Cellulose, hemicellulose and lignin are the main com­ponents found in lignocellulosic raw materials and the corresponding composition is dependent on the biomass resource. Production of sugar-rich hydrolysates from lignocellulosic biomass requires treatment with com­bined thermochemical treatment and enzymatic hydro­lysis. Previous studies on utilization of lignocellulosic resources have focused on hydrolysis of cellulose and hemicellulose fractions to simple sugars for microbial fermentation mainly aiming to bioethanol production. Nonetheless, given the interest arising in biopolymers production, bioethanol production could be combined with PHA production. Cellulose could be utilized for the production of bioethanol, while sugars from hemi — cellulose could be utilized for the production of PHAs. In this way, conventional processes employed for the production of bioethanol from lignocellulosic biomass could be upgraded into advanced biorefinery concepts.

Silva et al. (2004) screened 55 strains as potential PHB producers from xylose and identified Burkholderia sac — chari IPT 101 and Burkholderia cepacia IPT 048 that were subsequently evaluated via cultivations on xylose and bagasse hydrolysates. Intracellular PHB content reached 62% and 53% for the two strains, respectively, when grown on bagasse hydrolysates. Keenan et al. (2006) utilized detoxified hemicellulose hydrolysates from lignocellulosic resources for the production of P(3HB — co-3HV) with B. cepacia through supplementation with levulinic acid (0.25—0.5%) to achieve a P(3HB-co-3HV) concentration of 2 g/l, a P(3HB-co-3HV) content of 40% (w/w) and 3HV composition of 16—52 mol%. When xylose and levulinic acid were used in microbial biocon­versions with B. cepacia, the P(3HB-co-3HV) concentra­tion and 3HV composition achieved were up to 4.2 g/l and 61 mol%, respectively. Sugarcane bagasse hydroly­sates were evaluated for PHA synthesis via fermentation of R. eutropha (Yu and Stahl, 2008). The effect of inoc­ulum concentration, dilution of hydrolysate and imple­mentation of an adapted strain was studied regarding PHA accumulation, which reached up to 57% (w/w) polymer content. PHB was the major polymer accumu­lated, whereas copolymers could be also produced that presented high ductility.

PHA production could be incorporated in existing bioethanol production facilities from both sugar cane in Brazil and cereals, such as wheat and corn, in other countries worldwide. Sugar cane utilization for bio­ethanol production generates significant quantities of bagasse, a lignocellulosic raw material that could be used for combined production of ethanol from cellulose and PHAs from hemicellulose sugars (mainly xylose). Integration of PHA production in existing cereal-based facilities used for bioethanol production could be achieved by incorporating straw utilization as raw mate­rial for combined production of bioethanol and PHAs. Such integrated biorefinery concepts could improve the sustainability of first-generation bioethanol produc­tion plants.

Different combinations of the first three bioconversion steps have been investigated in order to reduce production costs, increase end-product yield and reduce time required for bioconversion. Sequential hydrolysis and fermentation provides the opportunity of optimizing each process separately, although it can result in the use of large amounts of enzymes such as b-glucosidase to overcome end-product inhibition during the hydrolysis making this a costly process (Blanch, 2012; Dashtban et al., 2009). Simultaneous saccharification and fermenta­tion (SSF) combines both steps into one reaction, which in theory allows direct fermentation of hydrolysates into bioethanol with a reduction in enzyme costs. However, involved both reactions and end-product yields can be compromised in SSF (Dashtban et al., 2009; Ong, 2004). Another method termed consolidated bioprocessing can be used to combine all three steps into one with the use of one or many microorganisms (Hasunuma et al., 2013; Matano et al., 2013; Amore and Faraco, 2012; Blanch, 2012; Hasunuma and Kondo, 2012; Girio et al., 2010; Dashtban et al., 2009). This particular process possesses the potential to reduce bioethanol production costs to competitive fuel levels. Although significant advances have been made with regard to CBP (Hansunuma et al., 2013; Hyeon et al., 2013; Matano et al., 2013; Olson et al., 2012), more research into the microbial cell fac­tories, enzymes and physicochemical and catalytic condi­tions (pH, temperature, and synergies) is required (Olson et al., 2012; Menon and Rao, 2012; Van Dyck and Pletschke, 2012).

However, key technologies are available to convert a variety of biomass into electricity, gas, or different liquid fuels (Table 2.3). These technologies use various types of feedstocks, and are produced in different ways (Farine et al., 2011).

Since the 1990s, bacteria, fungi and yeasts have been genetically engineered for the industrial production of biofuels and bioproducts. More conventionally, the improvement of microorganisms for biomass conversion has been done using classical chemical mutagenesis, a random approach followed by the screening and selec­tion of a desired trait. Nevertheless, with advancements in molecular biology and biotechnology approaches, the improvement of microorganisms via rational engi­neering of proteins and metabolic engineering of path­ways has become more prevalent (Strohl, 2001). This is due to the economic needs of the industry, which demands the development of strains that produce greater yields and a different variety of products. Specifically, in the bioconversion of biomass, researchers face challenges related to the substrate such as appropriate enzymes for conversion and microorganisms that produce them, fermentation of nonglucose sugars (i. e. xylose), and "consolidated bioprocessing", where the production of enzymes for biomass conversion (i. e. cellulose produc­tion), hydrolysis or modification of the biomass (i. e. cellulose hydrolysis), and fermentation of solubilized carbohydrates occur in a single step (Lynd et al., 1999). Therefore, prior to engineering microorganisms for biomass conversion it is important to select host organ­isms with desired characteristics; with emphasis on strains that can utilize low-cost substrates, have high product yield, competitive fitness, and are more robust to environmental stresses (Lynd et al., 1999). Once a good host has been selected based on targeted physiolog­ical characteristics and functionalities, one can identify the additionally desirable characteristic that will then be engineered into the host, whether targeting proteins such as enzymes through rational engineering or chang­ing the metabolism and/or metabolic flux through meta­bolic engineering (Zhang et al., 2009).

MET THROUGH EXOGENOUS REDOX MEDIATORS

Some microbes such as Escherichia coli, Pseudomonas sp., Proteus and Bacillus (Lovley, 2006a) cannot directly trans­fer electrons to the anode and must rely on mediators (Lovley, 2006a). When the oxidized mediators reach the surface of the microbes, they penetrate the cell mem­brane of the microbes, and they are reduced by electrons. The reduced mediators pass through the cell membrane again and reach the anode surface where then they are reoxidized (losing the electrons). In this fashion, electrons are transferred to the anode while the oxidized media­tors enter the microbes again, thereby continuing the redox cycle (Figure 9.3(a)) (Neto et al., 2010; Rabaey et al., 2005b).

Properties of good exogenous mediators should be the ability to (a) cross cell membranes with ease; (b) receive electrons from electron donors without interfering with other metabolic processes; (c) deliver electrons inside the cytoplasm for oxidation reactions and regenerate at rapid rates; (d) have good solubility and stability in both oxidized and reduced forms; (e) have no cytotoxicity; and (f) not be consumed by microbes in the biofilm as a nutrient (Bao and Wu,

2004) . These mediators include thionine, neutral red,

2- hydroxy-1,4-naphthoquinone, phenazines, quinines,

Fe(III) ethylenediaminetetraacetic acid, methylene blue, phenothiazines, phenoxazines and others (Choi et al., 2003b; Lovley, 2006a; McKinlay and Zeikus, 2004; Newman and Kolter, 2000; Osman et al., 2010; Park and Zeikus, 2000). However, these mediators are unsuitable for practical applications because they are costly and most of them are toxic and recalcitrant, harmful to the environment (Erable et al., 2010a; Lovley, 2006a).

MET THROUGH THE SECONDARY METABOLITES

Researchers have found that some microbes can trans­fer electrons without DET in the absence of exogenous redox mediators. These microbes such as S. putrefaciens, S. oneidensis, G. sulfurreducens, Pseudomonas aeruginosa, and Clostridium butyricum can produce their own media­tors (Angenent et al., 2004; Erable et al., 2010a; Fitzgerald et al., 2012; Newman and Kolter, 2000; Rabaey et al., 2005a). The presence of these microbes in the mixed cultures enhances electron transfer. These mediators mainly include phenazine derivatives like pyocyanine and 2-amino-3-carboxy-1,4-naphthoquinone (Osman et al., 2010).

In practical applications, the secondary metabolites (endogenous redox mediators) may be very important to MFCs because they can transfer the electron without the exogenous redox mediators (Schroder, 2007). The mechanism of electron transfer by the secondary metab­olites is similar to that of the exogenous electrochemical redox mediators (Figure 9.3(a)). The secondary metabo­lites can be reused, and one metabolite molecule can transfer thousands of electrons (Schroder, 2007). So a small amount of the secondary metabolites can single — handedly enhance the rate of electron transfer and thus increasing power density and improve the MFC performance without introducing costly exogenous mediators.

In batch-mode operations, these microbes are very suitable because the mediators will accumulate in the anodic chamber, thus improving the MFC performance (Osman et al., 2010). However, in continuous flow MFCs for wastewater treatment, the secondary metabo­lites can be insufficient due to diluted concentrations as a result of flow (Lee et al., 2003; Rabaey et al., 2005c), thus resulting in the decline of the performance after the flow starts (Lovley, 2006a; Osman et al., 2010).

MET THROUGH PRIMARY METABOLITES

The other endogenous redox mediators are primary metabolites. Some microbes can produce fermentation products such as hydrogen (H2), hydrogen sulfide (H2S), alcohols and ammonia (Erable et al., 2010a). When these primary metabolites reach the surface of the anode, they are oxidized, and the released electrons will be further transferred to the anode surface.

There are two types of anaerobic metabolism that can produce primary redox metabolites: one is anaerobic respiration, and the other is fermentation. Some microbes such as Proteus vulgaris, E. coli, P. aeruginosa and Desul — fovibrio desulfuricans can produce sulfide which may serve as the mediator to transfer electrons (Bullen et al., 2006; Schroder, 2007):

Cytoplasm : SO|~ + 9H+ + 8e~ / HS~ + 4H2O (9.3) Anode : HS~ + 4H2O/SO4~ + 9H+ + 8e~ (9.4)

This process relies on sulfate reducing bacteria (SRB) that cannot metabolize carbohydrates. A fermentation process can produce small organic acids and alcohols that can be used in anaerobic respiration (Schroder,

2007) . Many SRB degrade the substrates incompletely and this lowers the MFC power output. Electrode poisoning by sulfide due to its easy absorption on the electrode surface is also a major drawback (Reimers et al., 2006; Ryckelynck et al., 2005).

Fermentation also produces primary metabolites such as hydrogen, ethanol and formate. They can be oxidized directly by electrolysis on an anode such as platinum or tungsten carbide (Rosenbaum et al., 2006). For example, through electrocatalysis, the molecular hydrogen near and on an anode surface would be oxidized to H+, accompanying the electron transfer (Figure 9.3(b)). Molecular hydrogen is known to be used as an electron carrier used by hydrogenase — positive microbes such as some SRB in microbiologically influenced corrosion (Gu, 2012). Thus, it contributes to power generation.

Waste edible oil (WEO) is the waste product of cook­ing or frying foods. The disposal of WEO is difficult and thus the use of WEO as a biofuel would both alleviate the problem of disposal in addition to providing a renewable source of biodiesel. WEO has a high volume of FFAs, 0.5—15% in comparison with the 0.5% content of refined virgin vegetable oil, which cannot be con­verted to biodiesel using an alkaline catalyst as the FFAs undergo a saponification reaction with the catalyst thus reducing efficiency and yield (Knothe et al., 2005). The problem may be overcome by using a supercritical methanol transesterification for the transesterification process rather than an alkaline catalyst (Kusdiana and Saka, 2004). The volume of WEOs available is quite high with approximately 1 million tons produced in Europe each year while 10 million tons are produced annually in the United States (Gui et al., 2008). WEO is available two to three times cheaper than virgin vegetable oils (Phan and Phan, 2008) and the high volume of WEO available means it a viable method for biodiesel production. WEO has a higher estimated energy balance than rapeseed and soybean of 5.8; however, the value is lower than that of palm oil at 9.5 (Food and Agriculture Organization, 2008).

Pyrolysis of biomass is thermal depolymerization and decomposition of biomass (TDP) in the absence of air/ oxygen. The temperature generally used is in the range of 623—973K (Goyal et al., 2008). The products, charcoal or biochar, gaseous and liquid chemicals, depend on the biomass composition, the heating rate and the tempera­ture. According to the heating rate, pyrolysis is classified as slow pyrolysis, fast pyrolysis and flash pyrolysis.

Slow pyrolysis of biomass is conducted at slow heating rates (0.1—1 °C/s). In the relatively low temperatures of 573—673K, charcoal is the main product; when the tem­perature is are increased to >673K, the oil yield is increased (Putun et al., 2001; Ozbay et al., 2001; Onay and Kockar, 2004). By contrast, fast pyrolysis is conducted at higher heating rates (about 10—200 ° C/s) and intended to produce liquid bio-oil (Bridgwater, 2003). Flash pyroly­sis is conducted at heating rates >1000 °C/s within
reaction time of only several seconds or even less (Demi — rbas and Arin, 2002). Among these, fast pyrolysis is of most commercial interest for production of chemicals and liquid fuels (Zhou et al., 2011).

Fast pyrolysis is mainly intended to maximize the bio­oil yield as well as to increase the contents of the target compounds in it. To this end, there are needs to use a finely ground particle biomass feed of typically less than 3 mm, selective catalysts, a well-controlled pyrolysis temperature of around 773K, short hot vapor residence time of typically less than 2 s and rapid removal and cool­ing of the products (Bridgwater, 2012).

Aromatic compounds are important building blocks for many chemicals and polymers as well as components of fuel compositions. Furans, with their dienic structure, can replace aromatic compounds in several applications including polymers (e. g. Poly Ethylene Terephthalate by Poly Ethylene Furanoate), fuels (diesel) and pharmaceu­ticals (de Jong et al., 2012a; de Jong et al., 2013; Van Putten
et al., 2013a). In this paragraph we will discuss the forma­tion of furans from carbohydrates and the formation of aromatic compounds from lignin as an example how all major components of lignocellulosic biomass can be valorized by chemocatalytic routes. Some of the most important chemical transformations of carbohydrates are arguably the hydrolysis and subsequent dehydration of polysaccharides into the furan platform products, furfural and HMF (Dias et al., 2010; Van Putten et al., 2013a, b). Furfural has a wide industrial application pro­file and is considered as one of the top 30 building blocks that can be produced from biomass (Dias et al., 2010; Van Putten et al., 2013b; Lange et al., 2012; Bozell and Petersen, 2010; Zeitsch, 2000a; Hoydonckx et al., 2007). HMF is promising as a versatile, renewable furan chem­ical for the production of chemicals, polymers and bio­fuels, similar to furfural (Van Putten et al., 2013a; Bozell and Petersen, 2010). While furfural has been produced on an industrial scale for decades (Dias et al., 2010; Van Putten et al., 2013b), the production of HMF has not yet reached industrial scale (Van Putten et al., 2013a; Bozell and Petersen, 2010).

PGA can be produced only through the microbial route, unlike alpha poly glutamic acid, which can be chemically synthesized. Among PGA producers, a broad classification was established in the form of glutamic acid-dependent PGA producers and glutamic acid — independent PGA producers, depending on the manda­tory requirement or nonrequirement for glutamic acid as a major component in the nutrient medium. Examples of glutamic acid-dependent producers of PGA include B. subtilis IFO 3335, B. subtilis NX-2, and Bacillus licheniformis ATCC 9945A and examples of glutamic acid-independent PGA producers include B. subtilis TAM-4 and B. licheni — formis SAB-26. Initial studies for the production of PGA was carried out using the strain B. licheniformis ATCC 9945A. The production medium formulated for PGA production contains a relatively high concentration (20—1230 mM) of Mn+2 and the more it was, the amount of D-glutamic acid present as well (Leonard et al., 1958a, b). Bacillus subtilis IFO 3335 was originally isolated from natto, a traditional fermented food in Japan, which has mucilage containing PGA and a levan. PGA produc­tivity of this strain was higher than that of B. licheniformis ATCC 9945A. Bacillus subtilis IFO 3335 could produce PGA at levels of 9.6 g/l, with an optimized medium with major components including 30 g/l of L-glutamic acid, 20 g/l of citrate, and 5 g/l ammonium sulfate. It was found that L-glutamic acid merely stimulated the production of PGA, which could initiate PGA produc­tions at lower concentrations (0.1 g/l), without the addi­tion of 30 g/l of L-glutamic acid (Kunioka and Goto, 1994; Kunioka, 1995). Cheng et al. (1989) isolated B. lichenifor­mis A35 while looking for amino acid producer under denitrifying conditions. A strain with extremely high production rates of PGA was isolated from fermented bean curd (Shi et al., 2006). The strain was found to be B. subtilis ZJU7. In an optimized culture medium contain­ing 60 g/l sucrose, 60 g/l tryptone and 80 g/l L-glutamic acid and after cultivated at 37 °C for 24 h, the yield of g-PGA reached 54.4 g/l. In an interesting study, a cocul­ture of C. glutamicum S9114 along with B. subtilis ZJU7 was attempted to reduce the input costs of addition of L-glutamic acid into the medium, and it yielded 32.8 g/l of PGA after 24 h of fermentation (Shi et al., 2007).

Various studies regarding the fermentative production, downstream processing and characterization of PGA have been reported in the literature. The review by Bajaj and Singal (2011) provides updated information on fermentative production of PGA by various bacterial strains and effect of fermentation conditions and media component on production of PGA in submerged as well as solid state fermentation.

PGA can be extracted from the fermentation medium by two different methods. A crude method involves centrifuging cells followed by precipitation of PGA by methanol or ethanol (chilled) (Goto and Kunioka,

1992) . Subsequent purification steps, involving gel permeation chromatography followed by reprecipita­tion have to be used to get PGA in a pure form. Another method exploiting the specific interaction of Cu+2 ions with PGA, gives relatively purer PGA (Troy, 1973). Further, the purity of the polymer is checked through peptide hydrolysis, followed by thin layer chromatog­raphy, to assess the constituent amino acids. Large amounts of PGA are produced microbially by the Japanese company Meiji Seika Kaisha Ltd employing B. subtilis strain F-021.

The major argument for using cyanobacteria or eukaryote microalgae for biofuel production is the pos­sibility to directly couple photosynthesis with product formation. This strategy could have sustainable and economic advantages. The financial appeal is related to the production chain, with CO2 fixation directly produc­ing the desired fuel in a single organism. Thus, the bio­fuel is recovered at the production site, avoiding as a consequence the processing of photosynthetically pro­duced sugars in a second-stage microbial fermentation. This process also has great ecological and sustainability appeal since atmospheric CO2 is being recycled into fuels without using the conventional agriculture system, leaving arable land available for food crops. Neverthe­less, the inherently low value and high demand charac­teristics of fuels present a challenge for the development of biofuel production. The volume of fuel required to fulfill the needs of the transportation sector is massive, in contrast to their low market value, which must be at least as cheap as bottled water. The achievement of this goal requires the solution of major challenges in civil and mechanical engineering, chemistry, and biology.

In the biological arena, the main challenge is strain development. The ideal cyanobacterium for biofuel pro­duction would have a high quantum efficiency of photo­synthesis and well-defined carbon partitioning, where the CO2 fixed would be primarily directed to "house­keeping" metabolism and the targeted product. To achieve this goal, two main venues are being followed: high-throughput bioprospecting, which seeks naturally occurring species, enzymes and pathways adaptable for cultivation and economic exploitation, or the use of genetic engineering, where a model organism is geneti­cally modified to introduce and/or to enhance the pro­duction of a desired molecule. In this section the available tools are discussed as well as some paths toward the improvement of photosynthetic quantum efficiency.

Starch materials are also potential resources for the bio­ethanol production. Starch molecules are polysaccharides made up of long chains of glucose units covalently linked. Before the fermentation process, the starchy materials are broken into simple glucose molecules after which the sim­ple sugar units are easily fermented by the microbes. Examples of starchy materials used for bioethanol produc­tion include cereal grains, potato, sweet potato, beans, cas­sava, maize, wheat and cereal grains. As these materials are also too expensive and included in the human food

chain, wastes are collected from places where they are crushed into flour or from industries where they are used for various products (Kahn et al., 2011).

Table 3.2 reports the use of starchy residues in ethanol production. As starch is an important component of the food chain, it is expected that the wastes of the process­ing be more used for ethylic fermentation (Moukamnerd et al., 2010). Another important point that can be observed is the predominant use of simultaneous saccharification and fermentation (SSF) in the use of starch for energy generation and in both cases hydroly­sis was achieved by enzymes (Hong and Yoon, 2011; Moukamnerd et al., 2010). Hashem and Darwish (2010) reported the use of potato starch residue stream pro­duced during chips manufacture and the authors have used separate hydrolysis and fermentation (SHF) and a very low acid hydrolysis of the starch to reduce the cost associated with this necessary treatment.