1. Extracellular/free lipase lipases (i. e. recovered and purified from the cultivation broth): For industrial level production of extracellular lipases, bacteria, yeast and fungi are preferred. Lipases from different sources are able to catalyze the same reaction (Table 8.1). Bacterial and fungal lipases are mostly used in biodiesel production and recently, Streptomyces sp. was proved as an effective lipase producing microbe and the enzyme produced was appropriate for biodiesel production (Cho et al., 2012). Use of free or extracellular enzymes was limited due to their price. Cost of the enzyme was increased due to their specific separation and purification techniques. Extracellular lipases are soluble enzymes and they are dispersed in the solution and can move freely during the catalytic reaction, thus difficult to handle and reuse (Iso et al.,

2001) . One auspicious approach to overcome this

difficulty is to immobilize the enzyme in a way that can be separated and reused later by using simple separation methods like centrifugation and filtration (Iso et al., 2001; Cao, 2005).

2. Intracellular/immobilized lipases; i. e. lipases remain either inside or attached to the cell wall.

In this case, enzyme is immobilized (naturally) directly or together with the whole cell (intracellular). This strategy eliminates downstream operations and promises the recycling of enzymes. Alternatively lipases can be immobilized synthetically by different mechanisms. Immobilization restricts movement of the enzyme and constrains its location to an inert support or a carrier (Cao, 2005). Various methods like adsorption, covalent bonding, entrapment, and cross-linking are available for enzyme immobilization. The choice of method and support material is a protuberant factor for obtaining an efficient lipase (Table 8.2) (Sevil et al., 2012).

ADVANTAGES OF IMMOBILIZED LIPASE

1. Enzyme becomes more stable.

2. Immobilization of enzyme increases surface area of biocatalyst.

3. Option of regeneration and reuse of the immobilized lipase.

4. Protection from solvent inhibition.

5. Separation of product and enzyme is easier.

6. Avoids contamination of enzyme or whole cell.

7. Rigid external support expected to increase optimal temperature, thereby fasten reaction rate.

Realizing the oil-yielding potentialities with much faster growth rate and efficient CO2 fixation, microalgae appear to be the best option as a renewable source of biodiesel that has the potentiality to completely replace the petroleum diesel fuel. However, the lipid content in the selected microalga/strain is required to be high; otherwise the economic performance would be hard to achieve.

Each species of microalga produces different ratios of lipids, carbohydrates and proteins. Nevertheless, these tiny organisms have the ability to manipulate their metabolism by simple manipulations of the chemical composition of the culture medium (Behrens and Kyle, 1996); thus, high lipid productivity can be achieved. Physiological stresses such as nutrient limitation/defi — ciency, salt stress and high light intensity have been employed for directing metabolic fluxes to lipid biosyn­thesis of microalgae. Many reports are available, where attempts have been made to raise the lipid pool of various microalgal species. Table 11.1 summarizes those studies.

Exceptionally, an oil content of 86% of dry cell weight (dcw) was reported in the brown resting state colonies of Botryococcus braunii, while the green active state colonies were found to account for 17% only (Brown et al., 1969). However, the major obstacle in focusing B. braunii as an industrial organism for biodiesel production is its poor growth rate (Dayananda et al., 2007). Nitrogen limita — tion/deficiency has been found to raise the lipid content of a number of microalgal species profoundly. For instance, Piorreck and Pohl (1984) reported an increased lipid pool from 12% to 53% (dcw) in Chlorella vulgaris under nitrogen-limited condition. Unlike the green algae, the blue-green algae viz. Anacystis nidulans and Oscillato — ria rubescens contained the same quantities of lipid at different nitrogen concentrations. It was observed by Illman et al. (2000) that four species of Chlorella (Chlorella emersonii, Chlorella minutissima, C. vulgaris and Chlorella pyrenoidosa) could accumulate lipid up to 63, 57, 40 and 23% (dcw), respectively, in low N-medium. These values in control vessels were, respectively, 29%, 31%, 18% and 11% in the above order. In the same year, Takagi et al. (2000) observed an increase in intracellular lipid pool up to 51% (dcw) against 31% control in 3% CO2-purged cultures of Nannochloris sp. UTEX LB1999 grown in continuous low nitrate (0.9 mM)-fed medium. Chlorella protothecoides also showed a rise in lipid pool from 15% to 55% (dcw), when grown heterotrophically with glucose (1%) under reduced nitrogen concentration (Miao and Wu, 2004). Similarly, C. protothecoides depicted a lipid pool of 55% (dcw) when grown heterotrophically with corn powder hydrolysate under nitrogen limitation (Xu et al., 2006).

Gao et al. (2010) used sweet sorghum hydrolysate instead of corn powder for C. protothecoides culture, and lipid yield of 50% (dcw) was recorded. Nitrogen limitation/starvation also enhanced the lipid content in Neochloris oleoabundans, Nannochloropsis oculata,

C. vulgaris, Chlorella zofingiensis, Dunaliella tertiolecta and Thalassiosira pseudonana (Converti et al., 2009; Feng et al., 2011a; Gouveia and Oliveira, 2009; Jiang et al., 2012). However, the marine microalga Isochrysis zhang — jiangensis was found to accumulate lipid under high nitrate concentration, rather than limitation or depletion (Feng et al., 2011b).

Limitation of phosphate was also found to enhance lipid accumulation in Ankistrodesmus falcutus and Mono — dus subterraneus (Kilham et al., 1997; Khozin-Goldberg and Cohen, 2006). Rodolfi et al. (2009) screened 30 microalgal strains for lipid production, among which the marine genus Nannochloropsis sp. F&M-M24 emerged as the best candidate for oil production (50% under phosphorus deficiency against 31% control). Sobczuk and Chisti (2010) observed an increase in intra­cellular lipid content up to 60% (dcw) against 27% control in Choricystis minor under simultaneous nitrate and phosphate deficiencies. In Scenedemus obliquus, lipid accumulation up to 58% (dcw) was recorded when subjected to simultaneous nitrate and phosphate limita­tions in presence of sodium thiosulphate (against 13% under control condition, Mandal and Mallick, 2009). Simultaneous nitrate, phosphate and iron limitations have also been reported to stimulate lipid accumulation in a microalga C. vulgaris (57% against 8% control, Mallick et al., 2012).

In addition to nutrient limitations/deficiencies, other stress conditions may also enhance lipid accumulation in microalgae. Takagi et al. (2006) studied the effect of NaCl on accumulation of lipids and triacylglycerides in the marine microalga Dunaliella sp. Increase in initial NaCl concentration from 0.5 M (seawater) to 1.0 M resulted in a higher intracellular lipid accumulation (71% dcw). Damiani et al. (2010) studied the effects of continuous high light intensity (300 mmol photons/ m2 s) on lipid accumulation in Haematococcus pluvialis grown under nitrogen-sufficient and nitrogen-deprived conditions. A lipid yield of 33—35% was recorded under the high light intensity as compared to 15% yield in con­trol cultures. Nitrogen deprivation was, however, not found to raise the lipid content of H. pluvialis cultures.

Nutrient limitations/deficiencies or physiological stresses required for accumulation of lipids in microal­gal cells is associated with reduced cell division (Ratledge, 2002). The overall lipid productivity is there­fore compromised due to the low biomass productivity. For instance, Scragg et al. (2002) studied the energy recovery from C. vulgaris and C. emersonii grown in com­plete Watanabe medium and also in low nitrogen medium. The results showed that the low nitrogen medium, although induced higher lipid accumulation in both the test algae with high calorific values, the overall energy recovery was lower in comparison to Watanabe’s medium. A commonly suggested counter measure is to use a two-stage cultivation strategy, dedi­cating the first stage for cell growth/division in nutrient sufficient medium, and the second stage for lipid accu­mulation under nutrient starvation or other physiolog­ical stresses. To get maximal biomass and lipid yield, CO2 can also be utilized. Chiu et al. (2009) reported an increased accumulation of lipid (from 31% to 50% dcw) in the stationary phase cultures of N. oculata NCTU-3 grown under 2% CO2 aeration.

At present most methane from anaerobic digestion is used for cooking and in gas or dual fuel engines (diesel engines where part of the fuel is substituted by biogas) to generate electricity with an overall efficiency of 30—50%. In Western Europe part of the excess heat is used in residences and factories. In a few instances fuel cells are used to generate electricity and high — temperature process heat. It is, however, better to remove the carbon dioxide and to inject the gas into the natural gas grid. It can then be used to generate elec­tricity in 60% efficient combined cycle plants

In Europe and in several states of the United States there are requirements to gradually introduce biomass — derived fuels in the transport sector. Approximately 5 million cars currently run on compressed natural gas and could run on compressed methane from anaerobic digestion.

An alternative is to liquefy the gas and use the lique­fied gas as biofuel in vehicles. This is done in Snurrevar — den (Norway) and Gasum (Finland).

Anaerobic digestion and the use of compressed methane is more energy efficient than the hydrolysis of cellulose and hemicellulose to sugars and conversion of these sugars into alcohol. This alcohol has to be distilled in order for it to be used as a transportation fuel.

In areas where there is no natural gas infrastructure, methane in high-pressure bottles can replace bottled liq­uefied petroleum gas (LPG or propane). Energy den­sities of 20% of that of LPG bottles can be reached at a pressure of 4 MPa using bottles filled with activated carbon.

HMF is a very important building block for a wide range of applications. In this paragraph applications in the areas of polymers, fine chemicals, and fuels are sum­marized. When HMF is produced at high efficiency follow-up products will become an attractive option to replace petrochemical analogs. An interesting molecule that can be derived from HMF is FDCA. It can be obtained via the oxidation of HMF; several oxidation methods have been described in literature (Van Putten et al., 2013a). FDCA was identified by the US Department of Energy (Bozell and Petersen, 2010) to be a key bio­derived platform chemical, which in itself is the building block for polyesters, polyamides and plasticizers but FDCA can also serve as starting point for several other interesting molecules, including succinic acid, FDCA dichloride, and FDCA dimethyl ester. In addition to FDCA, other platform chemicals can be produced as well. 5-Hydroxymethylfuroic acid, 2,5-diformyl furan, the 2,5-diamino-methylfuran, and 2,5-bishydroxymeth — ylfuran are most versatile intermediate chemicals of high industrial potential because they are six-carbon monomers that could replace, for example, adipic acid, alkyldiols, or hexamethylenediamine in the production of polymers (Van Putten et al., 2013a). 2,5-Furandi — carboxaldehyde and 2,5-hydroxymethylfuroic acid can be considered intermediates to FDCA in the oxidation of HMF. De Vries, Heeres and coworkers (Buntara et al., 2011) have shown an interesting route to convert HMF into caprolactam, the monomer for nylon-6. In addition to applications in the polymer field HMF can also be used in many fine chemicals applications. In view of the rigid furan structure and the two substituents that can be easily modified, HMF has been used in quite a number of pharmaceutical studies (Van Putten et al., 2013a). HMF-derived 5-amino-levulinic acid (Binder et al., 2010) and its derivatives are herbicides. A synthesis route was published by Descotes in collaboration with Sudzucker (Schinzer et al., 2004).

The Maillard reaction between reducing carbohy­drates and amino acids is undoubtedly one of the most important reactions in the flavor and fragrance world, leading to the development of the unique aroma and taste as well as the typical browning, which contribute to the sensory quality of thermally processed foods, such as cooked or roasted meat, roasted coffee or cocoa.

Although numerous studies have addressed the struc­tures and sensory attributes of the volatile odor-active compounds, the information available on nonvolatile, sensory-active components generated during thermal food processing is scarce but HMF derivatives play an essential role (Van Putten et al., 2013a). HMF has also been linked to natural products, sugar derivatives (e. g. glucosylated HMF) and spiroketals (Van Putten et al., 2013a). HMF can also be a precursor of fuel components. HMF is a solid at room temperature with very poor fuel blend properties; therefore, HMF cannot be used and has not been considered as a fuel or a fuel additive. The Small Medium-sized Enterprise (SME) company Avantium is developing chemical, catalytic routes to produce furan derivatives "furanics" for a range of biofuel applications (de Jong et al., 2012a, b). Avantium targets biofuels with advantageous qualities, both over existing biofuels such as bioethanol and biodiesel as well as over tradi­tional transportation fuels. Another major goal is mini­mizing the H2 demand for their production. These C5-derived furanic monoethers and C6-derived furanic diethers have a relatively high energy density, and good chemical and physical characteristics, no difference in the engine operation was observed and strongly decreased smoke and particulates emissions. The use of furans, such as HMF and furfural, as precursors of liquid hydrocarbon fuels is also an option for the production of linear alkanes in the molecular weight range appropriate for diesel or jet fuel. The group of Dumesic has researched and evaluated the different strategies possible for upgrading HMF to liquid fuels (531 Alonso et al., 2010). HMF can be transformed by hydrogenolysis to 2,5-dimethyl furan. To form larger hydrocarbons, HMF and other furfural products can be upgraded by aldol condensation with ketones, such as acetone, over a basic catalyst (NaOH) already at room temperatures (West et al., 2008). Also several levulinic acid derivatives have been proposed for fuel applications, for instance ethyl levulinate, g-valerolactone, and MTHF (Geilen et al., 2010). The conversion of HMF to fuels has recently been reviewed (Maki-Arvela et al., 2012).

Alkaline organic compounds with an aliphatic, satu­rated carbon backbone having at least two primary amino groups, and a varying number of secondary amino groups are referred to as polyamines (Schneider and Wendisch, 2011). The polyamines were first discov­ered by Antonie van Leeuwenhoek (1678) when he iso­lated some "three-sided" crystals (sperminephosphate crystals) from human semen. The charge on the poly­amines is distributed along the entire length of the car­bon chain, making them unique and distinct from the point charges of the cellular bivalent cations. Their pos­itive charge enables polyamines to interact electrostati­cally withpolyanionicmacromolecules within the cell. Due to this they can modulate diverse cellular processes such as transcription and translation (Wallace et al.,

2003) , biosynthesis of siderophores (Brickman and

Armstrong, 1996), take part in acid resistance (Foster,

2004) , protect from oxygen toxicity (Jung et al., 2003), etc. They have a role in signaling for cellular differentia­tion (Sturgill and Rather, 2004) and are essential for pla­que biofilm formation (Patel et al., 2006). They are also found as a part of gram-negative bacterial outer mem­branes (Takatsuka and Kamio, 2004). Transgenic activa­tion of polyamine catabolism profoundly disturbs polyamine homeostasis in most tissues, creates a com­plex phenotype affecting skin, female fertility, fat de­pots, pancreatic integrity and regenerative growth (Janne et al., 2004). In the nucleosome, polyamine deple­tion results in partial unwinding of DNA and unmask­ing of sequences previously buried in the particle. These sequences are potential binding sites for factors regulating transcription (Morgan et al., 1987). This, together with the fact that polyamines favor the forma­tion of triplex DNA at neutral pH, may provide a mech­anism whereby polyamines regulate the transcription of growth regulatory genes such as c-myc (Hampel et al., 1991; Celano et al., 1992). Since polyamines play a wide range of activities in a living cell their relative intracellular concentrations may vary from species to species, and they can reach up to the millimolar range (Miyamoto et al., 1993).

The most common polyamines in bacteria and Archaea are putrescine (a diamine also named as

1,4- diaminobutane) and cadaverine (diamine also named 1,5-diaminopentane) (Figure 19.3). In addition to the above-mentioned polyamines, the pathways for the biosynthesis of 1,3-diaminopropane, norspermidine, homospermidine, and thermine are known in some bac­teria and Archaea (Tabor and Tabor, 1985). The poly­amine family also contains a number of uncommon longer or branched-chain polyamines, which were found in extremophiles and which seem to play an essential role for growth under such extreme conditions (Oshima, 2007). Polyamines are found in all living spe­cies, except two orders of Archaea, Methanobacteriales and Halobacteriales (Hamana and Matsuzaki, 1992).

Polyamines are used in a wide variety of commercial applications due to their unique combination of reactivity, basicity, and surface activity. With a few exceptions, they are used predominantly as intermediates in the produc­tion of functional products (e. g. polyamides/epoxy curing, fungicide, anthelmintics/pharmaceuticals, petro­leum production, oil and fuel additives, paper resins, chelating agents, fabric softeners/surfactants, bleach acti­vator, asphalt chemicals) (Kroschwitz and Seidel, 2004). The main commercial interest in biogenic polyamines is their use in the polymer industry. Today, the only example of an industrial polyamide containing a biogenic diamine, which can also be synthesized by bacteria, is nylon-4, 6. This polyamide is produced from putrescine and adipic acid (hexanedioic acid).

Hydrogenases in cyanobacteria have been studied for over 35 years (Benemann and Weare, 1974; Hallenbeck and Benemann, 1978) and many variations of hydroge — nases have been described in different bacterial phyla (Vignais and Billoud, 2007). These enzymes are frequently classified into three different groups: nitroge — nase, the reversible hydrogenase (Hox), and the uptake hydrogenase (Hup) (Ghirardi et al., 2007).

HUP—HYDROGEN UPTAKE ENZYME

Hup is a [NiFe] hydrogenase that occurs associated with the thylakoid membrane (Seabra et al., 2009). This enzyme shows the least sensitivity to oxygen among the three classes. Its function is in the oxidation of H2, returning the captured electrons to cellular electron transfer reactions. To date it has been found only in N2-fixing strains and appears to have an intimate rela­tionship with nitrogenase (Marreiros et al., 2013). Under natural conditions, nitrogenase functions to reduce atmo­spheric N2 to NH3, producing H2 in an unavoidable side reaction. It is thought that Hup functions to recycle the recently formed H2, which is oxidized back into protons or reacted with O2 in a respiratory oxyhydrogen reaction, protecting the nitrogenase from O2 inactivation, avoiding an excessive build up of H2 in the cell and recovering part of the ATP used in its formation (Bothe et al., 2010; Tamagnini et al., 2007). In the nitrogen-fixing cyanobacte­ria, transcription of the Hup-encoding genes hupSL is associated with the nitrogen depletion response and is under the regulation of the NtcA, the global nitrogen regulator (Weyman et al., 2008). Hup inactivation increases the production of H2 two — to threefold in most cyanobacteria (Ludwig et al., 2006; Tamagnini et al., 2007).

NITROGENASE—A GRATUITOUS HYDROGENASE

In nature this complex enzyme carries out a critical function, breaking the three covalent bonds of molecu­lar nitrogen (N2) providing ammonia to the cell and closing the nitrogen cycle. This process consumes a large amount of energy in the form of ATP and high — energy electrons (Eqn (22.1)), producing NH3 with the coproduction of hydrogen in an unavoidable side reaction.

N2 + 10H+ + 8e~ + 16ATP/2NH3 + H2 + 16ADP

(22.1)

The most common nitrogenase is the Mo-Fe nitrogenase, which is characterized by a complex iron-sulfur cluster containing molybdenum. While performing nitrogen fixation, up to one-fourth of the electron flux goes to­ward the reduction of hydrogen. Variations of this enzyme includes the substitution of the molybdenum by vanadium or iron (V-Fe and Fe—Fe nitrogenases, respectively), which, although a greater proportion of electrons are allocated to hydrogen production, in fact show a lower net flux of electrons to hydrogen since their overall reaction rates are much lower than that of the Mo-Fe enzyme, limiting the application of these variants in bioproduction systems. One option that is an interesting strategy for H2 production, to increase the electron flux into H2, is cultivation in the absence of N2, since nitrogenase turnover continues, but now the elec­tron flux goes totally toward hydrogen evolution. In addition, the growth arrest caused by the nutrient lim­itation is of interest as this decouples hydrogen evolu­tion from biomass production, therefore potentially leaving more energy available for H2 production (Benemann and Weare, 1974). Even so, the expression of an oxygen-sensitive enzyme in an O2 rich milieu is counter productive. To overcome this problem, temporal separation between N2 fixation and photosynthesis can be used, where during the day the photosynthetic machinery works toward the carbon fixation, which then can be consumed to power nitrogenase and consequently proton reduction. Interestingly, the peak of hydrogen production in indirect biophotolysis occurs when the cell is reilluminated, possibly due to a burst in ATP synthesis before the oxygen formed by PSII (Figure 22.1) reaches a toxic level for the nitrogenase.

Heterocyst forming species on the other hand can perform direct biophotolysis by carrying out nitrogen fixation in the differentiated cell during the day. The heterocyst can maintain an internal anoxic environment since the expression of PSII is repressed. Hydrogen production therefore is supported through the use of carbon compounds delivered by the neighboring vegetative cells.

REVERSIBLE HYDROGENASE (HOX)

In addition to nitrogenase, N2-fixing cyanobacteria can have a second hydrogen-evolving enzyme, the so-called reversible hydrogenase (Hox). This enzyme is a heteropentameric complex that is formed by a hydrog — enase module (HoxHY) and a diaphorase module (Hox — EFU), which transfers electrons from NAD(P)H to the hydrogenase module (Bothe et al., 2010). Like Hup, Hox is a [NiFe] hydrogenase, but in this case it shows a high sensitivity to O2. Its expression is totally indepen­dent from that of nitrogenase and varies among species. In some cases it is under the control of the circadian clock, where it is shown to promote hydrogen produc­tion in the dark (Hallenbeck and Benemann, 1978; Schmitz et al., 2001). The bidirectional hydrogenase is not taxon specific, being found in many different groups of cyanobacteria, and its location and organization in the chromosome are also heterogeneous. Recent studies regarding Hox transcription factors have elucidated many aspects of its regulatory mechanisms, which are reviewed elsewhere (Oliveira and Lindblad, 2009).

Consideration of the sustainability of biomass to bio­energy programs based on utilizing lignocellulosic feed­stocks is both timely and important in terms of the current plans for commercial valorization of this sector (Third International Conference on Lignocellulosic Ethanol; http: / /www. biofuelstp. eu/events/3rd-icle-

april-2013.pdf). Sustainability of second-generation bio­energy is also been driven and supported by European and International directives and certification programs, including the Renewable Energy Directive 2009/28/EC (EU-RED), International Sustainability and Carbon Cer­tification programs and standards, the Roundtable on Sustainable Biofuels and the Global Bioenergy Partner­ship (Scarlat and Dallemand, 2011). The sustainability of biomass to bioenergy programs has been a subject of great interest in Sweden, Canada and the western United States as well as in some Asian countries (Nguyen et al., 1999, 2000; Wu et al., 1999). The ecolog­ical and sustainable potential of biomass sources for fuel production is estimated to reach 130 TWh/year in Sweden by around 2020 (Parrika, 1997). Issues such as land use, environmental impact, logistics and resource management must be considered in terms of feedstock production. In addition, the sustainability of the biocon­version process(es) and downstream outputs, and the ability to meet REN and GHG emission targets must be carefully evaluated. High on the priority list of most national governments is the need to support rural development and sustain the local and national econo­mies. Consequently, biomass to bioenergy programs need to be subjected to detailed life cycle analysis (LCA), where all of the aforementioned considerations are evaluated. LCA can also help derisk biomass to bio­energy processes (Buonocore et al., 2012). The use of conventional crops for energy use can also be expanded, with careful consideration of land availability and food demand. For sustainable bioenergy development ligno — cellulosic crops (both herbaceous and woody) could be produced on marginal, degraded and surplus agricul­tural lands and, in theory, could provide the bulk of the biomass resource in the medium term along with aquatic biomass (algae) as a significant contribution in the longer term (Richardson, 2008). However, significant progress needs to be made to scale-up algal production and processing in an economic manner to make algal biomass to bioenergy a commercially viable option.

First-generation biofuels face both social and environ­mental challenges, largely because they use food crops that could lead to food price increases and possibly indi­rect land use change (ILUC). Nonfood biomass, e. g. lignocellulosic feedstocks such as organic wastes, forestry residues, high-yielding woody or grass energy crops and algae have the potential to provide possible solution to this problem, if developed and managed in a sustainable manner. The use of these feedstocks for second-generation biofuel production would signifi­cantly decrease the potential pressure on land use, improve GHG emission reductions when compared to some first-generation biofuels, and result in lower envi­ronmental and social risks (Bauen et al., 2009 IEA Report).

The environmental impacts of conventional crop pro­duction have been researched in far greater detail than those of lignocellulosic crop production. Technically, the potential supply of energy from lignocellulosic biomass depends largely on the amount of land that is available for growing energy crops. In parallel, the need to meet the growing worldwide demand for food, protect biodiversity, manage soil and water re­serves sustainably and fulfill additional socioeconomic objectives must be addressed. Bioenergy crop produc­tion can have positive impacts, for example, it can help to improve the soil structure and fertility of degraded lands. However, conversion of areas with sparse vegeta­tion to high-yielding lignocellulosic plantations or ILUC may lead to substantial reductions in ground water recharge and water supply, which may lead to deterio­rating conditions in water-scarce areas (Upham et al., 2011; Cabral et al., 2010; Smeets and Faaij, 2010). The cultivation of short rotation biomass crops may lead to nutrient removal or depletion (van den Broek et al.,

2000) , and important habitats may be lost through both land conversion and intensification (Pedroli et al., 2012). Aesthetic considerations also need to be consid­ered in terms of the impact of cultivating and harvesting short rotation bioenergy crops (Hardcastle, 2006). Sound agricultural methods exist that can achieve major in­creases in feedstock productivity in neutral or positive environmental conditions in order to provide a contin­uous supply of energy crops/biomass waste, which can support the important role of bioenergy chains in socioeconomic development (Figure 2.3; Dornburg et al., 2008). The issue of biomass logistics is also a factor that needs careful consideration in terms of feedstock supply, processing technology selection, sitting of com­mercial production facilities and overall sustainability (Stephen et al., 2010).

Recent studies have shown the potential of recycled wastewater for biomass production in an integrated nat­ural water treatment approach (Fedler and Duan, 2011), which suggests that through innovative and careful consideration of environmental impacts solutions can be found that have multiple potential benefits. It has been suggested that the application of strict sustainabil­ity criteria, standards and a requirement for certification (Scarlat and Dallemand, 2011; Schubert and Blasch, 2010; van Dam et al., 2010) of feedstocks, land use and

bioenergy programs globally could both alleviate concerns and provide a more harmonized framework globally for sustainable development of second — generation bioenergy (Cornelissen et al., 2012; Van Stappen et al., 2011).

The advantages of biological pretreatment include minimum facility cost, low energy requirement and mild environmental conditions. However, for practical application, there are two major disadvantages associ­ated with this process. First, fungi growth consumes hol — ocellulose as an energy source leading to significant carbohydrate loss; second, most biological pretreat­ments are long processes due to slow microbial growth and delignification reaction rates. Since lignin break­down in the biomass would lead to enzyme access to cel­lulose and hemicellulose, selective lignin degradation by white-rot fungi hold some promise for real application in biomass pretreatment if the procedure can be cut shorter and sugar consumption can be controlled to an insignificantly low level. However, not even white-rot fungi can use lignin as a sole carbon and energy source; fungi growth inevitably results in carbohydrate loss (Fan et al., 2012; Sanchez, 2009). Strategies taken to shorten biological pretreatment time and decrease car­bohydrate consumption include (1) selection for natu­rally occurring white-rot fungi that preferentially attack lignin (Ander Eriksson, 1977; Kirk and Moore, 1972; Lee et al., 2007; Muller and Trosch, 1986; Salvachua et al., 2011), (2) selection of cellulase-deficient mutants (Akin et al., 1993; Eriksson et al., 1980; Ruel et al., 1981), or (3) repression of cellulase and hemicellulase expression (Yang et al., 1980). As an example of strain se­lection, among 22 screened Basidiomycetes, mostly the white-rot fungi Pleurotus sp. "florida" preferentially at­tacks lignin in wheat straw to increase cellulose accessi­bility. After 90 days pretreatment with Pleurotus sp. "florida", the resulting biomass can release the same amount of glucose as Avicel, the lignin-free cellulose (Muller and Trosch, 1986). However, pretreatment using this strain is still time consuming.

Furthermore, there are many limitations to the strate­gies for strain improvement. First, carbohydrate con­sumption is needed for microbial growth; therefore, strains can only be selected for increased delignification and decreased sugar loss and not for minimal sugar loss. In addition, decreasing the secretion of carbohydrate hy­drolysis enzymes would lower the reaction rate and lead to even longer pretreatment time. Genetic modification of white-rot fungi to improve the required features may help resolve some of the drawbacks, but the tech­nical process is quite challenging (Fan et al., 2012).

Another way to improve the biological pretreatment process is through optimization of nutrients, tempera­ture, and preprocessing time to reach a balance between maximum sugar release and minimum sugar loss within the shortest possible time. Based on the enzymatic activity profile obtained in a 28-day pretreatment anal­ysis, switchgrass is pretreated with P. chrysosporium for 7 days. The pretreatment of switchgrass led to higher glucan, xylan, and total sugar yields than the unpre — treated sample, suggesting enzyme profile assays may be utilized for initial estimation of pretreatment time in or­der to enhance sugar yields and reduce sugar loss (Maha — laxmi et al., 2010). By monitoring compositional changes during biological pretreatment, a 15-day pretreatment time was selected for the pretreatment of the woody bio­masses Prosopis juliflora and Lantana camara with the white-rot fungus Pycnoporus cinnabarimus (Gupta et al.,

2011) . This 15-day pretreatment resulted in a relatively small weight loss in the pretreated feedstocks with decreased lignin and increased holocellulose contents. Enzymatic hydrolysis of the pretreated biomass led to sugar releases of 389 and 402 mg per gram of dried solid.

Alternatively, as a compromise, preliminary microbial pretreatment of biomass can be used in combination with downstream thermochemical, chemical or other pre­treatment. This procedure would reduce, for example, the amount of acid needed combined with lower temper­ature and shorter time, thus reducing energy and chemi­cal costs. In addition, there would be less biomass degradation and inhibitor production compared to con­ventional thermochemical pretreatment. Preliminary tests showed that after corn stover pretreatment with

P. chrysosporium, the shear forces needed to obtain the same shear rates of 3.2—7 rev/s were reduced 10- to 100-fold, respectively. The digestibility of C. stercoreus — pretreated corn stover showed a three — to fivefold improvement in enzymatic cellulose digestibility (Keller et al., 2003). Sawada et al. reported that combination of fungal pretreatment with less severe steam explosion maximizes enzymatic saccharification of beech wood meal (Sawada et al., 1995). Compared to steam explosion alone, combined pretreatments improve saccharification by 20—100% of the polysaccharide in the wood. However, 17% of the holocellulose was degraded during fungal pretreatment, and there was an unspecified holocellulose loss during steam explosion at optimum 215 °C for

6.5 min (Sawada et al., 1995). Pretreatment of wheat straw with P. juliflora followed by acid hydrolysis led to a reduc­tion in acid load and an increase in sugar release as well as ethanol yield (Kuhar et al., 2008).

Interestingly, a recent study showed that by simply changing the pretreatment sequence, i. e. when the wood Pimus radiata biomass was treated first with steam explosion followed by fungi pretreatment, a 10-fold increase in glucose yield was achieved after enzymatic hydrolysis (Vaidya and Singh, 2012). A combination of selected fungal pretreatment with a mild alkali treat­ment of wheat straw led to a maximum of 69% glucose yield and an ethanol yield of 62% with no inhibitor for­mation during the pretreatment (Salvachua et al., 2011). Also, a combination of the white-rot fungus Lenzites betulina C5617 pretreatment with LHW treatment enhanced the enzymatic hydrolysis of the poplar wood Populus tomentosa led to the highest hemicellulose removal of 92.33%, which was almost two times higher than that of LHW treatment alone and a 2.66-fold increase in glucose yield (Wang et al., 2012).

MET THROUGH EXOGENOUS REDOX MEDIATORS

Similar to bioanodes, the same exogenous mediators including neutral red, methyl viologen and the anthra — quinone-2,6-disulfonate can be used for biocathodes (Hatch and Finneran, 2008; Park and Zeikus, 1999; Stein — busch et al., 2010) to enhance MFC performance signifi­cantly. When mediators are added into the cathode chamber, they are reduced by the electrons donated by the cathode. The reduced mediators reach the microbial cell wall and then transfer the electrons through the wall while the mediators are oxidized. Subsequently, the oxidized mediators diffuse back to the cathodic sur­face for reuse. This cyclic process is illustrated in Figure 9.4(b). Usually one mediator molecule can accom­plish thousands of cycles. These mediators are relatively short-lived and costly, making their use unsustainable. Just like their use for bioanodes, these exogenous media­tors are used only in laboratory investigations of MFC mechanisms for academic purposes. Pili can also be used by microbes to transfer extracellular electrons to the cytoplasm (Zhou et al., 2013).

In manganese-oxidizing bacteria, manganese (IV) plays an important role in the electron transfer. This mechanism is similar to the exogenous mediator MnO2 on the biocathode surface. It is first reduced to MnOOH by the electrons donated from the cathode and then Mn2+ is released. Finally, with the help of manganese — oxidizing bacteria, Mn2+ was oxidized by dissolved ox­ygen to regenerate MnO2 (Nguyen et al., 2007). The power density can be improved by two orders of magni­tude, compared with the abiotic cathode (Rhoads et al.,

2005) , making it attractive for potential practical applications.

MET THROUGH SELF-EXCRETED REDOX MEDIATORS

Apart from exogenous redox mediators, some microbes can excrete metabolites that are redox active. For example, Pseudomonas spp. can produce phenazines (Venkataraman et al., 2010) and S. oneidensis can produce flavins (Marsili et al., 2008). These mediators can be used by biocathodes indirectly. In the presence of these medi­ators, the rate of electron transfer is enhanced. These mediators are more easily utilized by other microbes than their producers (Rosenbaum et al., 2011). Therefore, in biocathodes, the self-excreted mediators play an important role in a synergistic biofilm consortium cove­ring a cathode. Their mechanism of electron transfer is

similar to that used by exogenous redox mediators. Table 9.2 shows some reported microbial species for biocathodes.

Growing microalgae for biolipid production usually involves a lag phase of growth followed by a stationary phase induced by some sort of "stress" This "stress", often nitrogen depletion, induces a switch in the meta­bolism of the microalgae, which encourages the produc­tion of storage lipids in the form of triacylglycerides (TAGs) rather than cell division (Meng et al., 2009; Widjaja et al., 2009). Currently microalgae can be grown at industrial scale autotrophically in open raceway ponds (Sapphire Energy, 2013) or closed photobioreactor (PBR) systems (Solix BioSystems, 2013). In addition, many microalgae species have the ability to grow heterotrophically, in closed fermenters, given a suitable carbon source (Solazyme Inc., 2013). Open culture sys­tems, such as race way ponds, are significantly lower cost in terms of capital expenditure. They require greater land area than closed systems and are more prone to contamination by invasive species. Water loss due to evaporation can also be a significant problem when compared to closed systems (Chisti, 2007; Pulz, 2001; Sheehan et al., 1998). Closed systems, on the other hand, such as PBRs or fermenters are by their nature closed and thus less likely to be contaminated. Nutrient concentration can be more easily controlled and water loss through evaporation is negligible. However, some have argued that loss of cooling water, used to control temperature, negates any savings made from using a closed culture system. The tighter control over culture conditions facilitated by a closed culture system, along with more sterile cultures, results in PBRs producing much greater levels of microalgae biomass, when compared to raceway ponds. However, the increased production capability must be offset against the much larger capital cost involved in commissioning and main­taining a closed culture system (Carvalho et al., 2006; Pulz, 2001; Ugwu et al., 2008). Hybrid systems have also been proposed whereby a closed system is used for the log phase production of biomass and the nutrient depleted lag phase is allowed to occur in large raceway ponds. It is hoped that the relatively concentrated inoc­ulation of the raceway ponds will not allow any invasive species to become established (Greenwell et al., 2010; Huntley and Redalje, 2007; Rodolfi et al., 2008).

Microalgae present significant potential as a source of biolipids for bioenergy over more traditional sources of biolipids such as palm, soya or Jatropha for a number of reasons. Firstly, the oil content of microalgae as a percentage of the dry weight, shown in Table 12.3, is generally in the range of 20—70%, although levels above 40% are rarely observed (Borowitzka, 1988). Similarly, the potential yield of biolipids and derived biodiesel from microalgae per area far outweighs that of any current oilseed crop. For example, one of the best available studies of large-scale algae cultivation produced 0.1 g/l day or 20—23 g dry weight/m2 day. A conservative lipid content of 30% could therefore yield 24,000 l biodiesel/ha year (Moheimani and Borowitzka, 2006; Schenk et al., 2008). This compares extremely favorably with both Jatropha (18921 biodiesel/ha year) and oil palm (5950 l biodiesel/ha year) (Schenk et al., 2008).

The high potential yield of biodiesel from microalgae — derived biolipids is due to a number of factors including the growth rate of microalgae (Scott et al., 2010) all year round production capability (Schenk et al., 2008) and the higher photon conversion efficiency compared to terres­trial plants (Melis, 2009). Unlike algae-derived biofuels, first-generation biofuels directly competed with food crops for arable land sparking the "Food vs Fuel" debate (Gui et al., 2008). Although second-generation fuel crops such as Jatropha can grow on marginal land (Francis et al., 2005), microalgae are capable of growing on nonarable land ensuring competition for land with food crops is significantly reduced. Similarly, in terms of other resource demands, 1 kg of algae biomass requires 1.83 kg of CO2 to grow (Chisti, 2007) and much research has investigated the potential of indus­trial flue gases as a source of this CO2 (Bilanovic et al.,

2009) . This possibility of both sequestering excess CO2 from flue gases that would otherwise be released into the atmosphere, while also increasing the growth rate of microalgae to be used for bioenergy, offers both environmental and economic advantages (Pires et al., 2012; Yun et al., 1997). More recently, the apparent "peak phosphorus" problem has been identified whereby phosphorus will become a limiting resource in agriculture. As a result, the potential industrial scale culture of microalgae, which requires a phosphorus

and nitrogen source for growth, would also be affected (Cordell et al., 2009). Both phosphorus and nitrogen are available in plentiful supply within waste water streams (Sawayama et al., 1995; Yun et al., 1997).

Commercial harvesting of algae blooms from waste­water has already been demonstrated in New Zealand (Aquaflow, 2013) and the use of wastewater streams as a nutrient source in large-scale cultivation of microalgae
has been well studied and implemented. Similarly, in terms of water usage, microalgae cultivation, particu­larly in closed cultivation systems, demonstrates signif­icant water savings when compared to traditional biofuel crops. Many microalgae species are also capable of growing in brackish water most notably Dunaliella salina (Weldy and Huesemann, 2007).