K. Madhavan Nampoothiri*, Vipin Gopinatha, M. Anusreea,
Nishant Gopalan, Kiran S. Dhar

Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST),
CSIR, Trivandrum, Kerala, India, aEqual contributors
Corresponding author email: madhavan85@hotmail. com

OUTLINE

Amino Acids 337

Glutamic Acid 338

Lysine 339

Methionine 340

Threonine 340

Arginine 340

Aromatic Amino Acids 341

Aspartame 341

Poly(Amino Acid)s 341

Cyanophycin/Cyanophycin Granule Polypeptide 342

Production of Cyanophycin 343

Biodegradability of Cyanophycin 343

Applications for Cyanophycin 343

AMINO ACIDS

The amino acid industry has shown an exponential growth since its infancy in the 1950s. It has grown from extracting flavor enhancers from seaweeds, to fer­menting high-purity, optically active forms in hundreds and thousands tons. The isolation of a bacterial strain producing glutamic acid and an efficient screening method to identify the highest producer by the Japanese researchers of the Kyowa Hakko Kogyo Co. was the key event in the amino acid fermentation industry. Until
then, there was no suitable commercial process for the mass production of amino acids. Later on, it received further boost when the workers of the same organization reported a homoserine auxotrophic lysine producer. This discovery led to the development of a commercially viable fermentation process for lysine fermentation with a conversion efficiency of 26% from glucose. Bioprocess engineering and strain improvement methods have contributed to the massive growth of the industry.

The essential amino acids hold a major place in the global amino acid market, as these cannot be

Bioenergy Research: Advances and Applications http://dx. doi. org/10.1016/B978-0-444-59561-4.00019-X

synthesized in the organisms and have to be supplied externally. The annual demand for feed-grade amino acids globally is about 2.43 million tons with an esti­mated value of US $6 x 109. The global amino acid mar­ket is estimated to hit US $12.8 x 109 by the end of 2017 (Chapman, 2012). There has been a substantial increase in the demand for amino acids in the last 30 years with a steady growth rate in the market. It is estimated that in that last 10 years the market demand for amino acids has doubled with glutamic acid and lysine on the top of the chart. Corynebacterium glutamicum is generally used for amino acid production, with an estimated annual production of 2,160,000 tons of L-glutamate and

1,480,0 tons of L-lysine (Zahoor et al., 2012).

The commonly used methods for amino acid produc­tion are extraction, chemical synthesis, enzymatic con­version and fermentation. Selection of the best method depends on the cost of the raw materials used, overall production cost, purification methods adopted, market­ability and demand. Cost of production and environ­mental impact can further be reduced by using sugars from agricultural, industrial or municipal wastes than pure and refined sugars. Microbial amino acid process for biorefining applications will lead to a cleaner envi­ronment and lesser production costs.

For surviving under different environmental condi­tions, cyanobacteria have developed a number of different alternative electron transfer pathways, which at the same time decrease the photosynthetic and H2 pro­duction efficiency. In order to improve H2 photoproduc­tion from cyanobacteria, the competing alternative electron transport pathways should be diminished. This will guide future genetic and metabolic engineering ef­forts to modulate the major energetic pathways to avoid "wasteful" electron flow and to channel major electron flux to H2 production. Recent studies have confirmed that the alternative electron-transport routes in cyanobac­teria are also strong competitors for H2 production. Under Ci-deprivation conditions the Flv1/Flv3 proteins in cooperation with photorespiratory pathway might flux up to 60% of electrons to O2, functioning as a power­ful electron sink for the electrons originated from water­splitting PSII (Allahverdiyeva et al., 2011). In line with this, recent studies on deletion mutants of the respiratory electron transport complexes, terminal oxidases and the NdhB subunit of the NDH-1 complex have revealed increased hydrogenase activity and the production of H2 (Cournac et al., 2004; Gutthann et al., 2007).

Biodiversity

Cyanobacteria are very diverse organisms. For many decades only a limited number of cyanobacterial strains have been used as model laboratory organisms for H2 research and by the end of 2000 only a few attempts to screen large cyanobacterial culture collections for H2 production were recorded (Berchtold and Bachofen, 1979; Lambert and Smith, 1977).

Recently, more emphasis has been given to the biodi­versity of cyanobacteria in order to identify promising H2 producers with flexible metabolic pathways (Yeager et al., 2011; Yoshino et al., 2007; Allahverdiyeva et al., 2010). Screening of 400 cyanobacteria strains from the University of Helsinki Culture Collection revealed that about 50% of these strains produced easily detectable amounts of H2. Ten of them produced similar or up to four times as much of H2 as the uptake hydrogenase mu­tants of Anabaena PCC 7120 (Masukawa et al., 2002) and N. punctiforme ATCC 29133 (Lindberg et al., 2002), spe­cifically engineered to produce higher amounts of H2 (Allahverdiyeva et al., 2010). All 10 best H2 producers were N2-fixing, heterocystous filamentous strains. Notably, the changes in environmental parameters had differential effects on H2 production, depending on the strain. Therefore, it is necessary to test multiple environ­mental conditions when screening for superior H2- producing strains (Yeager et al., 2011). Optimization of culture conditions and genetic modification of new strains would enhance further H2 production.

As mentioned earlier, two important factors in bio­char production are the type of feedstock used and pyrolysis conditions as they affect the physical and chemical properties of the biochar that is produced. Depending on the origin of the feedstock (e. g. cellulose, lignin, lingocellulose, hemicellulose) the chemical and structural composition of pyrolyzed biochar can change, and thus when biochar is used as a soil amendment, its behavior, function and fate in soils could be different. For instance, Winsley (2007) showed that when wood — based feedstocks are pyrolyzed, coarse and resistant bio­chars were generated with nearly 80% carbon contents, because the rigid ligninolytic nature of the source mate­rial is still retained in the biochar residue. The following sections discuss the chemical, physical, and biological properties of soil amended with biochar with particular focus on how the various properties influence the soil — biochar interactions and consequently improve the soil’s biological health and crop productivity.

Chemical and Physical

In an effort to offset anthropogenic C emissions to the atmosphere, a number of geoengineering technologies have been proposed, which can be divided into the

two broad categories of solar radiation management and CO2 removal, with C sequestration through biochar incorporation into soil in the latter (Vaughan and Lenton,

2011) . What distinguishes biochar CO2 sequestration (Figure 25.1) from competing CO2 removal technologies, such as ocean iron fertilization and CO2 geological injec­tion, are two factors. First, biochar C sequestration relies on photosynthesizing plants to draw CO2 from the atmo­sphere and is stored in the form of charcoal, whereas methods such as CO2 geological injection rely on rela­tively new and untested storage methods, including me­chanically forcing supercritical CO2 into depleted fossil fuel reservoirs or deep sea sediments (Vaughan and Lenton, 2011). Second, charcoal storage in soils is already ubiquitous and the clearest challenges in the short term involve optimizing economic profitability, rather than projected efficacy (Spokas et al., 2012a).

C sequestration through pyrolysis for biochar pro­duction is a result of the conversion of C forms present in the feedstock into recalcitrant forms present in biochar. Pyrolysis consists of a succession of changes in chemical structural composition as temperature in­creases. Cellulose and lignin are degraded as volatile compounds are driven off between 250 °C and 350 °C, followed by lateral growth and coalescence of polyaro­matic graphene sheets, and culminating with carboniza­tion, constituted by the expulsion of the majority of non-C atoms above 600 °C (Verheijen et al., 2010). Addi­tionally, as pyrolysis temperature and residence time increase, H/C and O/C ratios of biochar decrease and
aromaticity, the extent to which aromatic rings are con­nected, increases, resulting in greater recalcitrance against degradation (Kookana et al., 2011). Recent research suggests that biochars intended for both soil improvement and C sequestration should possess recal­citrant carbon of >15%, O/C ratios of <0.4, H/C ratios of <0.6, polyaromatic hydrocarbon contents below back­ground soil values, and a surface area of >100 m2 g (Schimmelpfennig and Glaser, 2012). As pyrolysis tem­peratures increase, biochar specific surface area and microporosity analogously increase. Biochar chemical and physical properties are due to both the composition of the feedstock and the extent of the alterations under­gone during pyrolysis (Kookana et al., 2011). The diver­sity of biochar chemical and physical qualities achievable through varied pyrolysis conditions and feedstocks is reflected in the diversity of effects on soil biota that may be achievable. Biochar is sterile when produced, yet has been observed to have beneficial effects on soil microbes that play essential roles in nutrient cycling. These effects are complex; however, they are linked to the chemical and physical properties of the biochar employed, which can serve as both a source of nutrients and as a habitat for soil microbes (Lehmann and Rondon, 2006; Lehmann et al., 2011).

Microbiological Effects and Synergisms

Literature focused on the effects of biochar on soil biota is currently sparse relative to biochar chemical

and physical effects on soil (Lehmann et al., 2011). Pub­lished research fails to adequately address the diverse spectrum of biochars within a single study, typically evaluating the effects of a small number of biochars on microbial and root abundance for a small number of soil types. Depending on the feedstock from which bio­char is produced and the pyrolysis conditions involved in the feedstock conversion, biotoxic substances may persist through or be generated during pyrolysis. Stan­dardized methods, including germination rates of various crops and degree of earthworm avoidance, have recently been proposed in order to assess biochar toxicity to avoid inadvertent detrimental effects on crop production and the environment (Busch et al., 2012; Rogovska et al., 2012).

The addition of biochar to compost systems and the resultant effects on both the microbial community dy­namics and final compost quality have been evaluated, and the potential for synergistic effects and increased soil C stability are known (Fischer and Glaser, 2012). While the aromatic core of biochar has been observed to remain unchanged during the composting process with manure, C and plant-available nutrients are drawn into its pores and adhere to its surfaces, elevating the cation exchange capacity (CEC) and acid neutralizing capacity, and enhancing the functionalization of biochar surfaces, although the mechanisms responsible warrant additional research (Prost et al., 2013). Biochar has been observed to affect mycorrhizal symbioses, yet the mech­anisms responsible are still being determined (Warnock et al., 2007) and recently, it has been proposed that soil nitrogen may act as a switch controlling the proliferation of mycorrhizae and the subsequent oxidation of fresh biochar surfaces (LeCroy et al., 2013). Compared to compost, biochar is a much more stable soil amendment and the addition of biochar to composts has been shown to dramatically increase compost stability (Bolan et al.,

2012) .

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).

Hydrothermal Liquefaction

Hydrothermal liquefaction of biomass makes bio­mass react at high-temperature aqueous solutions under high vapor pressures. In the field of geochemistry and mineralogy, this method also was used for getting in­sights into the solubility of minerals in hot water under high pressure (Zhang et al., 2010; Wu et al., 2012; Tong et al., 2013). Hydrothermal liquefaction in­volves thermal depolymerization in an aqueous or organic medium. In this context, it might be called a depolymerization process using hydrous pyrolysis for decomsition of complex organic materials (for example, here biomass) into light crude oil. In this way, it is expected that under pressure and upon heat, long-chain lignocellulosic polymers decompose into short-chain petroleum-like hydrocarbons and chemicals. In this aspect, pyrolysis technologies are best suited for the conversion of dry feedstocks (<5% moisture), while hydrothermal liquefaction of biomass is ideal for processing high-moisture (i. e. wet) biomass.

Direct hydrothermal liquefaction involves convert­ing biomass to an oily liquid under elevated pressures (50—200 atm) and at low temperatures (473—673K) to keep water in either liquid or supercritical state. In a hot pressurized water for sufficient time, hydrother­mal treatment of biomass breaks down the solid biopolymeric structure to liquid components and even gases. Usually, the liquid product from the hydrothermal liquefaction of lignocellulosic biomass is a complicated mixture with a wide range of compo­sitions. It typically consists of glycoaldehyde dimers,

1,3- dihydroxyacetone dimers, anhydroglucose, soluble polyols, 5-hydroxy-methylfurfural (HMF), furfural, organic acids, phenolic compounds and even hydrocarbons.

Feedstock

Hydrothermal liquefaction has been applied to a wide range of biomass. One of the advantages of hydrothermal processing is the use of high-moisture biomass without the need for preliminary drying of the biomass. The feedstocks can be cellulose, hemicel — lulose, lignin, aquatic biomass such as duckweed, microalgae, microalgae, wastes animal manure and human sewage. In general, the presence of high cellu­lose and hemicelluloses content in biomass yields more bio-oil (Akhtar and Amin, 2011). For example, hardwood samples (cherry) produced more oils than softwood (cypress) due to the high lignin contents in the latter biomass (Bhaskar et al., 2008). Besides the oil yield, the oil composition is also different when different feedstocks are used. Karagoz et al. made analysis of oil compositions obtained from hydrother­mal treatment of sawdust, rice husk, lignin and cellu­lose at 553K for 15 min (Karagoz et al., 2005b). The conclusion was that the oil from the cellulose mainly consisted of furan derivatives, whereas lignin — derived oil mainly contained phenolic compounds. The compositions of oils from sawdust and rice husk contained both phenolic compounds and furans; how­ever, phenolic compounds were dominant. But rice husk-derived oil consists of more benzenediols than sawdust-derived oil.

In addition, hydrothermal liquefaction of algae biomass has also received much attention. The advan­tage of microalgae compared to terrestrial biomass is its much higher photosynthetic efficiency, which results in higher growth rates and improved CO2 mitigation (Brennan and Owende, 2009). Studies on the hydrother­mal processing of microalgae indicated that 30—60% of the algal biomass can be converted to bio-oils (Tsuka — hara and Sawayama, 2005; Patil et al., 2008). With different biochemical content of pristine microalgae, the oil from the hydrothermal liquefaction of microalgae is different. Biller and Ross liquefied microalgae and cyanobacteria with different biochemical contents (lipids, proteins and carbohydrates) under hydrother­mal conditions at 623K, ~ 200 bar in water (Biller and Ross, 2011). The results indicated that bio-oil formation followed the trend: lipids > proteins > carbohydrates, and proteins produced large amounts of nitrogen het­erocycles, pyrroles and indoles; carbohydrates pro­duced cyclic ketones as well as phenols while lipids were converted to fatty acids.

Reaction Conditions

Hydrothermal liquefaction of biomass need be accomplished with careful choices of time, temperature, pressure, catalyst and the use of reducing gases. Increasing temperature in a certain range is favorable. Temperature control is important because after reach­ing a maximum of the oil yield, further increase in tem­perature actually inhibits biomass liquefaction due to the secondary decomposition, Bourdard gas reactions and char formation (Mok and Antal, 1992; El-Rub et al., 2004; Zhong and Wei, 2004). The choice of temper­ature also depends on the biomass types. Rogalinski et al. carried out a kinetic study on hydrolysis of different biopolymers (Rogalinski et al., 2008). It was observed that cellulose hydrolysis rate in water at 25 MPa increased 10-fold between 513 and 583K and at 553K, a 100% of cellulose conversion was achieved within 2 min. Lignin showed a higher hydrother­mal liquefaction temperature than hemicellulose and cellulose. Zhang and Wei found that the optimal tem­perature of wood hydrothermal liquefaction shifted to a higher value as the lignin content increased (Zhong and Wei, 2004).

Pressure increases the density of solvent to facilitate solvent penetration into molecules of biomass compo­nents, which results in enhanced decomposition and extraction (Deshande et al., 1987). According to Le Chatelier’s principle, one would expect that the higher the pressure during liquefaction, the less liquid com­ponents are gasified. By maintaining pressure above the critical pressure of medium, the rate of hydrolysis and biomass dissolution can be controlled. This can be used to enhance favorable reaction pathways thermo­dynamically for the production of liquid fuels. How­ever, once supercritical conditions for liquefaction are used, pressure has little or negligible influence on the yield of liquid oil or gas yield because in the super­critical region influence of pressure on the properties of water or solvent medium becomes very weak small (Kersten et al., 2006; Sangon et al., 2006).

Reaction atmosphere also need to be considered. The use of reductive gases (e. g. CO and H2) generally im­proves oil yields with higher H/C ratios (He et al.,

2001) . The reducing gases stabilize the products of lique­faction by inhibiting the condensation, cyclization, or repolymerization of free radicals. Hence, they help reduce char formation (Xu and Etchevery, 2008). By using H2 instead of Ar atmosphere for liquefaction, Wang et al. found that both the conversion of sawdust and the oil yield were able to be increased (Wang et al., 2007a). Besides the oil yield, the quality of gaseous product is also improved by using H2; For example, CO and C1—C4 products increased and CO2 decreased.

Solvent

Water is the most common medium used for hydro­thermal liquefaction of biomass. The bio-oil obtained from hydrothermal liquefaction of lignocellulose in water is usually a viscous tarry lump with a high oxy­gen content and low heat. To make bio-oils with low viscosity and high yield, the use of organic solvents is an alternative. The tested ones include ethyl acetate (Demirbas, 2000), acetone (Liu and Zhang, 2008), methanol, ethanol, propanol, butanol, propylene gly­col, ethylene glycol, diethylene glycol and so forth (Mun and Hassan, 2004; KrZan et al., 2005).

Liquefaction of biomass with proper solvents is a pro­cess that can be integrated with optimized conditions to produce fuel and valuable chemicals. Liu et al. liquefied pinewood in the presence of various solvents (water, acetone and ethanol) in the conditions of temperature range 523—723 K, starting pressure 1 MPa, reaction time 20 min (Demirbas, 2000). The results showed that the highest oil yield reached 26.5% at 473 K in ethanol and the product distribution was strongly affected by the solvent type. The major compound was 2-methoxy — phenol (17.20%) for liquefaction in water, while it was 2-methoxy-4-methyl-phenol (8.23%) for liquefaction in ethanol and 4-methyl-1,2-benzenediol (9.49%) for liquefaction in acetone. Recently, it was found that co-solvents are a much more effective than the constitu­ent monosolvents alone due to the synergistic effects of different solvents. For example, biomass conversion in
100% ethanol and 100% methanol at 573K is 43% and 42%, respectively, producing a bio-oil yield at approxi­mately 26 and 23 wt%, while the liquefaction in the mixed 50 wt% methanol-water solution or the 50 wt% ethanol-water solution led to a conversion of biomass >95 wt% and a bio-oil yield of as high as 65 wt% at 573K (Cheng et al., 2010).

The use of donor hydrogen solvents is a new option to hydrogenate the biomass fragments. These solvents not only donate hydrogen but also act as hydrogen transport vehicle and it was found that the use of tetra — lin solvent enhanced liquid oil yield by suppressing the formation of asphaltenes, preasphaltenes and gases compared to toluene solvent (nonhydrogen donor) (Wang et al., 2007b). For example, Wang et al. observed that in the presence of solvent the yield of oil increased to 33.1% in toluene (nonhydrogen donor) and 48.4% in tetralin (Wang et al., 2007c). Besides, tetrahydrophe — nanthrene, octahydrophenanthrene, hexahydropyrene, hexahydrofluorene, and tetrahydroacenaphthene are also useful solvents for hydrogenation (Akhtar and Amin, 2011).

Catalyst

Table 15.3 summarizes some results of catalytically hydrothermal liquefaction of lignocellulosic biomass. Hydrothermal liquefaction of biomass was signifi­cantly affected by catalyst. Lignocellulosic biomass mainly contains cellulosic polymer and lignin poly­mer. The former readily interacts with acid; the latter readily interacts with alkali. In the presence of alkaline catalysts, liquefaction of lignocellulosic biomass mainly leads to oil-like products (Meszaros et al., 2004; Knill and Kennedy, 2003). The conversion and yield of liquid products decreases in the following order: K2CO3 > KOH > Na2CO3 > NaOH (Karagoz

et al., 2006; Akhtar et al., 2010).

Typically, the equipment corrosion by caustic hy­droxides is severely enhanced under subcritical and

supercritical water conditions. Therefore, in this aspect, alkali and alkaline earth carbonate salts are thought to be optional catalysts. Karagbz et al. found that the al­kali and alkaline salts enhanced bio-oil formation from wood hydrothermal processing and the catalytic activity of these catalysts shown a sequence of K2CO3 > KOH > Na2CO3 > NaOH > RbOH > CsCO3 > RbCO3 > CsOH based on heavy oil yield (Karaghz et al., 2004b, 2005a, 2005c). Jena et al. investigated the thermochemical liquefaction of the microalga Spirulina platensis over an alkali metal salt catalyst (Na2CO3), an alkaline earth metal salt (Ca3(PO4)2), and a transition metal oxide (NiO) and without a catalyst (Jena et al.,

2012) . Results showed that Na2CO3 was found to in­crease biocrude oil yield, resulting in 51.6% biocrude oil, which was ~29.2% higher than that under noncata­lytic conditions and ~71% and ~50% higher than those when NiO and Ca3(PO4)2 were used as catalysts, respectively.

Hydrothermal processing of biomass can also be car­ried out over halide catalysts. Lewis acid catalysts could exhibit good catalytic properties in hydrothermal lique­faction of lignocellulosic biomass while catalytic hydro­lysis is frequently conducted in the presence of Bronsted acid catalysts. Transition metal chlorides such as CrCl3, FeCl3, CuCl2 and АЮ3 (Zhang and Zhao, 2010; Li et al., 2009), including a pair of these metal chlorides (for example CuCl2 and CrCL) (Su et al., 2009), exhibited high catalytic activity.

In addition sulfates can also be used as catalysts for the catalytic liquefaction of lignocellulosic biomass. Kong et al. revealed, for example, that lactic acid can be produced from the catalytic hydrothermal liquefac­tion of lignocellulosic biomass in the presence of different transition metal ions like ZnSO4, NiSO4, CoSO4 or Cr2(SO4)3 (Kong et al., 2008).

Recently, natural minerals are used as catalysts in the hydrothermal liquefaction of biomass. Tekin et al. reported the effects of a natural calcium borate min­eral, colemanite, on the hydrothermal liquefaction of beech wood biomass (Tekin et al., 2012). The highest light bio-oil yield (11.1 wt%) and the highest heavy bio-oil yield (29.8 wt%) were obtained at 573K over colemanite catalysts. The total bio-oil yields were about 22 and 41 wt% at 573K without and with cole — manite, respectively.

CONCLUSION

Catalytically thermochemical technologies allowed the possibilities to convert biomass into fuels and chem­icals. The parameters such as temperature, pressure, feedstock, catalysts, and medium have been extensively studied. In the process of catalytic hydrothermal gasification, catalysts can be naturally occurring min­erals (dolomite and olivine); alkali metal catalysts; Ni, Fe, Co, and Cu-based catalysts and supported noble metal catalysts (Rh, Pd, Pt and Ru). Biomass gasification has been profiled as being CO2-neutral, having a poten­tial to produce hydrogen and syngas.

For pyrolysis, charcoal, gas and liquid are always pro­duced simultaneously. However, by adjusting process parameters (high heating rates and very high heat trans­fer rates, controlled pyrolysis reaction temperature at around 773K, short hot vapor residence time, rapid removal of product char and cooling of the pyrolysis va­pors), maximizing bio-oil yield could be achieved. Fast pyrolysis has now achieved a nearly commercial success and is being actively developed for producing liquid fuels. Catalytic pyrolysis of biomass could increase the content of the target compounds in the mixture prod­ucts. Besides, the catalytic pyrolysis of lignocellulosic biomass over zeolites, along with integrated hydropro­cesses, offer a new potential way to produce hydrocra — bon fuels from biomass.

Catalytically hydrothermal liquefaction of lignocel — lulosic biomass produces a very complex mixture of liquid products (typically consists of glycoaldehyde dimers, 1,3-dihydroxyacetone dimers, anhydroglucose, soluble polyols, 5-HMF, furfural, organic acids, phen­olic compounds and even hydrocarbons). Therefore, the novel technology for separation and extraction of downstream products from hydrothermal liquefaction of lignocellulosic biomass need to be developed (Miller et al., 1999).

Acknowledgments

The authors wish to acknowledge the financial support from the National Natural Scientific Foundation of China (21373185), the Distinguished Young Scholar Grants from the Natural Scientific Foundation of Zhejiang Province (ZJNSF, R4100436), ZJNSF (LQ12B03004), Zhejiang "151 Talents Project", and the projects (2010C14013 and 2009R50020-12) from Science and Technology Department of Zhejiang Provincial Government and the financial support by the open fund from breeding base of state key laboratory of green chemistry and synthesis technology.

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).