of organic matter takes place in sediments, waterlogged soils and animal intestinal tracts, in which the oxygen access is restricted; whilst in engineered environments it refers to the biotechnological process by which organic matter (i.e., organic waste, wastewater and/or a renewable resource) is degraded in the absence of oxygen for the commercial production of biogas that can be used as an eco-friendly energy source [109], thereby representing an important asset in times of decreasing fossil fuel supplies. According to [4], the anaerobic digestion from swine, bovine and poultry slurries resulted in the production of biogas at average rates of 0.30, 0.25 and 0.48 L/g volatile solids, respectively. Another valuable coproduct derived from this process is the anaerobic digested slurry, which can be applied as an organic amendment into soil either in agricultural and non-agricultural lands [110](section 3.1). It is for this reason along with the production of biogas and the reduction in greenhouse gas emissions [10] that anaerobic digestion is becoming increasingly popular as a methodological alternative for manure recycling, which in turn has increased the number of farm-scale anaerobic bioreactors up to 4200 in central and northern Europe [111]. Bacteria represent over 80% of the total diversity in anaerobic digesters [112], and they are mainly composed by the phyla Firmicutes, Proteobacteria and Bacteroidetes; whereas most of the archaeal representatives belong to the phylum Euryarchaeota, which includes all the known methanogens. Anaerobic eukaryotes - particularly, fungi and protozoa- have received less attention probably because they are slower growers than bacteria and, as such, their abundance is lower in anaerobic reactors. As occurs with composting, anaerobic digestion may be described as a four-phase microbiologically driven process. Briefly, the first and rate limiting step of the anaerobic food chain is the *depolymerization* and *hydrolysis* of complex biopolymers, such as polysaccharides, lipids, proteins and nucleic acids into their corresponding structural units (sugars, fatty acids, amino acids, purines and pyrimidines) through the joint action of a complex community of fibrolytic bacteria and fungi, which produce extracellular hydrolitic enzymes (i.e., cellulases, xylanases, proteases, lipases) responsible for the disassembling of such polymers. Since polysaccharides, mainly cellulose, are the most abundant structural and storage compounds of biomass, their hydrolysis is considered as the most determinant enzymatic process regarding the efficiency of anaerobic reactors. The rate and efficiency of cellulose hydrolysis depends greatly on the particular microbial species composition involved [113], and under anaerobic conditions it proceeds slowly due to the heterogeneity of forms in which cellulose is present in nature and to the complexity of the hemicelluloses and lignin matrices in which it is embedded [113]. Similarly, protein hydrolysis to peptides and amino acids takes place slowly, whilst lipid hydrolysis into glycerol and long-chain fatty acids occurs rapidly compared to their subsequent fermentation or β-oxidation. As mentioned above, bacterial populations are more abundant and diverse and, hence, they are responsible for the majority of hydrolytic reactions, being *Clostridium*, *Acetivibrio*, *Bacteroides*, *Selenomonas* or *Ruminococcus* common

examples of hydrolytic bacteria found in anaerobic reactors [112,114].

The monomeric compounds released after the hydrolysis of biopolymers can be taken up by microbial cells, in which they are either fermented or anaerobically oxidised into alcohols, short-chain fatty acids, CO2 and molecular hydrogen (H2). This step is known as *fermentation*

Furthermore, recent studies have demonstrated the presence of various bacteria, which are useful for different biotechnological purposes, in diverse vermicomposts [106-107], reinforcing that the biological component (i.e., the microbial community composition) of a vermicompost largely determines its usefulness in agriculture and other applications, such as soil restoration and bioremediation. For instance, Fernández-Gómez et al. [106] detected the presence of *Sphingobacterium*, *Streptomyces*, Alpha-Proteobacteria, Delta-Proteobacteria and Firmicutes in diverse vermicomposts, irrespective of the parent material used for the process, by applying DGGE and COMPOCHIP, thereby demonstrating the usefulness of both techniques to assess the potential of vermicomposts as bioactive organic materials. Indeed, disease suppressiveness is obviously linked to the microbiota added with the vermicompost, along with the biological and physicochemical characteristics of the native soil microbial community. However, despite the large body of scientific evidence showing the positive effects of vermicompost regarding the suppression of soil-borne plant fungal diseases (reviewed in [75,108]), it is still necessary to obtain a deeper understanding of the mechanisms involved and the main factors influencing such suppressing effects. According to the mechanisms proposed for compost [68-69], disease suppression by vermicompost may be attributed to either direct effects or to the induction of systemic resistance in the plant. Direct suppression of the pathogen by the vermicompost-associated microflora and/or microfauna may be general or specific, depending on the existence of a single suppressive agent or the joint action of several agents, and the proposed mechanisms are competition, antibiosis and parasitism. Some of the indirect effects of vermicompost have been related to changes in the microbiological properties of the soil or the potting media. Processing by earthworms during vermicomposting has a strong effect on the microbial community structure and activity of the initial waste [8]. Vermicompost therefore has a rather different microbial community structure than the parent waste, with lower biomass but enhanced metabolic diversity [18]. Application of such a microbiologically active organic substrate may thus have important effects on the microbial and biochemical properties of soil or greenhouse potting media thereby influencing plant growth. Moreover, vermicompost may affect directly the plant growth via the supply of nutrients, as it constitutes a slow-release fertiliser that supplies the plant with a gradual and constant source of nutrients, and/or through the supply of plant growth regulating substances [95].

### **3. Anaerobic digestion**

The process of anaerobic digestion (AD) has been extensively studied in natural and engineered ecosystems for more than a century. In natural habits, the anaerobic degradation of organic matter takes place in sediments, waterlogged soils and animal intestinal tracts, in which the oxygen access is restricted; whilst in engineered environments it refers to the biotechnological process by which organic matter (i.e., organic waste, wastewater and/or a renewable resource) is degraded in the absence of oxygen for the commercial production of biogas that can be used as an eco-friendly energy source [109], thereby representing an important asset in times of decreasing fossil fuel supplies. According to [4], the anaerobic digestion from swine, bovine and poultry slurries resulted in the production of biogas at average rates of 0.30, 0.25 and 0.48 L/g volatile solids, respectively. Another valuable coproduct derived from this process is the anaerobic digested slurry, which can be applied as an organic amendment into soil either in agricultural and non-agricultural lands [110](section 3.1). It is for this reason along with the production of biogas and the reduction in greenhouse gas emissions [10] that anaerobic digestion is becoming increasingly popular as a methodological alternative for manure recycling, which in turn has increased the number of farm-scale anaerobic bioreactors up to 4200 in central and northern Europe [111].

256 Biomass Now – Cultivation and Utilization

resource allocation within the plant. Different genotypes may therefore enhance root growth or modify root exudation patterns in order to increase nutrient uptake [100], and all of these strategies will determine the establishment of different interactions with the microbial communities at the rhizosphere level. Indeed, after the application of vermicompost to sweet corn crops, these authors found important differences in the rhizosphere microbial community of two genotypes from cultivars of maize, with the sugary endosperm mutation (*su1*) and with

Furthermore, recent studies have demonstrated the presence of various bacteria, which are useful for different biotechnological purposes, in diverse vermicomposts [106-107], reinforcing that the biological component (i.e., the microbial community composition) of a vermicompost largely determines its usefulness in agriculture and other applications, such as soil restoration and bioremediation. For instance, Fernández-Gómez et al. [106] detected the presence of *Sphingobacterium*, *Streptomyces*, Alpha-Proteobacteria, Delta-Proteobacteria and Firmicutes in diverse vermicomposts, irrespective of the parent material used for the process, by applying DGGE and COMPOCHIP, thereby demonstrating the usefulness of both techniques to assess the potential of vermicomposts as bioactive organic materials. Indeed, disease suppressiveness is obviously linked to the microbiota added with the vermicompost, along with the biological and physicochemical characteristics of the native soil microbial community. However, despite the large body of scientific evidence showing the positive effects of vermicompost regarding the suppression of soil-borne plant fungal diseases (reviewed in [75,108]), it is still necessary to obtain a deeper understanding of the mechanisms involved and the main factors influencing such suppressing effects. According to the mechanisms proposed for compost [68-69], disease suppression by vermicompost may be attributed to either direct effects or to the induction of systemic resistance in the plant. Direct suppression of the pathogen by the vermicompost-associated microflora and/or microfauna may be general or specific, depending on the existence of a single suppressive agent or the joint action of several agents, and the proposed mechanisms are competition, antibiosis and parasitism. Some of the indirect effects of vermicompost have been related to changes in the microbiological properties of the soil or the potting media. Processing by earthworms during vermicomposting has a strong effect on the microbial community structure and activity of the initial waste [8]. Vermicompost therefore has a rather different microbial community structure than the parent waste, with lower biomass but enhanced metabolic diversity [18]. Application of such a microbiologically active organic substrate may thus have important effects on the microbial and biochemical properties of soil or greenhouse potting media thereby influencing plant growth. Moreover, vermicompost may affect directly the plant growth via the supply of nutrients, as it constitutes a slow-release fertiliser that supplies the plant with a gradual and constant source of nutrients, and/or

the shrunken endosperm mutation (*sh2*), which differ in their C storage patterns.

through the supply of plant growth regulating substances [95].

The process of anaerobic digestion (AD) has been extensively studied in natural and engineered ecosystems for more than a century. In natural habits, the anaerobic degradation

**3. Anaerobic digestion**

Bacteria represent over 80% of the total diversity in anaerobic digesters [112], and they are mainly composed by the phyla Firmicutes, Proteobacteria and Bacteroidetes; whereas most of the archaeal representatives belong to the phylum Euryarchaeota, which includes all the known methanogens. Anaerobic eukaryotes - particularly, fungi and protozoa- have received less attention probably because they are slower growers than bacteria and, as such, their abundance is lower in anaerobic reactors. As occurs with composting, anaerobic digestion may be described as a four-phase microbiologically driven process. Briefly, the first and rate limiting step of the anaerobic food chain is the *depolymerization* and *hydrolysis* of complex biopolymers, such as polysaccharides, lipids, proteins and nucleic acids into their corresponding structural units (sugars, fatty acids, amino acids, purines and pyrimidines) through the joint action of a complex community of fibrolytic bacteria and fungi, which produce extracellular hydrolitic enzymes (i.e., cellulases, xylanases, proteases, lipases) responsible for the disassembling of such polymers. Since polysaccharides, mainly cellulose, are the most abundant structural and storage compounds of biomass, their hydrolysis is considered as the most determinant enzymatic process regarding the efficiency of anaerobic reactors. The rate and efficiency of cellulose hydrolysis depends greatly on the particular microbial species composition involved [113], and under anaerobic conditions it proceeds slowly due to the heterogeneity of forms in which cellulose is present in nature and to the complexity of the hemicelluloses and lignin matrices in which it is embedded [113]. Similarly, protein hydrolysis to peptides and amino acids takes place slowly, whilst lipid hydrolysis into glycerol and long-chain fatty acids occurs rapidly compared to their subsequent fermentation or β-oxidation. As mentioned above, bacterial populations are more abundant and diverse and, hence, they are responsible for the majority of hydrolytic reactions, being *Clostridium*, *Acetivibrio*, *Bacteroides*, *Selenomonas* or *Ruminococcus* common examples of hydrolytic bacteria found in anaerobic reactors [112,114].

The monomeric compounds released after the hydrolysis of biopolymers can be taken up by microbial cells, in which they are either fermented or anaerobically oxidised into alcohols, short-chain fatty acids, CO2 and molecular hydrogen (H2). This step is known as *fermentation*

(*acidogenesis*) and usually occurs through the production of an energy-rich intermediate that is used to synthesise ATP, rendering a fermentation product that is excreted out of the cell. Since these products are typically acidic substances, the fermentative reactions are accompanied by a decrease in the extracellular pH. This fact along with the increase in short-chain fatty acids represents the most common reasons for reactor failure. Thus, for maintaining the pH balance of the system, it is of great importance to bear in mind the equilibrium between fermentative-acidogenic bacteria and acid scavenging microbes. Typically, the bacteria from the same group that hydrolyze biopolymers take up and ferment the resulting monomers. For instance, *Clostridium* sp. and enteric bacteria are common sugar fermenters in anaerobic reactors. *Streptococcus* sp. and *Lactobacillus* sp. also ferment sugars, producing lactate or lactate and ethanol, plus CO2 and molecular hydrogen [114]. Fermentation of amino acids and purines and pyrimidines in anaerobic environments is mainly carried out by species of *Clostridium* [115].

Animal Manures: Recycling and Management Technologies 259

especially those related to the impact of the anaerobic digested slurry on the soil microbiota. In a recent work, Walsh et al. [120] observed that its application affected the fungal and bacterial growth in a very similar way to the application of mineral fertilizers in a 16- week greenhouse experiment. They found a pronounced shift towards a bacterial dominated microbial decomposer community, and such effects were consistent in different soils and different crop types. They conclude that mineral fertiliser could be thus exchanged for anaerobic digested slurry with limited impact on the actively growing soil microbial community, which is of great importance in the regulation of soil processes and consequently in soil fertility and crop yield. Recently, Massé et al. [4] gave an overview of the agronomic value of anaerobic digestion treated manures. In line with this, previous findings showed that the AD of animal slurries improved their fertilizer value [3], thereby leading to an increased forage yield and N uptake relative to raw liquid swine manure and mineral fertilizers [121]. Bougnom et al. [122] found a 20% increase in grass yield compared to conventional manure. Liedl et al. [123] also found that digested poultry litter resulted in similar or superior grass and vegetable yields versus N fertilizers. Therefore, all of the above provides evidence that the anaerobic digested slurry acts similarly to mineral fertiliser and should be considered as such in its application to land. Additionally, the enrichment of mineral fractions of N and P during digestion ultimately results in a higher concentration of plant-available nutrients compared with undigested manure and a subsequently elevated plant growth promotion ability, suggested to be similar to mineral fertilisers [4,123]. These latter authors also found that AD reduced swine manure total and volatile solid concentrations by up to 80% resulting in improved manure homogeneity and lowered viscosity allowing more uniform land application [4]. Nevertheless, the higher levels of mineral N found in the slurry, mainly ammonia, may also lead to an increase in the level of phytotoxicity of the slurry, thereby affecting seed germination and plant growth after land- spreading of this co-product into soils [124]. The presence of other phytotoxic substances, such as volatile fatty acids (i.e., acetic, propionic and butyric acids), as well as the high content of soluble salts may contribute to the slurry phytotoxicity [125]. Furthermore, Goberna et al. [126] found that amending soils with slurry resulted in greater nitrate losses during the first 30 d of a 100 d incubation period in 20 cm-depth lysimeters. In fact, around 23 and 45% of the total N contained in the soil (natural + added) was lost from soils amended with cattle manure and anaerobic slurry, respectively. Other authors also observed that N leaching was, along with NH3 volatilisation, one of the most important sources of N leakage to the environment in a field-scale experiment, after having quantified the amount of mineral N at 1.7 m depth from grass cultivated plots amended with anaerobic digested slurry and mineral fertiliser [127]. Thus, the use of this coproduct as an organic amendment should accurately match crop N demand because if not taken by the plants, nitrates could be drained to surface waters, leached to ground waters or

denitrified into gaseous forms and emitted to the atmosphere.

The presence of pathogenic bacteria in agricultural amendments also represents a potential threat and their screening is thus of great importance mainly in those produced from animal manures, as it has been shown that such pathogenic organisms constitute a common fraction of the microbial community in manure [128]. In fact, it has been shown that some

Then, during *acetogenesis*, the fermentation products are mainly oxidised to acetate, formate, CO2 and H2 by acetogenic bacteria, most of them belonging to the low G+C branch of *Firmicutes* [116]*.* Certain acetogenic reactions are thermodynamically unfavourable under standard conditions, which make necessary a syntrophic relationship between the acetogen and a H2-consuming methanogens in order to degrade the substrate and, in turn to obtain a net energy gain [117]. Finally, the last and most sensitive step during the anaerobic digestion of organic matter is the *methanogenesis*, i.e. the formation of methane from acetate, H2/CO2 and methyl compounds by the action of methanogenic organisms belonging to the phylum Euryarchaeota [118]. The orders Methanobacteriales, Methanococcales, Methanomicrobiales and Methanosarcinales include known methanogens commonly found in aerobic reactors. Members of the first three orders use CO2 and H2 as an electron acceptor and donor, respectively. Some species from these orders can also use formate and/or secondary alcohols (i.e., isopropanol or ethanol), but they cannot use acetate or C1 compounds such as methanol and methylamines (with the exception of the genus *Methanosphaera* from the order Methanobacteriales). However, Methanosarcinales are more diverse metabolically, and they can use acetate, hydrogen, formate, secondary alcohols and methyl compounds as energy sources. It is believed that the predominance of hydrogenotrophic or acetotrophic methanogens depends on the levels of their substrates and their tolerance to diverse inhibitors, including ammonia, hydrogen sulphide, or volatile fatty acids [119]. The aforementioned steps involved in the anaerobic digestion are explained in more detail by [109].

### **3.1. Influence of anaerobic digested slurry on the soil microbiota**

The changes that occur in the parent waste during the process of anaerobic digestion largely depend on the dynamics of the abovementioned microbial groups, ultimately influencing the quality of the final products, i.e. biogas and anaerobic digested slurry. As mentioned before, anaerobic slurries are rich in partially stable organic carbon and can be used as organic amendments for crop production. However so far, many environmental issues relevant when these co-products are applied to agricultural land still have to be studied, especially those related to the impact of the anaerobic digested slurry on the soil microbiota. In a recent work, Walsh et al. [120] observed that its application affected the fungal and bacterial growth in a very similar way to the application of mineral fertilizers in a 16- week greenhouse experiment. They found a pronounced shift towards a bacterial dominated microbial decomposer community, and such effects were consistent in different soils and different crop types. They conclude that mineral fertiliser could be thus exchanged for anaerobic digested slurry with limited impact on the actively growing soil microbial community, which is of great importance in the regulation of soil processes and consequently in soil fertility and crop yield. Recently, Massé et al. [4] gave an overview of the agronomic value of anaerobic digestion treated manures. In line with this, previous findings showed that the AD of animal slurries improved their fertilizer value [3], thereby leading to an increased forage yield and N uptake relative to raw liquid swine manure and mineral fertilizers [121]. Bougnom et al. [122] found a 20% increase in grass yield compared to conventional manure. Liedl et al. [123] also found that digested poultry litter resulted in similar or superior grass and vegetable yields versus N fertilizers. Therefore, all of the above provides evidence that the anaerobic digested slurry acts similarly to mineral fertiliser and should be considered as such in its application to land. Additionally, the enrichment of mineral fractions of N and P during digestion ultimately results in a higher concentration of plant-available nutrients compared with undigested manure and a subsequently elevated plant growth promotion ability, suggested to be similar to mineral fertilisers [4,123]. These latter authors also found that AD reduced swine manure total and volatile solid concentrations by up to 80% resulting in improved manure homogeneity and lowered viscosity allowing more uniform land application [4]. Nevertheless, the higher levels of mineral N found in the slurry, mainly ammonia, may also lead to an increase in the level of phytotoxicity of the slurry, thereby affecting seed germination and plant growth after land- spreading of this co-product into soils [124]. The presence of other phytotoxic substances, such as volatile fatty acids (i.e., acetic, propionic and butyric acids), as well as the high content of soluble salts may contribute to the slurry phytotoxicity [125]. Furthermore, Goberna et al. [126] found that amending soils with slurry resulted in greater nitrate losses during the first 30 d of a 100 d incubation period in 20 cm-depth lysimeters. In fact, around 23 and 45% of the total N contained in the soil (natural + added) was lost from soils amended with cattle manure and anaerobic slurry, respectively. Other authors also observed that N leaching was, along with NH3 volatilisation, one of the most important sources of N leakage to the environment in a field-scale experiment, after having quantified the amount of mineral N at 1.7 m depth from grass cultivated plots amended with anaerobic digested slurry and mineral fertiliser [127]. Thus, the use of this coproduct as an organic amendment should accurately match crop N demand because if not taken by the plants, nitrates could be drained to surface waters, leached to ground waters or denitrified into gaseous forms and emitted to the atmosphere.

258 Biomass Now – Cultivation and Utilization

is mainly carried out by species of *Clostridium* [115].

involved in the anaerobic digestion are explained in more detail by [109].

**3.1. Influence of anaerobic digested slurry on the soil microbiota**

The changes that occur in the parent waste during the process of anaerobic digestion largely depend on the dynamics of the abovementioned microbial groups, ultimately influencing the quality of the final products, i.e. biogas and anaerobic digested slurry. As mentioned before, anaerobic slurries are rich in partially stable organic carbon and can be used as organic amendments for crop production. However so far, many environmental issues relevant when these co-products are applied to agricultural land still have to be studied,

(*acidogenesis*) and usually occurs through the production of an energy-rich intermediate that is used to synthesise ATP, rendering a fermentation product that is excreted out of the cell. Since these products are typically acidic substances, the fermentative reactions are accompanied by a decrease in the extracellular pH. This fact along with the increase in short-chain fatty acids represents the most common reasons for reactor failure. Thus, for maintaining the pH balance of the system, it is of great importance to bear in mind the equilibrium between fermentative-acidogenic bacteria and acid scavenging microbes. Typically, the bacteria from the same group that hydrolyze biopolymers take up and ferment the resulting monomers. For instance, *Clostridium* sp. and enteric bacteria are common sugar fermenters in anaerobic reactors. *Streptococcus* sp. and *Lactobacillus* sp. also ferment sugars, producing lactate or lactate and ethanol, plus CO2 and molecular hydrogen [114]. Fermentation of amino acids and purines and pyrimidines in anaerobic environments

Then, during *acetogenesis*, the fermentation products are mainly oxidised to acetate, formate, CO2 and H2 by acetogenic bacteria, most of them belonging to the low G+C branch of *Firmicutes* [116]*.* Certain acetogenic reactions are thermodynamically unfavourable under standard conditions, which make necessary a syntrophic relationship between the acetogen and a H2-consuming methanogens in order to degrade the substrate and, in turn to obtain a net energy gain [117]. Finally, the last and most sensitive step during the anaerobic digestion of organic matter is the *methanogenesis*, i.e. the formation of methane from acetate, H2/CO2 and methyl compounds by the action of methanogenic organisms belonging to the phylum Euryarchaeota [118]. The orders Methanobacteriales, Methanococcales, Methanomicrobiales and Methanosarcinales include known methanogens commonly found in aerobic reactors. Members of the first three orders use CO2 and H2 as an electron acceptor and donor, respectively. Some species from these orders can also use formate and/or secondary alcohols (i.e., isopropanol or ethanol), but they cannot use acetate or C1 compounds such as methanol and methylamines (with the exception of the genus *Methanosphaera* from the order Methanobacteriales). However, Methanosarcinales are more diverse metabolically, and they can use acetate, hydrogen, formate, secondary alcohols and methyl compounds as energy sources. It is believed that the predominance of hydrogenotrophic or acetotrophic methanogens depends on the levels of their substrates and their tolerance to diverse inhibitors, including ammonia, hydrogen sulphide, or volatile fatty acids [119]. The aforementioned steps

> The presence of pathogenic bacteria in agricultural amendments also represents a potential threat and their screening is thus of great importance mainly in those produced from animal manures, as it has been shown that such pathogenic organisms constitute a common fraction of the microbial community in manure [128]. In fact, it has been shown that some

pathogenic bacteria can survive the process of anaerobic digestion and persist in the slurry, as previously reported by [129]. In line with this, those microorganisms with a sporeforming capacity such as *Clostridium* and *Bacillus* species, which are commonly found in the intestinal flora of most warm-blooded animals and can harbor some highly pathogenic members for animals and humans [12,129], cannot be reduced during the process [130]. Accordingly, Olsen and Larsen [131] observed that the spores of *Clostridium perfringens* were not inactivated in either mesophilic or thermophilic biogas digesters. Similar results were observed by other authors [132-133] in a reactor operating under mesophilic and thermophilic conditions, respectively. It is acknowledged that bacterial spores can survive in extreme conditions and germinate after long periods, when the conditions become more favourable [131]. The non-hygienic conditions of the storage/transporting tanks can also favour pathogen regrowth [134]. The composition of the substrate fed into the reactor, as well as the reactor conditions such as pH, digestion temperature, slurry hydraulic retention time, ammonium concentration, volatile fatty acids content and nutrient supply are expected to have a significant influence on the sanitation of the end-product [130]. This indicates that there exists a potential risk of spreading potentially pathogenic microbes after the application of anaerobic slurries into soil. Indeed, Crane and Moore [135] stated that amending soils with raw and treated manures, even with a low pathogenic load, still posed a threat for the environment because a period of regrowth of some pathogens including *Escherichia coli*, enterococci, faecal streptococci and *Salmonella enterica* have been shown after manure deposition to soil [136]. Goberna et al. [126] also found that the levels of *Listeria* in soils amended with either cattle manure or anaerobic slurry were significantly higher than those in the control treatment after having been incubated for a month. They observed, however, that the cultivable forms of *Listeria* in the studied soils could correspond to *L. innocua* instead of *L. monocytogenes*, as shown by the polymerase chain reaction assays. However, as recently summarised by [137], anaerobic digestion generally reduces the pathogen risk when compared to untreated substrates.

Animal Manures: Recycling and Management Technologies 261

**Figure 5.** Abundance of *Escherichia coli* and faecal coliforms in the original materials (manure (M), compost (C), vermicompost (Vc) and anaerobic digested slurry (AS)) and in the unamended and

Briefly, culturable forms of both faecal coliforms and *E. coli* were isolated from all the initial materials, although their levels were greatly lower in compost relative to the other substrates (Figure 5). This is not surprising taking into account that the composting process, unlike vermicomposting, involved a four-day thermophilic phase, during which the process reached a temperature of 70 °C. Those pathogens were also detected in the anaerobic digested slurry after 40 days of anaerobic digestion (Figure 5). This fact suggests that feeding the reactor with four to five m3 cattle manure d-1 could have provided enough nutrients to maintain a large population of the studied pathogens. Indeed, nutrient availability is one of the major factors influencing pathogen survival in biogas digesters, as previously reported by [130]. Once applied to soils, *E. coli* CFUs were detected in manureamended soils at the start of the experiment and after incubation for 15 d (Figure 5A); whilst faecal coliforms CFUs were recorded in both manure and slurry-amended soils in the shortterm, even though at lower values in comparison with the start of incubation (Figure 5B).

However, the spore-forming *C. perfringens* persisted in all the amended soils (Figure 6), which supports the fact that this bacterium has more resistance to environmental stresses and the capacity to outcompete the native soil microbiota. After 60 d CFU of *C. perfringens* were much closer to those in the control in the slurry-amended soils (Figure 6), which suggests that this time period could be considered as a safe delay between land-spreading

the product into soil and crop harvesting with respect to its pathogenic load.

amended soils at the three incubation times (0, 15 and 60 days). Values are means ± SE.

In a current study, we evaluated, at a microcosm level, the short and long-term effects of the anaerobic digested slurry on soil chemical and microbiological properties compared to its ingestate (i.e., raw manure) and the two widely-recognized products, compost and vermicompost. All of the organic substrates were mixed with soil by turning at a rate of 40 mg N kg-1 soil (dry weight). A control treatment that consisted of soil without the addition of any organic amendment was also included. A total of 45 experimental units (5 amendment levels x 3 incubation times x 3 replicates) were set-up in the present study. After an equilibration period of 4 days at 4 °C, 15 columns were dismantled and the sample was collected to analyze (incubation time 0 days). The remaining thirty columns were then maintained in a room at 22 °C, which is the average temperature of the hottest and wettest month in this area and the most suitable for the survival of pathogens. These columns were destructively sampled after 15 and 60 d incubation corresponding to short and mediumterm effects. The survival of selected pathogens was then determined according to standard protocols [138-140] (ISO 16649-2, 2001 for *Escherichia coli*; ISO 4832, 1991 for faecal coliforms; and ISO 7937, 2004 for *Clostridium perfringens*) in all the organic materials and amended soils.

pathogen risk when compared to untreated substrates.

soils.

pathogenic bacteria can survive the process of anaerobic digestion and persist in the slurry, as previously reported by [129]. In line with this, those microorganisms with a sporeforming capacity such as *Clostridium* and *Bacillus* species, which are commonly found in the intestinal flora of most warm-blooded animals and can harbor some highly pathogenic members for animals and humans [12,129], cannot be reduced during the process [130]. Accordingly, Olsen and Larsen [131] observed that the spores of *Clostridium perfringens* were not inactivated in either mesophilic or thermophilic biogas digesters. Similar results were observed by other authors [132-133] in a reactor operating under mesophilic and thermophilic conditions, respectively. It is acknowledged that bacterial spores can survive in extreme conditions and germinate after long periods, when the conditions become more favourable [131]. The non-hygienic conditions of the storage/transporting tanks can also favour pathogen regrowth [134]. The composition of the substrate fed into the reactor, as well as the reactor conditions such as pH, digestion temperature, slurry hydraulic retention time, ammonium concentration, volatile fatty acids content and nutrient supply are expected to have a significant influence on the sanitation of the end-product [130]. This indicates that there exists a potential risk of spreading potentially pathogenic microbes after the application of anaerobic slurries into soil. Indeed, Crane and Moore [135] stated that amending soils with raw and treated manures, even with a low pathogenic load, still posed a threat for the environment because a period of regrowth of some pathogens including *Escherichia coli*, enterococci, faecal streptococci and *Salmonella enterica* have been shown after manure deposition to soil [136]. Goberna et al. [126] also found that the levels of *Listeria* in soils amended with either cattle manure or anaerobic slurry were significantly higher than those in the control treatment after having been incubated for a month. They observed, however, that the cultivable forms of *Listeria* in the studied soils could correspond to *L. innocua* instead of *L. monocytogenes*, as shown by the polymerase chain reaction assays. However, as recently summarised by [137], anaerobic digestion generally reduces the

In a current study, we evaluated, at a microcosm level, the short and long-term effects of the anaerobic digested slurry on soil chemical and microbiological properties compared to its ingestate (i.e., raw manure) and the two widely-recognized products, compost and vermicompost. All of the organic substrates were mixed with soil by turning at a rate of 40 mg N kg-1 soil (dry weight). A control treatment that consisted of soil without the addition of any organic amendment was also included. A total of 45 experimental units (5 amendment levels x 3 incubation times x 3 replicates) were set-up in the present study. After an equilibration period of 4 days at 4 °C, 15 columns were dismantled and the sample was collected to analyze (incubation time 0 days). The remaining thirty columns were then maintained in a room at 22 °C, which is the average temperature of the hottest and wettest month in this area and the most suitable for the survival of pathogens. These columns were destructively sampled after 15 and 60 d incubation corresponding to short and mediumterm effects. The survival of selected pathogens was then determined according to standard protocols [138-140] (ISO 16649-2, 2001 for *Escherichia coli*; ISO 4832, 1991 for faecal coliforms; and ISO 7937, 2004 for *Clostridium perfringens*) in all the organic materials and amended

**Figure 5.** Abundance of *Escherichia coli* and faecal coliforms in the original materials (manure (M), compost (C), vermicompost (Vc) and anaerobic digested slurry (AS)) and in the unamended and amended soils at the three incubation times (0, 15 and 60 days). Values are means ± SE.

Briefly, culturable forms of both faecal coliforms and *E. coli* were isolated from all the initial materials, although their levels were greatly lower in compost relative to the other substrates (Figure 5). This is not surprising taking into account that the composting process, unlike vermicomposting, involved a four-day thermophilic phase, during which the process reached a temperature of 70 °C. Those pathogens were also detected in the anaerobic digested slurry after 40 days of anaerobic digestion (Figure 5). This fact suggests that feeding the reactor with four to five m3 cattle manure d-1 could have provided enough nutrients to maintain a large population of the studied pathogens. Indeed, nutrient availability is one of the major factors influencing pathogen survival in biogas digesters, as previously reported by [130]. Once applied to soils, *E. coli* CFUs were detected in manureamended soils at the start of the experiment and after incubation for 15 d (Figure 5A); whilst faecal coliforms CFUs were recorded in both manure and slurry-amended soils in the shortterm, even though at lower values in comparison with the start of incubation (Figure 5B).

However, the spore-forming *C. perfringens* persisted in all the amended soils (Figure 6), which supports the fact that this bacterium has more resistance to environmental stresses and the capacity to outcompete the native soil microbiota. After 60 d CFU of *C. perfringens* were much closer to those in the control in the slurry-amended soils (Figure 6), which suggests that this time period could be considered as a safe delay between land-spreading the product into soil and crop harvesting with respect to its pathogenic load.

Animal Manures: Recycling and Management Technologies 263

The remaining residuals may eventually be used for biogas production, a cascade process that utilizes the organic waste at its highest level. Furthermore, although an interest in vermicompost research and technology has been increasing over recent years, and the body of knowledge available is quite large, there are still some important topics to be investigated. During vermicomposting, earthworm activity helps microbial communities to use the available energy more efficiently and plays a key role in shaping the structure of the microbial communities during the process. Hence, it is of future interest to evaluate whether the changes in the composition of microbiota in response to earthworm presence are accompanied by a change in the microbial community diversity and/or function. Ultimately, this knowledge will help us to understand the functional importance of earthworms on the stabilization of organic matter from a microbial viewpoint, thereby contributing to minimize

the potential risks related to the use of animal manures an organic amendments.

*Departamento de Ecoloxía e Bioloxía Animal, Facultade de Bioloxía, Universidade de Vigo,*

Marina Fernández- Delgado Juárez is in receipt of a PhD fellowship from Doktoratsstipendium aus der Nachwuchsförderung der Universität Innsbruck. María Gómez Brandón is financially supported by a postdoctoral research grant from *Fundación Alfonso Martín Escudero*. The authors acknowledge Paul Fraiz for his highly valuable help in

[1] Moral R, Paredes C, Bustamante MA, Marhuenda-Egea R, Bernal, MP (2009) Utilisation of Manure Composts by High-Value Crops: Safety and Environmental Challenges.

[2] Faostat – Food and Agriculture Organization of the United Nations, FAO Statistical

[3] Holm-Nielsen JB, Seado TAl, Oleskowicz-Popiel P (2009) The Future of Anaerobic

[4] Massé DI, Talbot G, Gilbert Y (2011) On Farm Biogas Production: A Method to Reduce GHG Emissions and Develop more Sustainable Livestock Operations. Anim. Feed Sci.

Digestion and Biogas Utilization. Bioresour. Technol. 100:5478-5484.

*University of Innsbruck, Institute of Microbiology, Innsbruck, Austria*

, Marina Fernández-Delgado Juárez and Heribert Insam

**Author details**

Jorge Domínguez

language editing.

**6. References**

 \*

Bioresour. Technol. 100:5454-5460.

Tech. 166-167:436-445.

Corresponding Author

Databases (2003). Available: http://faostat.fao.org/.

*Vigo, Spain*

María Gómez-Brandón\*

**Acknowledgement**

**Figure 6.** Abundance of *Clostridium perfringens* in the original materials (manure (M), compost (C), vermicompost (Vc) and anaerobic digested slurry (AS)) and in the unamended and amended soils at the three incubation times (0, 15 and 60 days). Values are means ± SE.

### **4. Conclusions**

The intensity and concentrated activity of the livestock industry generate huge amounts of biodegradable wastes, which must be managed with appropriate disposal practices to avoid a negative impact on the environment. Composting is one of the best-known processes for the biological stabilization of solid organic wastes under aerobic conditions. Vermicomposting, i.e. the processing of organic wastes by earthworms under aerobic and mesophilic conditions, has also proven to be a low-cost and rapid technique. Although aerobic processes are thermodynamically more favorable, the manure treatment by anaerobic digestion has become increasingly important due to its energetic potential. A stabilised end product that can be used as an organic amendment is obtained under both aerobic and anaerobic conditions. A multi-parameter approach applying diverse methods constitutes the best option for evaluating the stability/maturity degree of the organic matter, which is of utmost importance for its safe use for the agriculture and the environment. During biodegradation all organic matter goes through the microbial decomposer pool and thus, further knowledge about the changes occurring during the process from a microbial viewpoint will contribute to further develop efficient strategies for the management of animal manures.

### **5. Outlook**

Waste management continues to be a topic of increasing importance. Deeper knowledge of the different biological processes involved in the recycling and recovery of waste components is thus of utmost importance in order to contribute towards more sustainable production and consumption systems. For example, biowaste may be used as a resource to produce high quality lactic acid and protein, as well as biogas in a cascade procedure. Briefly, biowaste is separated into two phases, i.e. a solid phase that is used to feed *Hermetia illucens* larvae that may be harvested as an excellent source of protein for feeding chicken or fish, and the liquid phase that is microbially fermented to the platform chemical lactic acid. The remaining residuals may eventually be used for biogas production, a cascade process that utilizes the organic waste at its highest level. Furthermore, although an interest in vermicompost research and technology has been increasing over recent years, and the body of knowledge available is quite large, there are still some important topics to be investigated. During vermicomposting, earthworm activity helps microbial communities to use the available energy more efficiently and plays a key role in shaping the structure of the microbial communities during the process. Hence, it is of future interest to evaluate whether the changes in the composition of microbiota in response to earthworm presence are accompanied by a change in the microbial community diversity and/or function. Ultimately, this knowledge will help us to understand the functional importance of earthworms on the stabilization of organic matter from a microbial viewpoint, thereby contributing to minimize the potential risks related to the use of animal manures an organic amendments.

### **Author details**

262 Biomass Now – Cultivation and Utilization

**4. Conclusions**

animal manures.

**5. Outlook**

**Figure 6.** Abundance of *Clostridium perfringens* in the original materials (manure (M), compost (C), vermicompost (Vc) and anaerobic digested slurry (AS)) and in the unamended and amended soils at the

The intensity and concentrated activity of the livestock industry generate huge amounts of biodegradable wastes, which must be managed with appropriate disposal practices to avoid a negative impact on the environment. Composting is one of the best-known processes for the biological stabilization of solid organic wastes under aerobic conditions. Vermicomposting, i.e. the processing of organic wastes by earthworms under aerobic and mesophilic conditions, has also proven to be a low-cost and rapid technique. Although aerobic processes are thermodynamically more favorable, the manure treatment by anaerobic digestion has become increasingly important due to its energetic potential. A stabilised end product that can be used as an organic amendment is obtained under both aerobic and anaerobic conditions. A multi-parameter approach applying diverse methods constitutes the best option for evaluating the stability/maturity degree of the organic matter, which is of utmost importance for its safe use for the agriculture and the environment. During biodegradation all organic matter goes through the microbial decomposer pool and thus, further knowledge about the changes occurring during the process from a microbial viewpoint will contribute to further develop efficient strategies for the management of

Waste management continues to be a topic of increasing importance. Deeper knowledge of the different biological processes involved in the recycling and recovery of waste components is thus of utmost importance in order to contribute towards more sustainable production and consumption systems. For example, biowaste may be used as a resource to produce high quality lactic acid and protein, as well as biogas in a cascade procedure. Briefly, biowaste is separated into two phases, i.e. a solid phase that is used to feed *Hermetia illucens* larvae that may be harvested as an excellent source of protein for feeding chicken or fish, and the liquid phase that is microbially fermented to the platform chemical lactic acid.

three incubation times (0, 15 and 60 days). Values are means ± SE.

María Gómez-Brandón\* , Marina Fernández-Delgado Juárez and Heribert Insam *University of Innsbruck, Institute of Microbiology, Innsbruck, Austria*

Jorge Domínguez *Departamento de Ecoloxía e Bioloxía Animal, Facultade de Bioloxía, Universidade de Vigo, Vigo, Spain*

### **Acknowledgement**

Marina Fernández- Delgado Juárez is in receipt of a PhD fellowship from Doktoratsstipendium aus der Nachwuchsförderung der Universität Innsbruck. María Gómez Brandón is financially supported by a postdoctoral research grant from *Fundación Alfonso Martín Escudero*. The authors acknowledge Paul Fraiz for his highly valuable help in language editing.

### **6. References**

<sup>\*</sup> Corresponding Author

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**Chapter 11**

© 2013 Gunawardena and Fernando, 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

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Gunawardena and Fernando, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

properly cited.

**Methods and Applications of**

**Deoxygenation for the Conversion**

Duminda A. Gunawardena and Sandun D. Fernando

Additional information is available at the end of the chapter

could lead to net CO2 emission reductions [4].

**1.1. Biomass production**

http://dx.doi.org/10.5772/53983

**1. Introduction**

**of Biomass to Petrochemical Products**

Depleting reserves, uncertain economics, and environmental concerns associated with crude oil have prompted an extensive search for alternatives for producing transportation fuels. Biomass has been given close scrutiny due to the emphasis on climate change and the ability of biomass based energy systems to mitigate greenhouse gas (GHG) emissions. Further, its ability to produce fuels and chemicals that are identical to those produced using petroleum resources, makes biomass an important alternative raw material [1, 2]. Biomass can be considered clean, as it contains negligible amounts of sulfur and nitrogen. Consequently, the emissions of SO2, NO*x* are extremely low compared to conventional fossil fuels. The overall CO2 emission is considered to be neutral, as CO2 is recycled by the plants through photosynthesis [3]. Moreover, substitution of fossil fuels with biomass-based counterparts

Biomass encompasses all plant and plant-derived materials as well as animal matter and animal manure. Due to its abundance biomass has to be considered as a vital source of energy to satisfy the global energy demand. Table 1 shows the breakdown of primary energy sources and their contribution to total world energy demand during 1973 and 2009. It is evident that the contribution from biomass is only 10% over this period and in the global scale bioenergy has not yet made any significant impact. However, according to a report published by the United States Department of Agriculture, biomass derived energy has become the highest contributor among all the renewable sources during 2003. According to US statistics 190 million dry tons of biomass is consumed per year, which is equivalent to

**Chapter 11**

## **Methods and Applications of Deoxygenation for the Conversion of Biomass to Petrochemical Products**

Duminda A. Gunawardena and Sandun D. Fernando

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53983

### **1. Introduction**

272 Biomass Now – Cultivation and Utilization

[125] McLachlan KL, Chong C, Voroney RP, Liu HW, Holbein BE (2004) Assesing the Potential Phytotoxicity of Digestates during Processing of Municipal Solid Waste by

[127] Matsunaka T, Sawamoto T, Ishimura H, Takamura K, Takekawa A (2006) Efficient Use of Digested Cattle Sslurry from Biogas Plant with respect to Nitrogen Recycling in

[128] Sidhu JPS, Toze SG (2009) Human Pathogens and their Indicators in Biosolids: a

[129] Bagge E, Sahlström L, Albihn A (2005) The Effect of Hygienic Treatment on the

[130] Sählström L (2003) A Review of Survival of Pathogenic Bacteria in Organic Waste

[131] Olsen JE, Larsen HE (1987) Bacterial Decimation Times in Anaerobic Digestions of

[132] Chauret C, Springthorpe S, Sattar S (1999) Fate of Cryptosporidium oocysts, Giardia cysts, and Microbial Indicators during Waste- Water Treatment and Anaerobic Sludge

[133] Aitken MD, Walters GW, Crunk PL, Willis JL, Farrell JB, Schafer PL, Arnett C, Turner BG (2005) Laboratory Evaluation of Thermophilic-Anaerobic Digestion to Produce Class A Biosolids. 1.Stabilization Performance of a Continuous-Flow Reactor at Low

[134] Pepper IL, Brooks JP, Gerba CP (2006) Pathogens in Biosolids. Advance. Agron. 90:1-41. [135] Crane SR, Moore JA (1986) Modeling Enteric Bacterial Die-Off—a Review. Water Air

[136] Sinton L W, Braithwaite R R, Hall CH, Mackenzie ML (2007) Survival of Indicator and Pathogenic Bacteria in Bovine Feces on Pasture. Appl. Environ. Microbiol. 73:7917–7925. [137] Franke-Whittle IH, Insam H. Pathogen Survival After the Composting, Anaerobic digestion and Alkaline Hydrolysis of Slaughterhouse Wastes: A Review. Critical

[138] ISO 4832 (1991) Microbiology-General guidance for the Enumeration of Coliforms-

[139] ISO 16649-2 (2001) Microbiology of Food and Animal Feeding Stuffs-Horizontal Method for the Enumeration of β-glucuronidase-positive *Escherichia coli*-Part 2: Colony-

[140] ISO 7937 (2004) Microbiology of Food and Animal Feeding Stuffs-Horizontal Method for the Enumeration of *Clostridium perfringens*-Part 2: Colony-count Technique, ISO,

Microbial Flora of Biowaste at Biogas Plants. Water Res. 39: 4879-4880.

Anaerobic Digestion: Comparison to Aerobic Composts. Acta Hort. 638: 225-230. [126] Goberna M, Podmirseg SM, Waldhuber S, Knapp BA, García C, Insam H (2011) Pathogenic Bacteria and Mineral N in Soils Following the Land Spreading of Biogas

Digestates and Fresh Manure. Appl. Soil Ecol. 49:18-25.

Grassland. International Congress Series 1293: 242-250.

Literature Review. Environ. International 35:187–201.

Used in Biogas Plants. Bioresour. Technol. 87:161-166.

Residence Time. Water Environ. Res. 77: 3019–3027.

Animal Slurries. Biol. Waste. 21:153-160.

Digestion. Can. J. Microbiol. 45:257–262.

Reviews in Microbiology (under review)

Colony Count Technique, ISO, Geneva.

count Technique, ISO, Geneva.

Geneva.

Soil Poll. 27: 411-439.

Depleting reserves, uncertain economics, and environmental concerns associated with crude oil have prompted an extensive search for alternatives for producing transportation fuels. Biomass has been given close scrutiny due to the emphasis on climate change and the ability of biomass based energy systems to mitigate greenhouse gas (GHG) emissions. Further, its ability to produce fuels and chemicals that are identical to those produced using petroleum resources, makes biomass an important alternative raw material [1, 2]. Biomass can be considered clean, as it contains negligible amounts of sulfur and nitrogen. Consequently, the emissions of SO2, NO*x* are extremely low compared to conventional fossil fuels. The overall CO2 emission is considered to be neutral, as CO2 is recycled by the plants through photosynthesis [3]. Moreover, substitution of fossil fuels with biomass-based counterparts could lead to net CO2 emission reductions [4].

### **1.1. Biomass production**

Biomass encompasses all plant and plant-derived materials as well as animal matter and animal manure. Due to its abundance biomass has to be considered as a vital source of energy to satisfy the global energy demand. Table 1 shows the breakdown of primary energy sources and their contribution to total world energy demand during 1973 and 2009. It is evident that the contribution from biomass is only 10% over this period and in the global scale bioenergy has not yet made any significant impact. However, according to a report published by the United States Department of Agriculture, biomass derived energy has become the highest contributor among all the renewable sources during 2003. According to US statistics 190 million dry tons of biomass is consumed per year, which is equivalent to

© 2013 Gunawardena and Fernando, 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. © 2013 Gunawardena and Fernando, licensee InTech. This is a paper 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.

3% of current energy consumption [5]. Even though it is hard to conceive that biomass can totally eliminate the use of fossil fuel consumption, it can complement the renewable energy sector together with other sources like wind, solar and geothermal energy.

Methods and Applications of Deoxygenation for the Conversion of Biomass to Petrochemical Products 275

as biodiesel using the transesterification process. This process converts triglyceride in the

Biochemical conversion primarily involves using microorganisms or enzymes to breakdown complex chemicals present in biomass into simpler sugars or alcohols. Biomass conversion to alcohols such as ethanol has attracted wide interest in the recent past. The corn to ethanol technology is mature and is commercial in the US. Nevertheless, this technology has created some debate in the context that corn is still a primary food to many around the world. To circumvent this issue, significant strides have been made in the use of lingo-cellulosic biomass as a source for ethanol. In this method enzymes are used to breakdown cellulose to its monomers and subsequently subjected to fermentation under anaerobic conditions using

Thermochemical conversion is another key process that uses heat to induce chemical transformations in the biomass constituents to produce energetically useful intermediate and/or end products. Conversion techniques available under this umbrella can be categorized into four main processes as represented in figure (1). Energy generation by combustion of biomass can be considered as the most archaic [7]. However, increasing demand for transport fuels has led to the development of other processes that involve converting biomass into liquid and gaseous products [8] such as gasification, pyrolysis and liquefaction. Gasification is the conversion of biomass to a mixture of gases called synthesis gas (or syngas) that primarily consists of hydrogen, carbon monoxide, carbon dioxide and methane. During gasification, biomass is heated under an oxygen-lean environment [9]. Synthesis gas can be directly used in an internal combustion engine or can be converted to liquid fuels using a method known as Fisher-Tropsch (FT) synthesis [7, 10]. Fisher-Tropsch process is considered to be quite energy intensive and therefore, is not yet believed to be economical to compete with petroleum fuels. Nevertheless, active research is still in progress to improve the process [11, 12]. Fermentation of synthesis gas to alcohols

(primarily ethanol) using microorganisms is also an active area of research [13].

reactor. The term bio-crude represents a more deoxygenated liquid product.

Pyrolysis and liquefaction are two closely related routes targeted towards producing liquids – called bio-oil or bio-crude [14, 15]. Although not universally accepted, the term bio-oil generally refers to the highly oxygenated liquid product that directly exits a pyroloysis

Pyrolysis, unlike gasification, takes place in an oxygen-free atmosphere. The most common technique for producing a large volume of condensable fraction is the fast pyrolysis process. Several reactor configurations used for fast pyrolysis include: ablative, entrained flow, rotating core, vacuum pyrolysis, circulating fluidized bed, and deep bubbling fluidized bed reactors [16]. During fast pyrolysis, biomass is heated at a very high heating rate (eg. 103– 104 K/s)[17]. The temperature at which the thermal scission of biomass material such as cellulose and lignin take place is around 450–550 °C. The bio-oil portion is originally in the vapor-phase and obtained by quenching the volatile output. The yields of the condensablefraction are reported to be as high as 70–80%. According to some kinetics studies conducted for different biomass, the frequency factor for pyrolysis varies between 109-1011 orders of

presence of alcohol to fatty acid alkyl esters .

microorganisms.

**Table 1.** The world's primary energy sources with its contribution. [6]

### **1.2. Biomass to energy conversions**

Biomass has been intimately connected with the everyday life of people since prehistoric times. With the advent of fire by rubbing splinters, humans began to exploit the chemical energy of biomass to produce heat. Ever since, various methods have been developed to convert chemical energy present in biomass to useful heat energy. These conversion methods can be summarized as shown in figure (1).

**Figure 1.** Different categories of biomass conversion

Physical conversion of biomass typically involves pressing the plant (or animal) matter to produce triglyceride oils. Triglycerides cannot be used directly as transport fuels and needs to be processed further. Triglycerides can be converted into a renewable fuel widely known as biodiesel using the transesterification process. This process converts triglyceride in the presence of alcohol to fatty acid alkyl esters .

274 Biomass Now – Cultivation and Utilization

3% of current energy consumption [5]. Even though it is hard to conceive that biomass can totally eliminate the use of fossil fuel consumption, it can complement the renewable energy

> Primary energy sources 1973 2009 The total world energy demand (Mtoe) 6111 12150 Oil 46.00% 32.80% Natural gas 16.00% 20.90% Nuclear 0.90% 5.80% Hydro 1.80% 2.30% Biofuels and waste 10.60% 10.20% Coal / peat 24.60% 27.20% other 0.10% 0.80%

Biomass has been intimately connected with the everyday life of people since prehistoric times. With the advent of fire by rubbing splinters, humans began to exploit the chemical energy of biomass to produce heat. Ever since, various methods have been developed to convert chemical energy present in biomass to useful heat energy. These conversion

Physical conversion of biomass typically involves pressing the plant (or animal) matter to produce triglyceride oils. Triglycerides cannot be used directly as transport fuels and needs to be processed further. Triglycerides can be converted into a renewable fuel widely known

sector together with other sources like wind, solar and geothermal energy.

**Table 1.** The world's primary energy sources with its contribution. [6]

**1.2. Biomass to energy conversions**

methods can be summarized as shown in figure (1).

**Figure 1.** Different categories of biomass conversion

Biochemical conversion primarily involves using microorganisms or enzymes to breakdown complex chemicals present in biomass into simpler sugars or alcohols. Biomass conversion to alcohols such as ethanol has attracted wide interest in the recent past. The corn to ethanol technology is mature and is commercial in the US. Nevertheless, this technology has created some debate in the context that corn is still a primary food to many around the world. To circumvent this issue, significant strides have been made in the use of lingo-cellulosic biomass as a source for ethanol. In this method enzymes are used to breakdown cellulose to its monomers and subsequently subjected to fermentation under anaerobic conditions using microorganisms.

Thermochemical conversion is another key process that uses heat to induce chemical transformations in the biomass constituents to produce energetically useful intermediate and/or end products. Conversion techniques available under this umbrella can be categorized into four main processes as represented in figure (1). Energy generation by combustion of biomass can be considered as the most archaic [7]. However, increasing demand for transport fuels has led to the development of other processes that involve converting biomass into liquid and gaseous products [8] such as gasification, pyrolysis and liquefaction. Gasification is the conversion of biomass to a mixture of gases called synthesis gas (or syngas) that primarily consists of hydrogen, carbon monoxide, carbon dioxide and methane. During gasification, biomass is heated under an oxygen-lean environment [9]. Synthesis gas can be directly used in an internal combustion engine or can be converted to liquid fuels using a method known as Fisher-Tropsch (FT) synthesis [7, 10]. Fisher-Tropsch process is considered to be quite energy intensive and therefore, is not yet believed to be economical to compete with petroleum fuels. Nevertheless, active research is still in progress to improve the process [11, 12]. Fermentation of synthesis gas to alcohols (primarily ethanol) using microorganisms is also an active area of research [13].

Pyrolysis and liquefaction are two closely related routes targeted towards producing liquids – called bio-oil or bio-crude [14, 15]. Although not universally accepted, the term bio-oil generally refers to the highly oxygenated liquid product that directly exits a pyroloysis reactor. The term bio-crude represents a more deoxygenated liquid product.

Pyrolysis, unlike gasification, takes place in an oxygen-free atmosphere. The most common technique for producing a large volume of condensable fraction is the fast pyrolysis process. Several reactor configurations used for fast pyrolysis include: ablative, entrained flow, rotating core, vacuum pyrolysis, circulating fluidized bed, and deep bubbling fluidized bed reactors [16]. During fast pyrolysis, biomass is heated at a very high heating rate (eg. 103– 104 K/s)[17]. The temperature at which the thermal scission of biomass material such as cellulose and lignin take place is around 450–550 °C. The bio-oil portion is originally in the vapor-phase and obtained by quenching the volatile output. The yields of the condensablefraction are reported to be as high as 70–80%. According to some kinetics studies conducted for different biomass, the frequency factor for pyrolysis varies between 109-1011 orders of magnitude. This is an indication of how fast the reaction would occur during a short residence time [18].

Methods and Applications of Deoxygenation for the Conversion of Biomass to Petrochemical Products 277

**Figure 3.** Different industrial applications of bio-oil derived from biomass (In formation adapted from

**Figure 4.** Relative distribution of chemical compounds in bio-oil (In formation adapted from Adjaye et

Bio-oil has already been tested in furnaces and gas turbines [21], as well as in space heaters and in boilers[24, 25], as represented in figure (3). Although bio-oil has potential as a crudeoil alternative, problems have been reported when bio-oil was used in such applications. These include, blocking filters by high levels of char particulates, high viscosity causing

pumping issues, and corrosion from the low pH [26].

Bridgwater et al. [21].)

al. [23].)

Since its chemical complexity, the biomass pyrolysis reaction has been studied by using model compounds like cellulose. A set of possible reactions paths under different heating conditions is presented in figure (2)[19, 20].

The composition of bio-oil is substantially different from crude petroleum due to the presence of high concentrations of oxygenates. Biomass to bio-oil pyrolysis stoichiometry can be represented using an empirical formula as shown in eq.(1).

**Figure 2.** Cellulose pyrolysis with the products at different heating conditions. (Information for scheme was adapted from Huber et al. [20])

Bio-oil properties are highly variable and depend heavily on the type of biomass. High fibrous biomass that contains high amounts of lignin is considered to be the most effective for the production of bio-oil for fuel applications. Further, the oil-yields and composition of bio-oils highly depend on the process (pyrolysis/ liquefaction) used. Fast pyrolysis yields higher amounts of bio-oil compared to slow pyrolysis whereas liquefaction produces low amounts of oxygenated compounds as compared to fast pyrolysis. Oxygenated compounds such as aldehydes, alcohols, ketones, and carboxylic acids can be seen in bio-oils in varying degrees. A typical product distribution of bio-oil is depicted in figure (4). It can be seen that bio-oil contains large amounts of ketones, carboxylic acids and aldehydes [22].

Methods and Applications of Deoxygenation for the Conversion of Biomass to Petrochemical Products 277

276 Biomass Now – Cultivation and Utilization

conditions is presented in figure (2)[19, 20].

can be represented using an empirical formula as shown in eq.(1).

residence time [18].

CO, CO2, H2O

O

OH

H2O oil

**Figure 2.** Cellulose pyrolysis with the products at different heating conditions. (Information for scheme

Bio-oil properties are highly variable and depend heavily on the type of biomass. High fibrous biomass that contains high amounts of lignin is considered to be the most effective for the production of bio-oil for fuel applications. Further, the oil-yields and composition of bio-oils highly depend on the process (pyrolysis/ liquefaction) used. Fast pyrolysis yields higher amounts of bio-oil compared to slow pyrolysis whereas liquefaction produces low amounts of oxygenated compounds as compared to fast pyrolysis. Oxygenated compounds such as aldehydes, alcohols, ketones, and carboxylic acids can be seen in bio-oils in varying degrees. A typical product distribution of bio-oil is depicted in figure (4). It can be seen that

O

HO

HO

magnitude. This is an indication of how fast the reaction would occur during a short

Since its chemical complexity, the biomass pyrolysis reaction has been studied by using model compounds like cellulose. A set of possible reactions paths under different heating

The composition of bio-oil is substantially different from crude petroleum due to the presence of high concentrations of oxygenates. Biomass to bio-oil pyrolysis stoichiometry

CH O 0.71CH O 0.21CH O 0.08CH O 1.46 0.67 1.98 0.76 0.1 0.15 0.44 1.23

Unsaturated polymer Coke

O HO O

O

Bio oil Char Gas

CO,CO2, CH4, H2O

> CO,CO2, CH4, H2O

> CO,CO2, CH4, H2O

(1)

Slow pyrolysis

C (0.1-1<sup>o</sup> C/s)

Fast pyrolysis

Flash pyrolysis

C (>1000 <sup>o</sup>

C/s)

C (10-200<sup>o</sup>

C/s)

< 300 <sup>o</sup>

300 - 500 o

>500 <sup>o</sup>

Tars oil

Coke

Coke

**Figure 3.** Different industrial applications of bio-oil derived from biomass (In formation adapted from Bridgwater et al. [21].)

**Figure 4.** Relative distribution of chemical compounds in bio-oil (In formation adapted from Adjaye et al. [23].)

Bio-oil has already been tested in furnaces and gas turbines [21], as well as in space heaters and in boilers[24, 25], as represented in figure (3). Although bio-oil has potential as a crudeoil alternative, problems have been reported when bio-oil was used in such applications. These include, blocking filters by high levels of char particulates, high viscosity causing pumping issues, and corrosion from the low pH [26].

Liquefaction is another approach to producing bio-oils. The concept of oil production using biomass in hot water surfaced in the early 1920's. However, a more robust and effective method was not available until Pittsburg Energy Research Center (PERC) in the 70's demonstrated the use of carbon monoxide, steam and sodium carbonate catalyst at Albany Biomass Liquefaction facility in Oregon USA. Detailed information on this method can be found elsewhere [27].

Methods and Applications of Deoxygenation for the Conversion of Biomass to Petrochemical Products 279

content are considered problematic [29, 30]. Additionally, the presence of oxygenates and water in the bio-oil reduces the heating value. Consequently, upgrading is necessary to

As shown in the figure (5) upgrading improves the H/C molar ratio together with increasing the heating value. In contrast, crude petroleum is lean in oxygenated compounds and therefore, most of the chemistries developed to date are based on adding functional groups to increase its activity. As a result, there is only limited knowledge on techniques available to remove functionality from highly oxygenated compounds. However the upgrading of bio-oil by removing of oxygen (deoxygenation) is considered as one of the most intricate challenges we face today in getting bio-oil to a form that is applicable as a fuel intermediate. As discussed above, chemical species present in biomass derived liquid bio-oil can be categorized into alcohols, aldehyde / ketones, carboxylic acids, phenols and furans. Therefore the upgrading process of bio-oil involves removal of various oxygenated species and can be collectively called as deoxygenation. The deoxygenation process predominantly involves three reaction classes, i.e., dehydration, decarbonilation and decarboxylation.

**Figure 5.** Change of characteristics before and after upgrading bio-oil derived from soybean stalk (

As previously mentioned, deoxygenation involves the removal of functionality of biomass constituents associated with -OH, -COOH, and -C=O. The bond dissociation energies (BDE) for these functional groups are quite high and can be written in descending order as follows: C-O (1076.5 kJ/mol) > C=O ( 749 kJ/mol) > C-C (610.0 kJ/mol) > O-H (429.99 kJ/mol) > C-H

make it useful as a fuel.

Information adapted from Li et al. [8]. )

(338.4 kJ/mol).

**2.2. Reactions involved in deoxygenation**

The quality of bio-oil from the liquefaction process is reported to be superior to that obtained from pyrolysis. Liquefaction of biomass using super -critical methods can be considered as one of the more recent techniques under investigation.

Conversion of biomass to a product that is compatible with existing petroleum refinery infrastructure is prudent in several fronts. First, this will allow biomass to be converted into fuels and chemicals that are identical to what we use today (such as gasoline, diesel and jet fuels). So, such fuels could be used in present-day automobiles with no engine modifications. On the other hand, usage of existing fuel production and distribution of infrastructure helps long-term sustainability of biomass-to-fuels technology [28].

The challenge of converting bio-oil into a hydrocarbon fuel has been effective for the removal of functional groups that contain oxygenates (–OH, -COH, -COOH, etc.). The oxygen content of biomass-derived bio-oil is estimated to be 35-40% with a heating value between 16 and 19 MJ/kg [8]. In the recent years, there has been an unprecedented growth of research and developmental efforts related to conversion technologies and the information is scattered. Accordingly, the overall objective of this review is to assimilate this information and compare the status of key deoxygenation technologies.

### **2. Deoxygenation**

The presence of oxygen in cellulose, hemicelluloses and lignin is the primarily reason for biomass derived bio-oil to be highly functionalized. Cellulose and hemicellulose have common structural building blocks which are glucose and xylose respectively. Cellulose which is the most abundant component in terrestrial biomass is a crystalline polymer, and therefore, is quite difficult to chemically transform. Lignin which is abundant in biomass is mainly an amorphous poly aromatic polymer. Because of this complex heterogeneity of biomass, the liquid derived from the pyrolysis or liquefaction process contains a variety of different chemical species which can be roughly estimated to be around 400.

### **2.1. Importance of deoxygenation**

The properties of bio-oil are significantly affected by its chemical composition. Unlike crude petrolium, bio-oil constituents have numerous functional groups as shown in figure (4). The high functionality decreases the stability of the oils and results in polymerization. A direct consequence is the increase of viscosity of bio-oils with time which in turn raises concerns about the feasibility of using bio-oil products as substitutes for petrochemical fuels. In addition to instability, low pH values and the presence of a high water content and ash content are considered problematic [29, 30]. Additionally, the presence of oxygenates and water in the bio-oil reduces the heating value. Consequently, upgrading is necessary to make it useful as a fuel.

As shown in the figure (5) upgrading improves the H/C molar ratio together with increasing the heating value. In contrast, crude petroleum is lean in oxygenated compounds and therefore, most of the chemistries developed to date are based on adding functional groups to increase its activity. As a result, there is only limited knowledge on techniques available to remove functionality from highly oxygenated compounds. However the upgrading of bio-oil by removing of oxygen (deoxygenation) is considered as one of the most intricate challenges we face today in getting bio-oil to a form that is applicable as a fuel intermediate.

As discussed above, chemical species present in biomass derived liquid bio-oil can be categorized into alcohols, aldehyde / ketones, carboxylic acids, phenols and furans. Therefore the upgrading process of bio-oil involves removal of various oxygenated species and can be collectively called as deoxygenation. The deoxygenation process predominantly involves three reaction classes, i.e., dehydration, decarbonilation and decarboxylation.

**Figure 5.** Change of characteristics before and after upgrading bio-oil derived from soybean stalk ( Information adapted from Li et al. [8]. )

### **2.2. Reactions involved in deoxygenation**

278 Biomass Now – Cultivation and Utilization

found elsewhere [27].

**2. Deoxygenation**

**2.1. Importance of deoxygenation**

Liquefaction is another approach to producing bio-oils. The concept of oil production using biomass in hot water surfaced in the early 1920's. However, a more robust and effective method was not available until Pittsburg Energy Research Center (PERC) in the 70's demonstrated the use of carbon monoxide, steam and sodium carbonate catalyst at Albany Biomass Liquefaction facility in Oregon USA. Detailed information on this method can be

The quality of bio-oil from the liquefaction process is reported to be superior to that obtained from pyrolysis. Liquefaction of biomass using super -critical methods can be

Conversion of biomass to a product that is compatible with existing petroleum refinery infrastructure is prudent in several fronts. First, this will allow biomass to be converted into fuels and chemicals that are identical to what we use today (such as gasoline, diesel and jet fuels). So, such fuels could be used in present-day automobiles with no engine modifications. On the other hand, usage of existing fuel production and distribution of

The challenge of converting bio-oil into a hydrocarbon fuel has been effective for the removal of functional groups that contain oxygenates (–OH, -COH, -COOH, etc.). The oxygen content of biomass-derived bio-oil is estimated to be 35-40% with a heating value between 16 and 19 MJ/kg [8]. In the recent years, there has been an unprecedented growth of research and developmental efforts related to conversion technologies and the information is scattered. Accordingly, the overall objective of this review is to assimilate this information

The presence of oxygen in cellulose, hemicelluloses and lignin is the primarily reason for biomass derived bio-oil to be highly functionalized. Cellulose and hemicellulose have common structural building blocks which are glucose and xylose respectively. Cellulose which is the most abundant component in terrestrial biomass is a crystalline polymer, and therefore, is quite difficult to chemically transform. Lignin which is abundant in biomass is mainly an amorphous poly aromatic polymer. Because of this complex heterogeneity of biomass, the liquid derived from the pyrolysis or liquefaction process contains a variety of

The properties of bio-oil are significantly affected by its chemical composition. Unlike crude petrolium, bio-oil constituents have numerous functional groups as shown in figure (4). The high functionality decreases the stability of the oils and results in polymerization. A direct consequence is the increase of viscosity of bio-oils with time which in turn raises concerns about the feasibility of using bio-oil products as substitutes for petrochemical fuels. In addition to instability, low pH values and the presence of a high water content and ash

different chemical species which can be roughly estimated to be around 400.

infrastructure helps long-term sustainability of biomass-to-fuels technology [28].

considered as one of the more recent techniques under investigation.

and compare the status of key deoxygenation technologies.

As previously mentioned, deoxygenation involves the removal of functionality of biomass constituents associated with -OH, -COOH, and -C=O. The bond dissociation energies (BDE) for these functional groups are quite high and can be written in descending order as follows:

C-O (1076.5 kJ/mol) > C=O ( 749 kJ/mol) > C-C (610.0 kJ/mol) > O-H (429.99 kJ/mol) > C-H (338.4 kJ/mol).

Higher BDE for a particular functional group implies that the activation energy required for dissociating this bond (deoxygenation) would also be high. This would dictate rigorous reaction conditions for particularly C-O and C=O bond scissions. The presence of large amount of C=O groups in the pyrolysis products can be related to the higher activation energies required for dissociation of these bonds. However, using appropriate catalysts, these high activation energies could be overcome.

Methods and Applications of Deoxygenation for the Conversion of Biomass to Petrochemical Products 281

O

Si Al Si +

the deposition of large molecular weight hydrocarbons known as coke blocking the catalyst

Dehydration of ethanol has also been studied extensively with other catalysts. A study of the effect of different additives such as ZnO, Ga2O3, Mo2C and Re, on ZSM-5 on deoxygenation of alcohols has provided new insights on renewable aromatic hydrocarbons production [38]. The product selectivities for different catalysts are depicted in figure (8). It is clear that Ga2O3 performed better in terms of selectivity toward benzene, toluene and

Bronsted acid site Lewis acid site

CH2

<sup>O</sup> <sup>O</sup>

base site acid site

**Figure 7.** The proposed adsorption of methanol on ZSM-5.( Information was adapted from Chang et al.

Ethanol dehydration has been further studied in order to make ethylene as the precursor to make ethyl-tetra-butyl-ether (ETBE) [39, 40]. Ethers have gained much attention as a substitute for petroleum diesel. A study on ethanol dehydration to ethylene over dealuminated modernite (DM) and metals such as Zn, Mn, Co, Rh, Ni, Fe and Ag loaded DM has shown that Zn/DM and Zn/Ag/DM gives the highest selectivity to ethylene formation [40]. This indicates that incorporation of single metal or metal combinations onto dealuminated modernite makes the catalyst more selective toward ethylene production. Further , the results suggest that such combinations of metal and support would lower coke

formation as high molecular weight compounds were not produced significantly.

<sup>O</sup> <sup>H</sup> <sup>H</sup>

H

pores[37].

[31] )

xylenes found in gasoline.

O O

H

**Figure 6.** The two form of acid sites that exist in zeolites.

Si Al **-** Si

### *2.2.1. Dehydration*

Bio-oil has significant amounts of –OH groups in its components that require dehydration, i.e., removal of oxygen in the form of water, to make hydrocarbons. Dehydration occurs even during HDO process but what is considered here is the spontaneous removal of oxygen as water in the absence of extraneous H2.

Studies on dehydration as a method to produce motor fuel initiated several decades ago. With the advent of new catalysts, this area grew into new heights. A wide range of studies have been carried out with oxygenates ranging from simple methanol, to polyols like glycerol. The presence of light-molecular-weight alcohols such as methanol and ethanol in bio-oil is less common while phenolic compounds and polyols can be considered to be more abundant. However, studies on light alcohols give good insights into the chemistry and will be discussed in more detail below.

Dehydration of methanol to produce gasoline products such as benzene, toluene and xylene has been reported by different research groups [31-35]. In this regard, different heterogeneous catalysts have been studied. In particular, the molecular sieve ZSM-5 has received great attention for dehydration reactions. It has been widely accepted that Brǿnsted acid sites of the ZSM-5 catalyst play a crucial role for the dehydration reaction. Acid sites donates protons to the hydroxyl group of the oxygenate as shown in figure (7) to form water instigating dehydration.

Addition of metal oxides onto the catalyst framework was reported to increase the acidity of the support material. As a result, this would enhance dehydration reactions in turn facilitating the formation of higher molecular weight hydrocarbons. For example, methanol conversion to gasoline range hydrocarbons over metal oxides (such as ZnO and CuO) supported on HZSM-5 at temperature 400 OC and 1 atm pressure was reported [36]. The results indicate that pure HZSM-5 produced the lowest yields compared to metal-oxidepromoted catalyst such as CuO/ HZSM-5, CuO /ZnO/HZSM-5, ZnO/HZSM-5. The presence of CuO significantly increased the yields of aromatics. It was further concluded that addition of ZnO over CuO significantly reduced the catalyst deactivation potential [36]. Studies conducted to find the effect of different CuO loadings on methanol conversion indicate that the highest conversion of 97% was obtained when the CuO loading was at 7%. It was observed that further increase of oxide loading decreased methanol conversion. This behavior was attributed to loss of acid sites on the support. A subsequent catalyst deactivation study indicated that the catalyst deactivation increased with the increase in CuO loading. It was concluded that the deactivation of the catalyst occurred mainly due to the deposition of large molecular weight hydrocarbons known as coke blocking the catalyst pores[37].

Dehydration of ethanol has also been studied extensively with other catalysts. A study of the effect of different additives such as ZnO, Ga2O3, Mo2C and Re, on ZSM-5 on deoxygenation of alcohols has provided new insights on renewable aromatic hydrocarbons production [38]. The product selectivities for different catalysts are depicted in figure (8). It is clear that Ga2O3 performed better in terms of selectivity toward benzene, toluene and xylenes found in gasoline.

280 Biomass Now – Cultivation and Utilization

*2.2.1. Dehydration*

these high activation energies could be overcome.

oxygen as water in the absence of extraneous H2.

be discussed in more detail below.

instigating dehydration.

Higher BDE for a particular functional group implies that the activation energy required for dissociating this bond (deoxygenation) would also be high. This would dictate rigorous reaction conditions for particularly C-O and C=O bond scissions. The presence of large amount of C=O groups in the pyrolysis products can be related to the higher activation energies required for dissociation of these bonds. However, using appropriate catalysts,

Bio-oil has significant amounts of –OH groups in its components that require dehydration, i.e., removal of oxygen in the form of water, to make hydrocarbons. Dehydration occurs even during HDO process but what is considered here is the spontaneous removal of

Studies on dehydration as a method to produce motor fuel initiated several decades ago. With the advent of new catalysts, this area grew into new heights. A wide range of studies have been carried out with oxygenates ranging from simple methanol, to polyols like glycerol. The presence of light-molecular-weight alcohols such as methanol and ethanol in bio-oil is less common while phenolic compounds and polyols can be considered to be more abundant. However, studies on light alcohols give good insights into the chemistry and will

Dehydration of methanol to produce gasoline products such as benzene, toluene and xylene has been reported by different research groups [31-35]. In this regard, different heterogeneous catalysts have been studied. In particular, the molecular sieve ZSM-5 has received great attention for dehydration reactions. It has been widely accepted that Brǿnsted acid sites of the ZSM-5 catalyst play a crucial role for the dehydration reaction. Acid sites donates protons to the hydroxyl group of the oxygenate as shown in figure (7) to form water

Addition of metal oxides onto the catalyst framework was reported to increase the acidity of the support material. As a result, this would enhance dehydration reactions in turn facilitating the formation of higher molecular weight hydrocarbons. For example, methanol conversion to gasoline range hydrocarbons over metal oxides (such as ZnO and CuO) supported on HZSM-5 at temperature 400 OC and 1 atm pressure was reported [36]. The results indicate that pure HZSM-5 produced the lowest yields compared to metal-oxidepromoted catalyst such as CuO/ HZSM-5, CuO /ZnO/HZSM-5, ZnO/HZSM-5. The presence of CuO significantly increased the yields of aromatics. It was further concluded that addition of ZnO over CuO significantly reduced the catalyst deactivation potential [36]. Studies conducted to find the effect of different CuO loadings on methanol conversion indicate that the highest conversion of 97% was obtained when the CuO loading was at 7%. It was observed that further increase of oxide loading decreased methanol conversion. This behavior was attributed to loss of acid sites on the support. A subsequent catalyst deactivation study indicated that the catalyst deactivation increased with the increase in CuO loading. It was concluded that the deactivation of the catalyst occurred mainly due to

Bronsted acid site Lewis acid site

**Figure 7.** The proposed adsorption of methanol on ZSM-5.( Information was adapted from Chang et al. [31] )

Ethanol dehydration has been further studied in order to make ethylene as the precursor to make ethyl-tetra-butyl-ether (ETBE) [39, 40]. Ethers have gained much attention as a substitute for petroleum diesel. A study on ethanol dehydration to ethylene over dealuminated modernite (DM) and metals such as Zn, Mn, Co, Rh, Ni, Fe and Ag loaded DM has shown that Zn/DM and Zn/Ag/DM gives the highest selectivity to ethylene formation [40]. This indicates that incorporation of single metal or metal combinations onto dealuminated modernite makes the catalyst more selective toward ethylene production. Further , the results suggest that such combinations of metal and support would lower coke formation as high molecular weight compounds were not produced significantly.

Methods and Applications of Deoxygenation for the Conversion of Biomass to Petrochemical Products 283

RCO H RH CO 2 2 (2)

gave the highest yield of phenol. They concluded that vanadium, as an early transition metal, has the oxophilic property and had helped the efficient removal of oxygen from guaiacol in the form of water. Nevertheless, water formed during dehydration has a

Bio-oil contains considerable amounts of acids such as acetic, formic, and butyric acid, resulting in low pH values (c.a. pH 2-3). The presence of these acids creates various practical challenges for bio-oil applications. Highly corrosive nature makes bio-oil not suitable for applications with metals and rubber. Further, the presence of acids would increase O/C ratio and make bio-oil more reactive. Accordingly, it is critical to develop chemistries that can deal with carboxylic acids when upgrading bio-oil. Decarboxylation refers to the removal of oxygen in the form of CO2 from a carboxylated compound and can be given in a general

Insights on effective catalysts for removing oxygen in carboxylic acids can be obtained from studies on decarboxylation of model systems that include stearic, palmetic, benzoic, and heptanoic acids. Equations (3) and (4) depict the thermodynamic favorability of the decarboxylation reactions. The negative values for Go implies that the decarboxylation reactions of acetic and benzoic acids have a significant activation energy barrier to

Biodiesel research is an important area to look for information related to decarboxylation. Fatty acids and fatty acid methyl esters from biodiesel industry have been subjected further deoxygenation with the intension of obtaining higher quality liquid fuels [45, 46]. In one such study, Pd has been identified to be an active metal for decarboxylation of fatty acids present in plant oil. In this study, the ability to convert heptanoic acid to octane was investigated using Pd/SiO2 and Ni/Al2O3 catalysts [47-50]. It was reported that 98% acid conversion was obtained with Pd/SiO2 at 330oC but only 64% was reported by Ni/Al2O3 [44]. Pd supported on active carbon has also been tested as the catalyst for decarboxylation of stearic acid at 300oC. The results indicated that the reaction was selective toward *n*heptadecane [47]. Further, they claimed that conducting the reaction in the presence of hydrogen increased the rate of decarboxylation. A comparative study performed with thermal and catalytic deoxygenation of stearic acid further proved that catalytic deoxygenation is highly selective toward hydrocarbons. In this study, 5%Pd supported on mesoporous silica, SBA-15, MCM-41and zeolyte-Y has been used as the catalyst. It was reported that SBA-15 had a selectivity of 67% for n-pentadecane [48]. The study further

<sup>o</sup> CH CO H CH CO G 68.5 kJ / mol 32 4 2 (3)

<sup>o</sup> C H CO H C H CO G 54.3 kJ / mol 65 2 66 2 (4)

tendency to adsorb onto acid sites dramatic decreasing the catalyst activity.

*2.2.2. Decarboxylation*

equation as follows (eq.(2) ):

overcome[44].

**Figure 8.** (a) Selectivity towards different products from ethanol dehydration. (b) The catalysts and respective reaction conditions for each reaction.(Prepared with data from Barthos et al.[38])

There has been some studies on phenol dehydration [41, 42]. In once such study where HZSM-5 was used it has been observed that the reactivity of phenol and 2-methoxyphenol has been very low and that the catalyst had a greater tendency to form coke [42]. The rate of deactivation of the catalyst by coke formation reduced with increased water formation. In a separate study, lignin derivative guaiacol was attempted to be transformed to phenol at a temperature of 350 oC and 1atm pressure. In this study, first row transition metals (V to Zn) supported on alumina was tested [43]. Results indicate that vanadium oxide on alumina gave the highest yield of phenol. They concluded that vanadium, as an early transition metal, has the oxophilic property and had helped the efficient removal of oxygen from guaiacol in the form of water. Nevertheless, water formed during dehydration has a tendency to adsorb onto acid sites dramatic decreasing the catalyst activity.

### *2.2.2. Decarboxylation*

282 Biomass Now – Cultivation and Utilization

**Figure 8.** (a) Selectivity towards different products from ethanol dehydration. (b) The catalysts and respective reaction conditions for each reaction.(Prepared with data from Barthos et al.[38])

There has been some studies on phenol dehydration [41, 42]. In once such study where HZSM-5 was used it has been observed that the reactivity of phenol and 2-methoxyphenol has been very low and that the catalyst had a greater tendency to form coke [42]. The rate of deactivation of the catalyst by coke formation reduced with increased water formation. In a separate study, lignin derivative guaiacol was attempted to be transformed to phenol at a temperature of 350 oC and 1atm pressure. In this study, first row transition metals (V to Zn) supported on alumina was tested [43]. Results indicate that vanadium oxide on alumina Bio-oil contains considerable amounts of acids such as acetic, formic, and butyric acid, resulting in low pH values (c.a. pH 2-3). The presence of these acids creates various practical challenges for bio-oil applications. Highly corrosive nature makes bio-oil not suitable for applications with metals and rubber. Further, the presence of acids would increase O/C ratio and make bio-oil more reactive. Accordingly, it is critical to develop chemistries that can deal with carboxylic acids when upgrading bio-oil. Decarboxylation refers to the removal of oxygen in the form of CO2 from a carboxylated compound and can be given in a general equation as follows (eq.(2) ):

$$\text{RCO}\_2\text{H} \rightarrow \text{RH} + \text{CO}\_2\tag{2}$$

Insights on effective catalysts for removing oxygen in carboxylic acids can be obtained from studies on decarboxylation of model systems that include stearic, palmetic, benzoic, and heptanoic acids. Equations (3) and (4) depict the thermodynamic favorability of the decarboxylation reactions. The negative values for Go implies that the decarboxylation reactions of acetic and benzoic acids have a significant activation energy barrier to overcome[44].

$$\text{CH}\_3\text{CO}\_2\text{H} \rightarrow \text{CH}\_4 + \text{CO}\_2 \qquad\qquad \qquad \Lambda\text{G}^0 = \text{--} 68.5 \text{ kJ/mol} \tag{3}$$

$$\text{C}\_6\text{H}\_5\text{CO}\_2\text{H} \rightarrow \text{C}\_6\text{H}\_6 + \text{CO}\_2 \qquad \qquad \qquad \Lambda\text{G}^\bullet = -54.3 \text{ kJ/mol} \tag{4}$$

Biodiesel research is an important area to look for information related to decarboxylation. Fatty acids and fatty acid methyl esters from biodiesel industry have been subjected further deoxygenation with the intension of obtaining higher quality liquid fuels [45, 46]. In one such study, Pd has been identified to be an active metal for decarboxylation of fatty acids present in plant oil. In this study, the ability to convert heptanoic acid to octane was investigated using Pd/SiO2 and Ni/Al2O3 catalysts [47-50]. It was reported that 98% acid conversion was obtained with Pd/SiO2 at 330oC but only 64% was reported by Ni/Al2O3 [44].