Wang et al. [30] used a magnetic field concentrated to a small area and observed that it helped to regulate the anti-oxidant defense system of Chlorella vulgaris at a threshold magnetic flux intensity of 10-35 mT. The authors proposed that this is probably due to the free radicals altered by the magnetic field, which accelerated the relative biologi­cal reactions. The analysis of hydroxyl radical (-OH) showed that it increased simultaneously with increasing magnetic flux density sug­gesting an oxidative stress induced by the exposure compared to the control.

6.3.1.3.2 BIODEGRADATION

A study using airlift reactors showed that the influence of magnetic fields enhanced the degradation of phenolic waste liquors by submersed micro­organisms at a magnetic field intensity of 22 mT [23].

Neochloris oleoabundans (UTEX LB 1185) was obtained from the Uni­versity of Texas at Austin Algal Culture Collection and KAS 603, a saltwater species of Chlorella, was provided by Kuehnle Agro Systems. Neochloris was cultivated in freshwater Bold 3N (B3N) medium [30] us­ing an airlift bioreactor illuminated with cool white fluorescent lights on a 12:12 light:dark cycle and aerated with ambient air using an oil-free diaphragm pump. KAS 603 was cultivated in the same way using f/2 saltwater medium [31]. For nitrogen starvation experiments, Neochloris was grown for 14 days in B3N media, harvested by centrifugation, resuspended in sodium nitrate-free B3N, and cultured for an additional 21 days. For resin binding and FAME synthesis, algae were harvested by centrifugation and resuspended in distilled water to concentration of 0.4 g/L.

Microalgae may utilize one or more of the three major metabolic pathways depending on light and carbon conditions: photoautotrophy, heterotrophy, and mixotrophy [6]. Most microalgae are capable of photoautotrophic growth. Photoautotrophic cultivation in open ponds is a simple and low — cost way for large-scale production; however the biomass density is low because of limited light transmission, contamination by other species or bacteria, and low organic carbon concentration [7]. Some microalgae can make use of organic carbons and O2 to undergo rapid propagation through heterotrophic pathway. Heterotrophic cultivation has drawn increasing attention and it is regarded as the most practical and promising way to increase the productivity [8-10]. Currently, research on heterotrophic cul­tivation of microalgae is mainly focused on Chlorella. Cell densities as high as 104.9 gL-1 (dry cell weight, Chlorella pyrenoidosa) have been

reported [11]. Microalgae can adapt to different organic matters such as sucrose, glycerol, xylan, organic acids in slurry after acclimatization [12]. The ability of heterotrophic microalgae to utlize a wide variety of organic carbons provides an opportunity to reduce the overall cost of microalgae biodiesel production since these organic substrates can be found in the waste streams such as animal and municipal wastewaters, effluents from anaerobic digestion, food processing wastes, etc. On the basis of hetero­trophic cultivation, researchers have carried out studies of mixotrophic cultivation which can greatly enhance the growth rate because it realizes the combined effects of photosynthesis and heterotrophy. After examin­ing the biomass and lipid productivities characteristics of 14 microalgae, Park et al. [13] found that biomass and lipid productivities were boosted by mixotrophic cultivation. Andrade et al. [14] studied the effects of mo­lasses concentration and light levels on mixotrophic growth of Spirulina platensis, and found the biomass production was stimulated by molasses, which suggested that this industrial by-product could be used as a low-cost supplement for the growth of this species. Bhatnagar et al. [15] found the mixotrophic growth of Chlamydomonas globosa, Chlorella minutissima and Scenedesmus bijuga resulted in 3-10 times more biomass production compared to that obtained under phototrophic growth conditions. The max­imum lipid productivities of Phaeodactylum tricornutum in mixotrophic cultures with glucose, starch and acetate in medium were 0.053, 0.023 and

0. 020 gL-1day-1, which were respectively 4.6-, 2.0-, and 1.7-fold of those obtained in the corresponding photoautotrophic control cultures [16].

Increased concentrations of carbon dioxide (above atmospheric concen­tration) have been proven to improve the productivity [45-47] of algal cultivation. Production of synthetic CO2 however is too energy-intensive to generate and a source of waste carbon dioxide is required. Many studies have proven the advantages of using CO2 injection combined with algal cultivation [45, 47, 50, 51, 99]. As producing CO2 synthetically is not sus­tainable, it is necessary for an existing source of CO2 to be situated near to the algae growth ponds. Researchers have considered the plant flue gas from coal-fired power stations as an ideal source of CO2 [48, 50] and flue gases have been shown to be successful as a source of CO2. Nevertheless barriers would need to be overcome to implement the concept in a scaled — up system. It is evident from literature that CO2 concentrations that are too high (above 15%) will cause a decrease in biomass productivity and potentially death of the cells. This may limit the number of possibilities for use of flue gas, although it must be noted that generally flue gases contain CO2 concentrations lower than this [5]. It is not only the CO2 that could be lethal to the cells: other toxins may also negatively impact the biomass. SO2 can have a great impact upon the biomass and the pH of the water and high SO2 concentration cause the pH to drop to very low levels. pH can be adjusted using NaOH but this requires additional materials and energy. In addition, the temperature of flue gas is generally above that of normal culture conditions and is likely to be too high to allow biomass growth. Cooling would be necessary to reduce the temperature to an acceptable level thus requiring water and additional energy for pumping.

Clearly there are many issues related to the use of waste flue gas as a source of CO2 that must be addressed to allow implementation on a larger scale. It may be the case that transporting and treating flue gas prior to in­jection would require too much energy compared to the benefit that could be gained.

The Experimental Case is comprised of five batches, ranging in vol­ume from 970 L to 2000 L each. A marine species of Chlorella (KAS 603, provided by Kuehnle AgroSystems, Inc.) was used for all batches and was grown in four different growth stages: flasks, airlift photobioreactors, greenhouse tanks, and covered raceway ponds.

This growth process provided a stable method for scaling up cultiva­tion volumes, although, the inherent inefficiencies of operating at lab — scale required high energy and material inputs (an artifact described in detail by Beal et al. [14]) and yielded relatively low biomass and lipid productivities, as listed below. Energy and material consumption were measured throughout the entire cultivation process and these data have been reported previously [14]. The amounts of resources consumed in the smaller growth volumes (e. g., energy required for bioreactor lighting) were allocated to the larger growth volumes as the algae were transferred through the system during scale-up (cf. Appendix 4A of [19] for details).

— — — — — Highly Productive Case EROI Highly Productive Case QA EROI and FROI

• — • — Experimental Case EROI

——————- Experimental Case QA EROI and FROI *

The algal biomass was tracked during each batch by measuring the dry cell weight of multiple samples collected throughout the production pathway. These samples were centrifuged and the pellet was rinsed three times to remove salts. Then, the samples were maintained at 70 °C until a constant weight was obtained. High performance liquid chromatography (HPLC) was used to calculate the lipid content and lipid composition for each batch according to methods developed at The University of Texas at Austin [21], which are refinements of standard methods [22-24].

All five of the experimental batches were processed using a centrifuge for harvesting, electromechanical cell lysing, and a microporous hollow — fiber membrane contactor for separations. While the energy and materials consumed during each of these steps, and the associated uncertainty, has been described in detail by Beal et al. [14,19], this study, combines these data with monetary costs, water impacts, and resource constraints for each input.

The application of the nanosized voltmeter, used to measure the electric fields throughout the interior of cellular structures, has indicated that the theoretical calculation of electric field penetration into a cell’s cytosol

arising from the membrane and mitochondrial potential do not match the empirically measured values. It is proposed that this may be due to the traditional model using saline solution to simulate the physical proper­ties of the cytoplasm, where alternatively the cytoplasmic structure has been described as having a complex gel-like composition [86,87]. One such possibility for a heterogeneous substance with distinct microdomains is liquid crystal. Liquid crystals are phases of matter that are exhibited by anisotropic organic materials as they undergo cascades of transitions between solid and the liquid states [88]. These mesophases possess sym­metry and mechanical properties of long-range orientational order inter­mediate between those of liquids and of solid crystals. Liquid crystals can undergo rapid changes in orientation of phase transition upon electric or magnetic exposure, or changes in temperature, pH, pressure, hydration, and concentrations of inorganic ions. These properties are ideal for organ­isms, and it has been found that lipids of membranes, DNA in chromo­somes, all proteins, especially cytoskeletal proteins are liquid crystalline in nature [89]. Ho’s group observed that electrodynamic activities might be acting on endogenous non-equilibrium electrodynamic processes in­volved in phase ordering and patterning domains of liquid crystals [65]. Their findings support that organisms are polyphasic liquid crystals where different mesophases may have important implications for biological or­ganization and function [90].

1. Cell Membrane

• Magnetic field oscillations may increase membrane permeability under ion cyclotron resonance

• Increased circulation and selective enhancement of ion flow may affect the rate of biochemical reactions

• Alter the rate of binding of calcium ions to enzymes or receptor sites

• Change distribution of protein and lipid domains, and conformational changes in lipid-protein associations

• Change internal molecular distribution of electronic charge inside lipid molecule in the membrane bilayer

• May play the primary role in the stochastic resonance amplification process

2. Chloroplast

• May modulate the quantity of pigments, such as chlorophyll, phycocyanin, and beta-carotene

3. Nucleus/DNA

• Magnetic field affects specific gene expression

• Individual DNA sequences may function as antennae

• Leads to changes in DNA conformation

• May activate different DNA sequences depending on field intensity

• Can affect enzyme activity

4. Proteins:

• Breathing motions are the source and receiver of multipole EMF

• Potential coupling mechanism for external multipolar influences

5. Protoplasm

• Static magnetic fields influence the speed of protoplasm movement, miotic activity, and quantity of organic acids in plants

6. Whole Cell

• Biophotonic emission and interaction with nearby cells

• Endogenous electric field modulation may alter natural processes

Lipid determination in qualitative and quantitative analysis is crucial for identification of suitable strains for biodiesel production. Conventional methods such as solvent extraction or gravimetric methods have been used by Bligh and Dyer [30]. Separation and profiling of lipid components re­quire elaborate techniques in order to satisfy criteria of biodiesel qual­ity and includes thin layer chromatography (TLC), gas chromatography — mass spectroscopy (GC/MS) and/or high pressure liquid chromatography (HPLC) [31]. These methods are time-consuming for lipid extraction and analysis, especially for a large number of samples. Thus, a rapid screening for lipid content in organisms or cells is necessary and important for high — throughput screening. Nile red (9-diethylamino-5-benzo[a] phenoxazi — none), a lipophilic stain, maybe used for this purpose. It was first synthe­sized by Thorpe in 1907 by boiling Nile blue with sulfuric acid, and in the same year, Smith reported the use of Nile red for detecting lipids in human cells [32]. The application of Nile red for lipid staining in microorganisms such as bacteria, yeasts and microalgae is now a common practice that allows a rapid qualitative determination of lipids in cells (Figure 1) [33].

Although Nile red can be applied for rapid lipid screening, this meth­od has not been successful in some particular microalgae species due to variables such as staining time, temperature, rigid cell walls, etc. [34]. Thus, Nile red dye concentrations applied for lipid staining are different for particular microalgae species. To improve staining efficiency some factors can be considered. For instance, microwaves applied for staining were first introduced by Leong and Milios and then improved by Chiu et al. in 1987 [34-36]. Microwave exposure time was optimized for pro­cesses of pretreatment and staining. Results of this research showed that microwave-assisted staining increased remarkably fluorescence intensity

using a spectrofluorometer from 476 to 820 arbitrary units (a. u.) for Pseu- dochlorococcum sp. and from 662 to 869 a. u. for Chlorella zofingiensis after 50 s of microwave exposure in a pretreatment process and after 60 s of staining. Dimethyl sulfoxide (DMSO) has also been used for enhancing lipid staining effectiveness [33,34,37] and maybe used in low quantities instead of acetone as a solvent to allow viability of cells after staining. This means that Nile red staining can not only be used as a preliminary quantitative fluorometric assay for relative comparisons among closely — related strains, but potentially also for mutant screening and selection of high lipid-yielding strains.

Continuous production systems are a critical element in large scale com­mercial production of algal biomass. While we should continue to improve open pond operations, significant efforts should be invested in develop­ment of photobioreactors for high density cultivation of microalgae. Pho­tobioreactors with a real time smart on-line monitoring system which can maintain optimal conditions for the growth of microalgae is very promis­ing to realize high growth rate and cell density [55]. In order to address the issues with high capital and operational costs, innovative photobioreactor designs must be developed. Such designs may incorporate cost effective lighting techniques and renewable power combining solar energy, biogas, wind energy, waste heat.

STIJN CORNELISSEN, MICHELE KOPER, and YVONNE Y. DENG

3.1 INTRODUCTION

To reduce human dependence on fossil fuels and to reduce climate change, we need to make a switch to a fully renewable energy system with no or low associated greenhouse gas emissions. In our Ecofys Energy Scenario modelling, we describe the transition to such a system towards 2050 reach­ing 95% renewable energy without a major reduction in activity levels [1]. In the Ecofys Energy Scenario we describe how energy efficiency options and non-bioenergy renewable options can accommodate the majority of this transition. However, after applying these options, there is still a large demand that needs to be met with sustainable bioenergy options. This is illustrated in Fig. 1.

From Fig. 1 it becomes clear that the majority of this remaining de­mand consists of energy carriers that cannot be easily provided by renew­able options other than bioenergy. These include:

• Transport fuels where energy storage density is often a crucial factor; es­pecially:

о Long distance road transport о Aviation о Shipping

• Industrial fuels where electric or solar heating is insufficient; especially: о Long distance road transport

о Applications that require a specific energy carrier, e. g. a gaseous fuel or solid fuel. One example is the steel industry where the structural strength of a solid fuel is required.

In this study we answer the question: can we meet the remaining en­ergy demand in a fully renewable system like the Ecofys Energy Scenario using only sustainable bioenergy options?

In literature, there have been several attempts to quantify the potential of biomass available for energy supply with varying degrees of sustain­ability constraints. Estimates can differ within a very large range, depend­ing on whether the study takes a holistic view at land management and how stringent the applied sustainability criteria are. Not many of these studies look at the end uses for this biomass potential in detail [2], [3], [4], [5], [6], [7], [8], [9], [10] and [11]. Other studies postulate the use of biomass to fill a demand need, but do not always specify in detail where this biomass would come from [12], [13] and [14]. None of these studies is as comprehensive, as stringent in the applied sustainability criteria and as detailed on both the supply potential and the demand side use of biomass as the study we present here.

This is represented by our detailed bioenergy potential modelling ap­proach that acknowledges that:

• Bioenergy requires a thorough analytical framework to analyse sustainabil­ity, as cultivation, harvesting and processing of biomass and use of bioen­ergy have a large range of associated sustainability issues.

• Bioenergy encompasses energy supply for a multitude of energy carrier types, e. g. heat, electricity and transport fuels, using a multitude of differ­ent energy sources. Therefore a detailed framework of conversion routes is needed.

■ Fossil & Nuclear

■ Remaining demand tor bioenergy: Transport fuels

■ Remaining demand for txoenergy: Industry heat A fuels

1 Remaining demand for bioenergy: Building heat

Remaining demand for bioenergy: Electricity

Other renewables

2000 2010 2020 2030 2040 2050

The outline of this approach is described in Section 2. For clarity, Sec­tion 3 presents detail on the steps of the followed methodology alongside the results obtained from the analysis. In Section 4 we discuss the sensitiv­ity of our results and compare them to bioenergy potential values found in literature. In Section 5 we draw conclusions from our work and answer whether sustainable bioenergy options can meet the remaining demand in the Ecofys Energy Scenario.

3.2 APPROACH

From the demand modelling and supply modelling of all non-bioenergy options in the demand scenario, we find a remaining energy demand that needs to be met with bioenergy options. We applied technological choices (Section 2.1) and sustainability criteria (Section 2.2) to establish how this demand could be met.

For ideal conditions, roughly 70 g of nitrogen are required for each kg of algal biomass [15,17,44]. In the experiments, 0.20 kg of nitrogen was con­sumed per kLp. This amount translates to 77 kg of nitrogen per L of bio­oil produced (which is 769 g of N per kg of algal biomass). In the Highly Productive scenario (with 1 kg of algal biomass/kL of processed volume, 70 g of N per kL of processed volume, and 0.26 L of bio-oil per kLp) 0.27 kg of N are required per L of bio-oil produced, or 5.1 * 109 kg of N would be required for 19 GL of bio-oil (5 Bgal), which is 45% ofthe total amount of nitrogen fertilizer consumed in the U. S. annually [43].

5.3.6.2 ELECTRICITY

In the Experimental Case, 2.4 GJ of electricity were consumed per kLp, resulting in ~0.92 * 1012 J of electricity consumption per L of bio-oil. In the Highly Productive Case, 2.59 MJ of electricity is consumed per kLp, which yields 0.26 L of bio-oil. Thus, 9.9 MJ of electricity would be con­sumed per L of bio-oil, or 0.19 EJ per year for 19 GL of bio-oil per year (5 Bgal/yr). This amount is 1.3% of the annual U. S. electricity generation in 2009 [41].