradable, and produces lower exhaust emissions than petroleum-based fuels, mak­ing it is one of the most attractive forms of alternative energy [2,3]. Ad­ditionally, when derived from plant sources such as algae, biodiesel has the potential of a near-neutral carbon footprint [4], though considerable work must be done in order to realize this goal in an economically and environmentally-feasible manner [5].

While algae are promising, there are technical challenges that currently make it prohibitively expensive as a source of fuel. Algae grow at dilute concentrations, generally less than 1 g/L [6], so it must be concentrated before it can be processed. Most concentration processes require pumping the dilute algal suspension and may involve other energy intensive steps such as centrifugation [7], compressors for dissolved air flotation, or else

treatment of large volumes of water with chemicals (flocculation). Cen­trifugation, for example, could account for 30% of processing costs [8].

Once algae have been harvested, there are additional processing steps that often include lysis and drying of the algae followed by extraction with organic solvents to obtain the neutral lipids, primarily TAGs, that can then be converted to biodiesel [3]. Drying can be expensive but it facilitates the interaction between solvent and algae to improve extraction efficiency. However, the extraction solvent must be removed and recovered prior to conversion of lipids to biodiesel and there are attending questions about pollution of the air and contamination of the biomass with solvents. This is also considered a large part of the cost in processing algae [5].

Once the lipids have been isolated, they can be converted to biodiesel by either acid — or base-catalyzed transesterification, typically with metha­nol, to yield fatty acid methyl esters (FAMEs) and glycerol [9]. The trans­esterification reaction is sensitive to water [10-14] but this is not normally a problem since oils, such as would be obtained by hexane extraction, nor­mally contain little water. When the entire processes is analyzed for cost and energy expenditure, some current projections suggest that the energy spent in the cultivation, harvest, and extraction of oil from algae could be greater than that gained from the product [5] along with the possibility of significant environmental impact [15]. It is generally agreed that for algae to be economically feasible, improvements in technology will be needed.

Efforts to reduce cost and simplify processing of algal biomass to bio­fuel have led to studies of direct conversion of dry or even wet algal bio­mass to biodiesel. Previous studies have shown that algal lipids can be transesterified in situ by adding reagents to a dried sample of algae [11,16­19]. One study examined a two-step procedure where the acyl groups of component lipids were hydrolyzed with base and then re-esterified in ex­cess sulfuric acid/methanol [20]. This procedure gave greater amounts FAME than obtained by lipid extraction followed by transesterification. Another study showed that direct acid-catalyzed transesterification of wet biomass can produce FAME yields similar to that of dried biomass, al­though FAME compositions differed [11].

In this study we show that anion exchange resins such as Amberlite can concentrate and dewater algae (i. e., harvest algae) and then be eluted with 5% sulfuric acid/methanol reagent. The eluted algae appear to dissolve in the sulfuric acid reagent and esterified fatty acids are converted to FA­MEs (biodiesel). This one step harvesting and transesterification process can potentially eliminate many of the costly steps of processing algae to biofuel.

Probably the most effective method of biomass removal, with very high recovery rates, is centrifugation. As with the other alternative methods, centrifugation was considered a feasible option in early algal biomass de­watering work in the 1960s. Golueke and Oswald [4] investigated various means of dewatering algae further to provide a biomass with a sufficiently low moisture content. One of the methods they looked at was centrifuga­tion and three of the four centrifuges that they tested proved to be extreme­ly effective producing a maximum removal of 79% and a biomass with solids content of 11.5% and maximum of 18.2%. Further research was conducted by Mohn [63] in the area of harvesting algal biomass using cen­trifugation and he focussed on suitability of algal strains, cost and energy use. In accordance with Golueke and Oswald, Mohn found centrifuges to be very effective for the removal of Scenedesmus and Coelastrum, partic­ularly the Westfalia self-cleaning plate separator and the Westfalia nozzle centrifuge [63]. The centrifuges provided biomass with total solids con­tent of 2-22% with a minimum energy consumption of 0.9 kWh per m3. Table 5 provides an overview of Mohn’s findings indicating the possible harvesting methods, effectiveness, energy requirements and reliability of several harvesting methods. Mohn’s results suggest filtration provides the best harvesting strategy in terms of high concentration of solids with low energy requirements [63].

Despite centrifugation being an effective method of concentrating biomass, the energy requirements are much higher than that of filtration. However clearly the choice of harvesting depends heavily upon the bio­mass type, if the cell size is large enough, then filtration is likely to be the most effective and economically viable option. Otherwise it is likely that a process stream involving flocculation, sedimentation, flotation or cen­trifugation is necessary. There is little parallel between the effectiveness of common flocculants for harvesting algae in research conducted. It can be observed that there are many effective flocculants for algae removal however suggested optimal dosages vary significantly between studies. Ferric chloride can be considered a viable option potentially combined with chitosan to improve yield and reduce time and material input. Further research is necessary for individual scenarios to choose the most effective method of flocculation and consequent harvesting.

Sections 3.1 through 3.5 describe how we obtained the results for sustain­able bioenergy availability. We summarise our results on availability here and compare them to the use found in the demand side scenario [1].

Our sustainable biomass availability only includes bioenergy supply that meets the sustainability criteria in Section 2.2. Fig. 5 shows that the demand side scenario [1] is capable of meeting demand with bioenergy using the available sustainable biomass and rain-fed land for energy crop­ping identified in this study.

3.3.2 GREENHOUSE GAS EMISSION SAVINGS

We have performed a life cycle analysis of the greenhouse gas (GHG) emissions associated with bioenergy use in the Ecofys Energy Scenario. We have included GHG emissions from six different contributors in the bioenergy life cycle as displayed in Table 5.

TABLE 5: Types of greenhouse gas emissions included in bioenergy life cycle analysis in the Ecofys Energy Scenario.

Type of emissions

Emissions from land use change when land is converted to bioenergy [31] cropland.

Emissions from the production and application of nitrogen fertiliser for [30], [31] and [32] bioenergy crops and algae.

Emissions from agricultural fuel inputs for cultivation of bioenergy crops. [33] Emissions from transport of biomass to the processing site. [34]

Emissions from energy inputs during bioenergy conversion. [35]

Emissions from transport of the bioenergy carrier to the end use location. [34]

FIGURE 5: Bioenergy potential versus use. Overview of the Ecofys Energy Scenario’s sustainable bioenergy use and rain-fed land use for energy cropping versus sustainable potential found in this study.

Most of these contributors include emissions associated with energy use. As the Ecofys Energy Scenario drastically increases the share of re­newable energy technologies that have low or no GHG emissions, we have made two separate calculations: in one the emission factors for the energy inputs were extracted from the IPCC fossil fuel references [36] and in the other, they were taken from Ecofys Energy Scenario data. Therefore, we present results as a range.

Fig. 6 shows the results of the life cycle analysis. For 2050, we have calculated that the GHG emissions in CO2 equivalent associated with bio­energy are 12-18 g MJ-1 final energy use. The values for the correspond­ing fossil references in CO2 equivalent are 70-80 g MJ-1. This means that even for the most conservative calculation (with fossil reference for energy inputs) average GHG emission savings are ~75%. When the cor­responding Ecofys Energy Scenario values are used, average GHG emis­sion savings are ~85%. This level of emission saving is consistent with an overall emission reduction from the energy system which would result in global average temperature increase of less than 2 °C above pre-industrial levels [1], [37] and [38].

3.4 DISCUSSION

In our work we have shown that the Ecofys Energy Scenario is capable of meeting demand with bioenergy within the sustainable potential we identi­fied in this study. In this section we discuss this result in two ways: first, we compare our results on bioenergy use and the associated land use to energy and land potentials found in other studies. Then we show the sen­sitivity of our results to developments in the agricultural and food sectors such as diet and yield changes.

Stimulation in the growth of immobilized E. coli cells by 140% over con­trol, was reported by Chang et al. [10], which was attributed to the en­hanced removal of inhibitory products from the gel through electro-osmo­sis and electrophoresis as well as an augmented glucose supply. Kerns et al. [19] reported growth stimulation in Trichoderma reesei by using pulsed EMF’s for electroporation via inductively coupled electric currents from a Helmholtz coil. The use of electric fields has also been investigated with yeasts in either a static mode or an oscillating/pulsed mode. The survival rate of Saccharomyces cerevisiae was investigated under bipolar electric field pulses from 0-1.5 kV/cm by measuring plating efficiency. The maxi­mum growth after plating appeared at 0.85 kV/cm which demonstrated a 100% increase over the control [55]. An electrostimulation in S. cerevisiae from electric field application at 10 mA DC and 100 mA AC resulted in an increase in growth rate by 60% in AC mode and 50% in DC along with an increase in the production of the acetic acid, lactic acid and acetaldehyde. The results suggest that the acceleration of growth rate from a DC expo­sure stimulated cell budding during the early stages of cultivation, which could be due to a 60% decrease in inhibitory concentration of dissolved

CO2 and other chemical modifications of the culture medium [38]. Zrimec et al. [12] have shown that external AC electric fields of low intensity stimulated membrane bound ATP synthesis in starving E. coli cells with electric field amplitudes of 2.5-50 V/cm and a frequency optimum at 100 Hz. The model of electro conformational coupling was used to analyze the frequency and amplitude responses of ATP synthesis. Two relaxation frequencies of the system were obtained at 44 and 220 Hz, and an estimate of roughly 12 elementary charges was obtained as the effective charge displacement for the catalytic cycle of ATP synthesis.

An actinomycetous eubacterium Streptomyces noursei used for antibi­otic production was electrostimulated via PEMF’s using a pair of Helm­holtz coils via inductive-coupling producing 5 ms bursts of 220 ps dura­tion in intervals of 60 ms by Grosse [20]. The process resulted in a mean inductive electric field strength of approximately 1.5 mV cm-1. An increase was observed in the formation of the product but only during the first 50 hours of the starting phase although the exposed culture exhibited an over­all increase in O2 consumption and glucose utilization.

Electric field stimulation may also be used to improve the substrate uti­lization efficiency in microbial processes. Cells when subjected to electric field pulses of 0.25 kV for 10 ms in the presence of the enzyme cellobiose showed enhanced utilization of cellobiose and conversion of substrate into ethanol by a thermotolerant yeast, Kluyveromyces marxianus. As a result, ethanol yield increased by nearly 40% over the control [43].

Kerns et al. [19] showed that pulsed EMF’s at 1.5 mVcm-1 bursts for 115 hours used for electroporation lead to ~60% increase in cellulase activ­ity and ~80% increase in cellulase secretion in Trichoderma reesei. They concluded that the effect occurred inside the cells on either the formation of the cellulase enzyme complex at the genetic level or the secretion into the medium via altered membrane permeability. A 62% increase in biosorption of uranium was observed using pulsed electric fields of 1.25 to 3.25 kV cm-1, suggesting that the application of short and intense pulses might enhance the biosorption of toxic metals and radionuclides from wastewater streams [24].

For algae binding and FAME conversion studies, 10 mL polypropylene columns were loaded with 2 g of Amberlite resin and then washed with 1M hydrochloric acid followed by distilled water. To load the resin, 200 mL algal suspension containing 80 mg algae (a slight excess; based on the OD680 of the algae suspension) was passed through the resin. The OD680 of the flow through was then measured to determine the DCW of algae bound to the resin.

For transesterification and elution of algae from the resin, excess water was removed from column by vacuum, followed by the addition of 100 mL of transesterification reagent. For elution of algae and transesterifi­cation, reagents tested included 5% w/v sulfuric acid in either methanol or ethanol, 5% w/v potassium hydroxide in methanol, or 0.1 M sodium methoxide in methanol. The eluate was collected and the transesterifica­tion reaction continued at room temperature for 12 h. FAME was extracted sequentially with two 20 mL portions of hexane. The combined hexane extracts were then dried by rotary evaporation and resuspended in 1 mL hexaneiisopropanol (3:1, v/v) for HPLC analysis. For comparison, FAME was generated directly from dried algal biomass, based on the method described by O’Fallon et al. [26]. Algal pellets containing approximately 30 mg algae in preweighed tubes were dried using a SpeedVac concentra­tor (Savant) for 12 h at 10-1 Torr with heat. Final tube weight was then measured to obtain an accurate algal DCW. The dried algal pellet was suspended in methanol and transferred to a glass centrifuge tube where methanol was added to a final volume of 5.3 mL. Lipids were then saponi­fied by adding 0.7 mL of 10 N potassium hydroxide and heating at 55 °C for 1.5 h. Once the samples cooled, lipids were re-esterified by adding 0.6 mL of concentrated sulfuric acid to the same methanol suspension and heating again at 55 °C for 1.5 h. After cooling, FAME was extracted with 2 mL of hexane using a centrifuge to force the separation of the mixture into two layers. The upper hexane layer was then transferred to a vial for HPLC analysis.

It is easy to increase the chemical composition of microalgae by changing the environmental conditions and other factors. Many studies indicate that the composition, including lipid content, of microalgae is closely related to the environmental conditions and medium composition. Improving the lipid content in microalgae is a focus of commercial production of mi­croalgae biomass. Current studies on high lipid content of microalgae are focused mainly on selection of microalgae species, genetic modification of microalgae, nutrient management, metabolic pathways, cultivation con­ditions, and so on.

To be considered sustainable, as a fuel source, it is essential that the over­all process provide a positive energy balance with minimal environmental and social impacts whilst maintaining economic viability. Improving the energy balance is likely to improve the other areas. For example, reducing energy consumption requires less electricity generation which in turn will reduce environmental impacts whilst lessening production cost. If optimal process configurations can be designed for the production of algal bio­fuels maximising energy yield whilst minimising consumption the con­cept, could in the future, become a method of producing a sustainable fuel. As a result, many current studies are focussing upon grand scale systems centred around large power plants for CO2 with potential utilisation of wastewater if available for nutrient provision/water treatment. Although research is heading in the right direction by reducing energy consumption through combining wastewater treatment for nutrient provision and car­bon abatement with the use of flue gases, perhaps the future lies in more flexible, localised solutions which are adaptable to unique conditions.