During pyrolysis or liquefaction, biomass undergoes a series of complex conversion processes producing bio-oil. Bio-oils are highly diverse in its composition and has a spectrum of oxygenates. Due to the presence of a collective of different oxygenated compounds, an upgrading process to remove oxygen would likely involve a series of reactions. This review introduces several possibilities for upgrading bio-oil including dehydration, decarboxylation and decarbonylation. A summary of the best catalyst contenders for each reaction class is given in the Table 3.

Hydrodeoxygenation has been the most frequently studied and one of the most reliable methods that could be used for deoxygenation of oxygenates. However, the drawbacks of hydrodeoxygenation include: the need for continuous replenishment of sulfur (for sulfided catalysts), and the non-selective deoxygenation of all bio-oil chemical moieties resulting in a spectrum of short-chained and long-chained hydrocarbons that are less useful as liquid fuels. The need of hydrogen makes resulting biofuels less competitive with existing petroleum fuels. More research is needed on development of effective non-sulfided hydrodeoxygenation catalysts. Preliminary investigations with Ni-Cu on CeO2 or ZrO2 may provide new directions on this front. Further research should be directed on identifying materials and processes will allay the need for using direct hydrogen.

According to the analysis, metal promoted or unpromoted HZSM-5 proves to be one of the most versatile catalysts that is capable of catalyzing all three deoxygenation reaction classes, i. e., dehydration, decarboxylation and decarbonylation. Nevertheless, the nanopore structure of HZSM-5 (~5.4-5.5 A), while assisting size selectivity for gasoline range hydrocarbons, also promotes pore blockages resulting in rapid catalyst deactivation. Developing catalysts of the same class, with larger and consistent pore structure and/or higher activity/functionality should be closely looked at.

Hydropower contributes about 90 per cent of electricity generated in Uganda with sugarcane based bagasse bioelectricity, fossil fuel and solar energy among other sources of power. Although the current generation of 800 MW "in [18] ". has boosted industrial growth, the capacity is still lagging behind the demand that is driven by the robust growth of the economy.

The low pressure boilers of 45 bar currently generate 22 MW of which 10 MW is connected to the grid. However, Kakira sugar estate has a target of generating 50 MW of electricity with the installation of higher pressure boilers of 68 bar in 2013. This target can be surpassed given the abundance of the bagasse (Table 6 and 7).

Putting into consideration the productivity norms at Kakira estate and outgrowers (Table 8), with a potential of producing 908.9 m tons of sugarcane, Uganda has a potential of producing bio-electricity that surpasses the nation’s demand by far. Much of this electrical power can be exported to the region, greatly expanding on Uganda’s export base.

Edmilson Jose Ambrosano, Heitor Cantarella, Glaucia Maria Bovi Ambrosano, Eliana Aparecida Schammas, Fabio Luis Ferreira Dias, Fabricio Rossi,

Paulo Cesar Ocheuze Trivelin, Takashi Muraoka, Raquel Castellucci Caruso Sachs, Rozario Azcon and Juliana Rolim Salome Teramoto

Additional information is available at the end of the chapter http://dx. doi. org/10.5772/53825

1. Introduction

Healthy soils are vital to a sustainable environment. They store carbon, produce food and timber, filter water and support wildlife and the urban and rural landscapes. They also preserve records of ecological and cultural past. However, there are increasing signs that the condition of soils has been neglected and that soil loss and damage may not be recoverable [1]. Soil is a vital and largely non-renewable resource increasingly under pressure. The importance of soil protection is recognized internationally.

In order to perform its many functions, it is necessary to maintain soil condition. However, there is evidence that soil may be increasingly threatened by a range of human activities, which may degrade it. The final phase of the degradation process is land desertification when soil loses its capacity to carry out its functions. Among the threats to soil are erosion, a decline in organic matter, local and diffuse contamination, sealing, compaction, a decline in bio-diversity and salinisation.

The authors in [2] made the interesting observation when study of nine great groups of New Zealand soils, that although many soil quality indicators will be different between soil types differing in clay and organic mater contents, land use had an overriding effect on soil quality: agricultural systems could be clearly differentiated from managed and natural forests, and grass land and arable land were also clearly separated.

Regular additions of organic matter improve soil structure, enhance water and nutrient holding capacity, protect soil from erosion and compaction, and support a healthy community of soil organisms. Practices that increase organic matter include: leaving crop

residues in the field, choosing crop rotations that include high residue plants, using optimal nutrient and water management practices to grow healthy plants with large amounts of roots and residue, applying manure or compost, using low or no tillage systems, using sod — based rotations, growing perennial forage crops, mulching, and growing cover crops as green manure [3].

Addition to being a frequent addition of organic matter should be diversified. Diversity cropping systems are beneficial for several reasons. Each plant contributes a unique root structure and type of residue to the soil. A diversity of soil organisms can help control pest populations, and a diversity of cultural practices can reduce weed and disease pressures. Diversity across the landscape can be increased by using buffer strips, small fields, or contour strip cropping. Diversity over time can be increased by using long crop rotations. Changing vegetation across the landscape or over time not only increases plant diversity, but also the types of insects, microorganisms, and wildlife that live on your farm [3]. In addition a dedicated management approach is needed to maintain or increase the soil organic matter content.

The incorporation of plant materials to soils, with the objective of maintaining or improving fertility for the subsequent crop is known as green manuring. The inclusion of a legume fallow within a sugarcane cropping cycle is practiced to reduce populations of detrimental soil organisms [5, 6], provide nitrogen (N) through biological fixation [7,8] and for weed suppression [9, 10].

Interest in the use of green manure’s biomass has revived because of their role in improving soil quality and their beneficial N and non-N rotation effects [11]. Because of its nitrogen fixation potential, legumes represent an alternative for supplying nutrients, substituting or complementing mineral fertilization in cropping systems involving green manuring. This practice causes changes in soil physical, chemical and biological characteristics, bringing benefits to the subsequent crop both in small-scale cropping systems and in larger commercial areas such as those grown with sugarcane [12].

The area cropped with sugarcane (Saccharum spp.) in Brazil shows rapid expansion, with most of the increase for ethanol production. The area cultivated with sugarcane is now 9.6 Mha, with an increase of 5 Mha from 2000 and over 8.6 Mha of fresh sugarcane harvested per year [13]. Sugarcane crops in Brazil are replanted every five to ten years. In southeastern Brazil, the interval between the last sugarcane harvest and the new plantings occurs during the spring-summer season, under high temperature and heavy rainfall (almost 1,000 mm in six months) [14].

Green manure fertilization of the soil with legumes has been recommended before a sugarcane field is replanted. This practice does not imply on losing the cropping season, does not interfere with sugarcane germination, and provides increases in sugarcane and sugar yield, at least during two consecutive cuts [12]. Additionally, it protects the soil against erosion, prevents weed spreading and reduces nematode populations [15, 16]. Legumes usually accumulate large quantities of N and K, the nutrients which are taken up in the highest amounts by the sugarcane plants.

Residue incorporation studies of legumes using 15N label indicate that 10 to 34% of the legume N can be recovered in the subsequent rye or wheat crop, 42% in rice, 24% recovery from Velvet bean by corn crop, around 15% of N recovery from sunn hemp by corn plants in no-till system, 30% by maize [51],and 5% of N recovery from sunn hemp by sugarcane [12], and ranged from 19 to 21% when the recovery was observed from sunn hemp by two sugarcane harvest [17].

The authors suggested that legume residue decomposition provided long-term supply of N for the subsequent crops, by not supplying the nutrient as an immediate source.

There are many benefits to the sugarcane crop of leguminous plants rown in rotation in sugarcane renovation areas; these include the recycling of nutrients taken up from deep soil layers by the rotational crop, which may prevent or decrease leaching losses, and the addition of N from biological fixation. Leguminous plants can accumulate over 5 t ha-1 of dry mass during a short period of time during the summer and take up large amounts of N and K. Most of this N comes from the association of legumes with rhizobia. In this way crop rotation with legumes can replace partially or totally N mineral fertilization of sugarcane, at least for the first ratoon [18, 12].

Another important microbial association is that of mycorrhizal fungi and plant roots. These fungi are present in over 80% of plant species [19]. In contrast with the large diversity of plants, which includes sugarcane, that have their roots colonized by mycorrhizas, only 150 fungi species are responsible for that colonization [19]. A crop whose roots are colonized by mycorrhizal fungi can raise the soil mycorrhizal potential which can benefit plants which are responsive to this fungi association and that are cultivated in sequence. This could be particularly useful for the nutritional management of crops in low nutrient, low input — output systems of production [20].

This study evaluated biomass production, N content, characterizing the biomass and the natural colonization of arbuscular mycorrhizal fungi (AMF) of leguminous green manure and sunflower (Helianthus annuus L.) in rotation with sugarcane and their effect in the soil. Their effect on stalk and sugar yield and nematode that occur on sugarcane cv. IAC 87-3396 grown subsequently was also studied. The economic balance considered the costs of production and revenues of the rotational crops as well as three or five harvests of sugarcane was too evaluated.

1.2. Species and biomass yields

In NGP, species evaluated for biofuel crops include switchgrass, big bluestem, Indian grass, tall wheatgrass, intermediate wheatgrass, wild rye, alfalfa and sweet clover [11, 26-33]. Switchgrass still remains in most of the studies in NGP. In South Dakota, switchgrass has been evaluated under both conventional farmland and CRP land, and the biomass yield ranged from 2 to 11 Mg/ha [28-30]. In North Dakota, cultivars of switchgrass have been tested in western and central areas in small research plots (Dickinson and Mandan) and biomass yield ranged between 2 to 13 Mg/ha, depending on cultivar [26-27]. In another site (Upham), biomass yield of switchgrass ranged from 2.4 to 10.8 Mg/ha [32]. In an on-farm scale trial, switchgrass yield ranged from 4.6 to 9.9 Mg/ha in Streeter and Munich [8, 34].

For selecting species for biofuel crops, switchgrass still has more advantages than any other species. This is because: (1) the species has been studied extensively in the US in last two decades and the germplasm pool is larger than other species; (2) it is a warm season species and has greater water use efficiency and drought resistance; (3) it is native to North America and there are no concerns about the invasiveness; (4) there are many environmental benefits for growing switchgrass.

In addition to species, environmental factors (e. g., precipitation, temperature, soil type etc.) have large effects on yield and quality in biofuel crops. To address the interactions of species and environment, a ten-year long-term study was initiated and established in 2006 to evaluate ten cool and warm season grasses and mixtures across North Dakota [11]. The 10 entries of species and mixtures were shown in Tables 3. These grasses/mixtures were grown in six environments in five locations across North Dakota. Among the five locations, long term growing season precipitation varies from 318 mm at Williston in the west to 431 mm at Carrington in the east. In general, western ND has a semi-arid environment but eastern ND is more humid [11, 35].

Initial biomass yield data indicated Basin and Altai wildrye showed lower biomass yields than either switchgrass or wheatgrass species (Table 4). Tall wheatgrass and intermediate wheatgrass performed well across environments in North Dakota. In contrast, performance of switchgrass was largely related to environment, particularly the seasonal precipitation. For dryland conditions, studies are still needed to address both establishment and persistence of switchgrass in the future.

The inclusion of a hauling contractor in the business plan provides the best opportunity for all of the technology developed for other logistics systems to be applied to feedstock logistics. The information technologies applied include a GPS receiver in every truck and a bar code on every load as well as geographic mapping and routing management tools. Thus, time and location information can be observed at every SSL and the weigh-in scale at the receiving facility. The data collected can be used to optimize asset utilization in real time, and it will also feed needed data into the accounting software to compensate the farmgate and haul contracts.

It is expected that the collected data will be presented in real time. The information will provide a "Feedstock Manager," maps showing the location of all assets with updates at suitable time intervals. The goal is to provide an opportunity for the Feedstock Manager to make optimization decisions in real time. Examples are: trucks rerouted to avoid traffic delays, assets redeployed during breakdowns, increase at-plant inventory when inclement weather is forecasted, and a turn-down of plant consumption when a delay in feedstock deliveries cannot be avoided.

Some perspective of the logistics complexity, as presented to the Feedstock Manager, can be gained from the following sample parameters. This example presumes that operations will be in the Upper Southeast of the United States where switchgrass is harvested over an 8- month harvest season. Suppose the supply area has 199 SSLs within a 48-km radius of the plant, and each SSL has a different amount of material stored as shown in Figure 6. The producer desires to fill their SSLs at least twice during the harvest season to minimize per — Mg SSL investment. Suppose there are five SSL crews under contract and each of them wants the same opportunity to earn income (total Mg mass hauled per year). The Feedstock Manager’s job is to treat all farmgate and haul contractors fairly.

The huge superficial area and rich porosity of the activated carbon provide a preferable habitat for microbe and prompt the formation of biological membrane. According to the research, the synergy effects of activated carbon adsorption and biodegradation enable O3- BAC to realize the removal of organics in water (see 3.4 for details). Meanwhile, as the duration of BAC use gets longer, the action of organisms plays a more and more predominant role[50-51]. Quite a few researchers point out that the range of bacteria using the activated carbon as a carrier to habitat includes aerobic, anaerobic and facultative bacteria[52-53]. In this chapter the variation of bacteria communities and the characteristic of the community structure will be discussed, and the system chosen here is down-flow O3- BAC system shown in Fig. 14.

Figure 14. Pilot test setup

Maria Gomez-Brandon, Marina Fernandez-Delgado Juarez, Jorge Dominguez and Heribert Insam

Additional information is available at the end of the chapter http://dx. doi. org/10.5772/53454

1. Introduction

Many environmental problems of current concern are due to the high production and local accumulations of organic wastes that are too great for the basic degradation processes inherent in nature. With adequate application rates, animal manure constitutes a valuable resource as a soil fertilizer, as it provides a high content of macro — and micronutrients for crop growth and represents a low-cost, environmentally — friendly alternative to mineral fertilizers [1]. However, the intensification of animal husbandry has resulted in an increase in the production of manure — over 1500 million tonnes are produced yearly in the EU-27 [2] as reported by Holm-Nielsen et al. [3]- that need to be efficiently recycled due to the environmental problems associated with their indiscriminate and untimely application to agricultural fields. The potentially adverse effects of such indiscriminate applications include an excessive input of harmful trace metals, inorganic salts and pathogens; increased nutrient loss, mainly nitrogen and phosphorus, from soils through leaching, erosion and runoff-caused by a lack of consideration of the nutrient requirements of crops; and the gaseous emissions of odours, hydrogen sulphide, ammonia and other toxic gases [4]. In fact, the agricultural contribution to total greenhouse gas emissions is around 10%, with livestock playing a key role through methane emission from enteric fermentation and through manure production. More specifically, around 65% of anthropogenic N2O and 64% of anthropogenic NH3 emissions come from the worldwide animal production sector [5].

The introduction of appropriate management technologies could thus mitigate the health and environmental risks associated with the overproduction of organic wastes derived from the livestock industry by stabilizing them before their use or disposal. Stabilisation involves the decomposition of an organic material to the extent of eliminating the hazards and is normally reflected by decreases in microbial biomass and its activity and in concentrations

© 2013 Gomez-Brandon et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of labile compounds [6]. Composting and vermicomposting have become two of the best — known environmentally appropriate technologies for the recycling of manures under aerobic conditions [6-7], by transforming them into safer and more stabilised products (compost and vermicompost) with benefits for both agriculture and the environment. Unlike composting, vermicomposting depends on the joint action between earthworms and microorganisms and does not involve a thermophilic phase [8]. However, more than a century had to pass before vermicomposting was truly considered a field of scientific knowledge or even a real technology, despite Darwin [9] having already highlighted the important role of earthworms in the decomposition of dead plants and the release of nutrients from them.

Although microbial degradation under oxygen is usually faster and, as such aerobic processes are thermodynamically more favorable than anaerobic processes, in recent years, anaerobic digestion (AD) has become an upcoming technology for the treatment of animal manures [3, 10-13]. On the one hand, pretreatment of manure by anaerobic digestion can involve some advantages including malodor reduction, decreased biochemical oxygen demand, pathogen control, along with a reduction in the net global warming potential of the manure [4,14]. AD reduces the risk of water pollution associated with animal manure slurries (i. e., eutrophication) by removing 0.80-0.90 of soluble chemical oxygen demand and it improves human/farm cohabitation in rural regions by reducing odor emissions by 70­95% [4]. This process has other direct advantages beyond these, which are related to biogas production for renewable energy and the enrichment of mineral fractions of N and P during digestion [4,10], resulting in a more balanced nutrient mix and increased nutrient bioavalability for plants compared with undigested manure [15].

Therefore, the purpose of this chapter is to give an overview of the three major management technologies of manure recycling, including the aerobic processes of composting and vermicomposting and the anaerobic digestion for biogas production. The main changes that occur in the substrate from a chemical and microbial viewpoint during the specific phases of each degradation process are addressed, as such changes determine the degree of stability of the end product and in turn its safe use as an organic amendment. Different methods that have been proposed to evaluate compost stability are summarised. Also, the influence of the end products derived from each process on the soil microbiota and disease suppressiveness are discussed.

The composition and biomass of phytoplankton are very important parameters for understanding the structure and tropic level of aquatic systems. Phytoplankton cell size, carbon content and functional structure are investigated by many researchers. Phytoplankton communities can have cell size from a few microns to a few milimeters depending on the groups they belong to. Biovolume measurements are estimated by

automatic or semi-automatic methods. For example, morphometric methods, holographic method etc. In addition to these, the commonly used one is geometric method [12]. In addition to these methods, the most common method for calculationg biomass is measuring chlorophyl a value. Chlorophyll a is an important photosynthetic pigment for plant organisms. Environmental factors affect the amount of ambient phytoplankton and chlorophyll a value changes depending on the amount of phytoplankton [13].

Coulter Counter method is based on measuring electrical conductivity among cells. Electric current flowing through the cells placed in physiological saline varies depending on the cell size. Thus, the cell size is determined.

Morphometric methods is used in determinining the quantitative properties of cells. With this method, cell density is calculated depending on cell wall, chloroplast, vacuole, depot material and cytoplasmic content [14].

Holographic scanning tecnology which is used in conjunction with one curved mirror to passively correct focal plane position errors and spot size changes caused by the wavelength instability of laser diodies [15].

Geometric method is based on estimating biomass of phytoplankton via geometric shapes and mathematical equations. This model was found by Kovada and Larrance. 20 geometric models developed by Hillebrand et al. [16] are used for calculating biomass of algae. Each model was designed depending on cell structure with the shapes like sphere, cone, triangle etc. This method was applied to phytoplankton species found in sea waters in China and 31 geometric models were developed in this study [12].

Phytoplankton communities show vertical changes from time to time. Chlorophyll a is a pigment used for estimating biomass of phytoplankton. Seasonal changes cause variation in chlorophyll a value. For this reason, water affects the production of column light transmittance, hence, the value of chlorophyll a [18]. In determining chlorophyll a, fiber glass filter papers used for filtering water samples are waited for 3-4 hours then they are decomposed and kept in 10 ml 90% acetone one night, centrifuged and optical density of the extract is made by reading from spectrophotometer with 630, 645 and 665 nm wavelength [17].

The first step for calculating phytoplankton biomass is to store and protect the phytoplankton samples.

Recently, the structures and mechanisms for assembly of multienzyme complexes, cellulosomes, in anaerobic cellulolytic microorganisms are clear (Bayer et al., 2004, 2007; Doi & Kosugi, 2004). Generally, the key feature of the cellulosome is a scaffoldin that integrates the various catalytic subunits into the complex by self-assembly by cohesion-dockerin interaction. However, the structure and mechanism of the multienzyme complex produced by a facultatively anaerobic bacterium, such as P. curdlanolyticus B-6 is still unknown. In order to describe features of the multienzyme complex system produced by strain B-6, the multienzyme complex was purified by four kinds of chromatography (cellulose affinity, gel filtration, anion-exchange and hydrophobic-interaction chromatographys) (Fig. 7).

The multienzyme complex of P. curdlanolyticus strain B-6 with molecular mass of 1,450 (G1) was isolated from culture supernatant at the late stationary growth phase through cellulose affinity and Sephacryl S-300 gel filtration chromatographys (Pason et al., 2006b). Basically, the individual cellulosomes from anaerobic bacteria show 600 kDa to 2.1 MDa complexes size and show cohesion-dockerin domain as a signature protein (Bayer et al., 2004; Doi & Kosugi, 2004). While, multienzyme complexes from aerobic microorganisms, were range in mass from about 468 kDa to 2 MDa (with contained 5-12 protein subunits) (Table 3) and has no report of cohesion-dockerin domain. Here, the multienzyme complex produced by strain B-6 under aerobic conditions was the first report on characterization.

Elucidation of the purified multienzyme feature of P. curdlanolyticus strain B-6 was followed by anion-exchange and hydrophobic-interaction chromatographys (Pason et al., 2010). The complex G1 from gel filtration chromatography (1,450 kDa) was purified by anion-exchange chromatography and showed at least five large protein complexes or aggregates, namely F1-F5. Among the fractions obtained from anion-exchange chromatography, F1 was apparently the most suited fraction to study on the organization and function of the multienzyme system of strain B-6 because F1 formed one clear band on the top of native PAGE, had the highest xylanase activity, and its subunit composition was clearly shown on SDS-PAGE. In the final step, complex F1 was separated to one major complex (H1) and two minor protein components (H2 and H3) by hydrophobic — interaction chromatography. The multienzyme complex (H1) was composed of a 280 kDa protein with xylanase activity, a 260 kDa protein that is a truncated form on the C — terminal side of the 280 kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and CMCase activities (Fig. 8). The two components (280 and 40 kDa) of the multienzyme complex has characteristics similar to the cellulosome of C. thermocellum in that it is composed of a scaffolding protein and a catalytic subunit (Bayer et al., 1998; Demain et al., 2005). The 280 kDa protein resembled the scaffolding proteins of the multienzyme complex based on its migratory behavior in polyacrylamide gels and as a glycoprotein. The 280 kDa protein and a 40 kDa major xylanase subunit are the key components of multienzyme complex of the strain B-

S1 protein: From the early research, the 280 kDa subunit (S1) plays a role of scaffoldin in assembling the enzyme complex and shows xylanase activity (Pason et al., 2010). The S1 gene consists of 2,589 nucleotides and encodes 863 amino acids with a molecular weight of 91,000 Da, indicating that the 280 kDa subunit is highly glycosylated. Sequence analysis revealed that S1 did not have significant homology with any proteins in the databases except for two surface layer homology (SLH) domains in its N-terminal region. Surprisingly, the recombinant S1 exhibits xylanase activity, and cellulose — and xylan-binding ability, suggesting that the S1 should be a novel xylanase and CBM(s) with new functions (unpublished data).

Xylanase Xyn10A: The xyn10A gene consists of 3,828 nucleotides encoding a protein of 1,276 amino acids with a predicted molecular weight of 142,726 Da. Xyn10A is a multidomain enzyme comprised of nine domains in the following order: three family-22 CBMs, a family-10 catalytic domain of glycosyl hydrolases (GH), a family-9 CBM, a glycine-rich region, and three SLH domains. Xyn10A can effectively hydrolyze insoluble xylan and natural biomass without pretreatment such as sugarcane bagasse, corn hull, rice bran, rice husk and rice straw. Xyn10A binds to various insoluble polysaccharides such as cellulose, xylan and chitin. The SLH domains functioned in Xyn10A by anchoring this enzyme to the cell surfaces of P. curdlanolyticus B-6. Removal of the CBMs from Xyn10A strongly reduced the ability of binding and plant cell wall hydrolysis. Therefore, the CBMs of Xyn10A play an important role in the hydrolysis of native biomass materials (Waeonukul et al., 2009a).

Xylanase Xyn10B: The xyn10B gene consists of 1,047 nucleotides encoding a protein of 349 amino acids with a predicted molecular weight of 40,480 Da. Xyn10B consists of only a family-10 catalytic of GH. Xyn10B is an intracellular endoxylanase (Sudo et al., 2010).

Xylanase Xyn10C: The xyn10C gene consists of 957 nucleotides and encodes 318 amino acid residues with a predicted molecular weight of 35,123 Da. Xyn10C is a single module enzyme consisting of a signal peptide and a family-10 catalytic module of GH (unpublished data).

Xylanase Xyn10D: The xyn10D gene consists of 1,734 nucleotides and encodes 577 amino acid residues with a calculated molecular weight of 61,811 Da. Xylanase Xyn10D is a modular enzyme consisting of a family-10 catalytic module of the GH, a fibronectin type-3 homology (Fn3) module, and family-3 CBM, in that order, from the N terminus. The CBM3 in Xyn10D has an affinity for cellulose and xylan, and plays an important role in hydrolysis of arabinoxylan and native biomass materials (Sakka et al., 2011).

Xylanase Xyn11A: The xyn11A gene consists of 1,150 bp and encodes a protein of 385 amino acids with a molecular weight of 40,000 Da. Xyn11A is composed of two major functional domains, a catalytic domain belonging to family-11 GH and a CBM classified as family-36. A glycine- and asparagine-repeated sequences existed between the two domains. Xyn11A has been identified to be one of the major xylanase subunit in the multienzyme complex of strain B-6 (Pason et al., 2010).

Based on both biochemical and molecular biological findings, a simplistic schematic view of the enzyme system from P. curdlanolyticus B-6 and its interaction with substrate and cell surface was created and presented in Fig. 9. In this assessment, the S1 protein did not have significant homology with any proteins in the databases except for two S-layer homology domains in its N-terminal region. However, the S1 protein that exhibits xylanase activity and cellulose — and xylan-binding ability, and contains cell anchoring function, seems remarkable. The multifunctional protein S1 is also responsible for forming the enzyme subunits into the complex and anchoring the complex into cell surface via the SLH domain. The interaction between S1 protein and enzyme subunits should be a mechanism distinct from the cohesion-dockerin interaction known in cellulosome of anaerobic microorganisms, since cohesion — or dockerin- like sequences were not observed in the S1 protein or the major xylanase subunit, Xyn11A. In addition, strain B-6 also produces cell bound multimodular xylanase Xyn10A that contains the numerous CBMs and SLH domains. Xyn10A can bind to the plant cell wall through CBM, whereas the catalytic module (GH10) is able to access its target substrate. Thus, the CBM greatly increases the concentration of the enzyme in the vicinity of the substrate, leading to the observed increase in polysaccharide hydrolysis. Besides, the presence of the functional CBMs and SLH domains in Xyn10A allows the cells to attach to substrate. Although, the overall structure of the enzyme complex system of the strain B-6 is not entirely clear, the enzyme complex has unique characteristics distinct from multienzyme complex cellulosome of anaerobic microorganisms. However, the mechanism for complex formation, interaction between the S1 protein as scaffoldin and enzyme subunits, needs to be further investigated.

Figure 4 compares the FTIR spectra of the purified PHB granules and the original oven-dried PHB-containing cell mass. The peaks of amide I band at ~1650 cm-1 and amide II band at ~1540 cm-1 (N-H bend) are characteristic infrared radiation absorption of the proteins in cell mass [25,26]. They disappeared in the spectrum of purified PHB granules. This was confirmed with a pure PHB prepared with solvent extraction (the spectrum not shown here). It was also noticed that the native amorphous PHB granules became crystallized during the process of purification, which will be further discussed. This structural change in PHB matrix was also reflected in the absorption of infrared radiation at wave numbers of 1180, 1210, and 1280 nm-1 [27].

The purified PHB granules were subjected to repeated heating and cooling in a differential scanning calorimeter (DSC), and the results are presented in Figure 5. In the first heating (solid blue line), two melting peaks were observed, around 156 oC and 167 oC, respectively, indicating that the PHB powder had two types of crystalline structures. The melted polymer was re-crystallized again during the first cooling (dotted blue line), starting at 100 oC and ending at 80 oC. When the crystallized PHB was subjected to the second heating (solid black line), it was noticed that the relatively small melting peak at 156 oC (or crystalline structure) observed in the first heating disappeared. Only one melting peak (crystalline structure) was observed, starting at 160 oC and ending at 181 oC with a peak around 174 oC. This phenomenon indicates that the first melting peak in the first heating represents a type of crystalline structure that could not be formed at a cooling rate of 5 oC/min. The whole endothermic event in the second heating absorbed 87J/g PHB. Based on a theoretical melting enthalpy of 100% crystalline PHB (146 J/g) [28], it can be estimated that about 60% of PHB matrix was crystallized during the first cooling (Eq. 1).

Where Xc is the PHB crystallinity, AHm and AHt are the melting enthalpies of PHB powder and a theoretical PHB crystal [28].

In contrast to the thermal plastic behavior of PHB powders, the oven-dried cell mass containing 73% PHB could not be melted till carbonization. This fact reveals a complicated interaction between the biopolyester and the residual biomass. It also shows that a composite of 73% PHB and 27% cellular mass is not a thermoplastic material, but a rigid composite. The role of cellular mass in the PHB composite is not clear yet.