Currently, there is not a single industrial-scale syngas fermentation-to-biofuel plant. Gas — to-liquid mass transfer is still considered as the major bottleneck for the commercialization of syngas fermentation technology. Keegan (2008) reported the construction of 40,000 gal per year pilot plant and a 40- to 100-million gal per year commercial-scale syngas fermentation facility. Kundiyana et al. (2010) reported a successful operation of a 100-L pilot scale syngas fermentation facility. Regardless of the recent developments in reactor designs, process optimizations, and microbial catalysts selection, the ethanol concentration from syngas fermentation is still just under 30 g/L. This leads to a high cost of ethanol recovery. For cost-effective ethanol recovery, its concentration should be around 15% (v/v). Therefore, in order to reduce the recovery cost, thus improving the overall economy of the process, industrial-scale syngas fermentation should focus on achieving higher ethanol concentration. This requires significant research and development in process microbiology.

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Pradeep Chaminda Munasinghe, Samir Kumar Khanal

Department of Molecular Biosciences and Bioengineering (MBBE), University of Hawai’i at
Manoa, Agricultural Science Building 218, 1955 East-West Road, Honolulu, Hawaii 96822.
*Corresponding author: E-mail: khanal@hawaii. edu

1 BACKGROUND

Research on lignocellulosic biomass such as agri-residues (e. g., corn stover, wheat and barley straws, etc.), agri-processing byproducts (e. g., corn fiber, sugarcane bagasse, seed cake, etc.), and energy crops (e. g., switch grass, poplar, Napier grass, Miscanthus, etc.) has received considerable attention for bioenergy production, especially liquid transportation fuel in recent years. Lignocellulose is a renewable, nonfood feedstock with an annual availability of around 200 x 1012 kg (220 billion metric tons) globally. The United States alone has the potential of producing 1.3 billion dry tons of biomass annually, which could substitute more than 30% of the nation’s petroleum consumption (United States Department of Agriculture (USDA) and United States Department of Energy (USDOE) Joint Report, 2005). Thus, ligno­cellulosic biomass could play an important role in the bio-based economy to produce a variety of biofuels and bio-based products. Lignocellulosic biomass consists of 40-50% cellulose, 20-40% hemicellulose, and 10-30% lignin. Although multiple conversion technologies are avail­able for producing biofuels from biomass, there are two major pathways, namely biochemical and thermochemical. In biochemical conversion, the biomass is subjected to a combination of physical and chemical pretreatments to destruct the biomass structure. These pretreatments make the biomass accessible to enzymes. The pretreated biomass is subjected to enzyme hydro­lysis to obtain fermentable sugars, which are then fermented to biofuels (Takara and Khanal,

2011) . The biochemical route, however, has several drawbacks such as high pretreatment and enzymes costs, generation of inhibitory soluble compounds (acetic acid, furan derivatives, and various phenolic compounds), degradation of sugars, and low biomass to fuel conversion ratios (Lewis et al., 2010). On the other hand, in thermochemical conversion, the biomass is gasified to produce synthesis gas or syngas in short (a gas mixture predominantly consisting of CO, CO2, and H2). The syngas can be converted into liquid biofuels through Fischer-Tropsch (FT) synthesis (using metal catalysts) or direct microbial fermentation known as syngas fermen­tation (using microbial catalysts) (Henstra et al., 2007; Munasinghe and Khanal, 2010 (a)). The FT synthesis usually utilizes metal catalysts such as cobalt (Co), ferrous (Fe), copper (Cu), aluminum (Al), zinc (Zn), molybdenum (Mo), nickel (Ni), rubidium (Ru), and ruthenium (Rh) (Demirbas, 2007; Subramani and Gangwal, 2008). The major drawbacks of FT synthesis are the high costs of the metal catalyst, a fixed H2:CO ratio (2:1), catalyst poisoning due to inert gases and contaminants containing sulfur, and high operating temperature and pressure (Phillips et al., 1994; Vega et al., 1990; Worden et al., 1991).

Syngas fermentation via biocatalysts (such as Clostridium Ijungdahlii, C. autoethanogenum, C. carboxydivorans, Butyribacterium methylotrophicum, Methanosarcina barkeri, and Rhodospirillum rubrum) produces liquid/gaseous biofuels, and offers several advantages over the biochemical approach and the FT process. Some of the merits of syngas fermentation are the elimination of the need of expensive metal catalysts, a higher specificity of the biocatalysts, the independence of the H2:CO ratio for bioconversion, the operation of bioreactors at ambient conditions, and the elimination of issues concerning noble metal poisoning (Bredwell et al., 1999; Klasson et al.,

1990) . Poor solubility of syngas in the aqueous phase and low product yield are the major limitations of syngas fermentation. These limitations have been the bottlenecks to commercial­ization of the syngas fermentation process.

This chapter critically reviews the existing literature on biomass-derived syngas fermenta­tion into biofuels. Furthermore, relevant background information including pathways, micro­bial aspects, mass transfer, reactor design, and factors affecting syngas fermentation are also briefly discussed. In addition, the current developments, challenges, and future research directions in syngas fermentation to biofuels are also included.

There are several methane-fermenting microorganisms including Methanobacterium thermoautotrophicum, Methanothermobacter thermoautotrophicus, M. barkeri, Methanosarcina acetivorans strain C2A, R. rubrum, and M. formicum (O’Brien et al., 1984) that have been isolated for biomethane production from syngas. In syngas-to-methane fermentation, CO acts as an electron donor and CO2 as an electron acceptor, which gets reduced to methane (CH4). O’Brien et al. (1984) reported hydrogen production during the growth of M. barkeri on CO when the CO partial pressure exceeded 20 kPa. The authors further revealed a net consump­tion of hydrogen below CO partial pressure 20 kPa. Kluyver and Schnellen (1947) reported the production of intermediates such as H2 and CO2 in their suggested CO to methane pathway. Several studies reported the low growth rates of M. barkeri and M. thermoautotrophicus on CO compared to the growth on H2 as the electron donor (O’Brien et al., 1984). The possible chemi­cal reactions and the relevant Gibbs free energy contents of the conversion of CO to methane are given in Equations (8) and (9).

From 100% CO, 4CO + 2H2O! CH4 + 3CO2 AG° = -53.0kJ/mole CO (8)

From H2 and CO, CO + 3H2CH4 + H2O AG° = -151.0kJ/mole CO (9)

Sipma et al. (2003) reported the use of several granular anaerobic sludges to produce meth­ane from CO at 30 and 55 °C. The authors found a significant increase in the CO to methane conversion efficiency (up to 90%). But the authors did not fully characterize the microbial communities in the sludge. According to some studies, methanogenesis is highly sensitive to CO concentration in the liquid phase (Klasson et al., 1990). However, successive transfers could enhance the ability of the microorganisms to grow on 100% CO (O’Brien et al., 1984). CO fermentation to methane opens up new area of syngas bioconversion to methane gas, which may overcome some of the challenges of syngas-to-ethanol fermentation.

The ultra fast high-temperature pyrolysis will be carried out in a high-temperature fluid — wall reactor which can withstand working temperatures of up to 2200° C. The biomass is fed to the top of the reactor (rate of 1.0-1.8 kg/min). The feed falls and, at the same time is very quickly heated by radiation to the reaction temperature. The estimated heating rate is on the order of 106 °C/s for reactant surfaces. The fluid wall, produced by a nitrogen flow through the 30-cm diameter porous reactor core, prevents both reactants and products from reaching the reactor wall. The product distribution at the reactor exit has been determined for different operating conditions. The influence of reactor temperature, biomass feed rate, and biomass particle size on the product distribution and on the heating value of the exit gas has been investigated (Corella et al., 1988).

4.6 Syngas Quality

Gasification of biomass produces a gas mixture containing additional constituents such as tars, ash, particulate matter, higher hydrocarbons (e. g., C2H2, C2H4, and C2H6), and gaseous compounds containing sulfur and nitrogen other than CO, H2, and CO2. Many studies highlighted the adverse effects of these impurities on syngas fermentation including process upset, cell dormancies, and inhibition of enzymes. Therefore, syngas should be free from these impurities before entering into the fermenting process. The commonly adopted gas clean-up methods include cyclones, various types of filters and scrubbers, and rotating par­ticle separators. Post gas clean-up operations always contribute to high operational and main­tenance costs. Turn et al. (2003) proposed a pretreatment protocol combining milling and leaching to reduce S, N, and Cl compounds from biomass feedstocks from sugarcane family. Takara and Khanal (2011) reported the elimination of nitrogen compounds from biomass feedstocks by adopting wet or green processing through upfront juicing and clean fiber utili­zation for biofuel production.

1.1 Gasification of Biomass

Synthesis gas (syngas in brief) is a gas mixture of predominantly CO and H2. Gasification of biomass feedstocks produces syngas through partial oxidation. Syngas quality largely depends on the compositions of biomass feedstock, gasifier types, and the gasifying agents. Other than the major constituents—CO and H2, gasification of biomass also produces methane (CH4), nitrogen (N2), carbon dioxide (CO2), water vapor, trace amounts of sulfur containing compounds, tar, higher hydrocarbons such as ethane (C2H6), ethylene (C2H4) and acetylene (C2H2), and particulate matter (Datar et al., 2004).

Gasifiers are mainly divided into two categories, namely, fixed-bed and fluidized-bed gasifiers. The fixed-bed gasifiers are characterized by the stationary reaction zone. Typically, in these gasifiers, biomass is fed from the top. Depending on the direction of biomass feeding and the oxidant employed, fixed-bed gasifiers are further divided into updraft and downdraft gasifiers. Fluidized-bed gasifiers use sand, ash, or char as moving media to increase the heat transfer and the gasification efficiency. Generally, the gasification of biomass takes place at high temperatures (fluidized bed: 750-900 °C and fixed bed: 1000-1200 °C). Table 1 shows the compositions of the produced gas mixtures from various gasification techniques and biomass feedstocks.

Bioconversion of syngas to organic acids (e. g., acetic and butyric acids) and alcohols (e. g., ethanol and butanol) follows the acetyl-CoA pathway (Henstra et al., 2007; Klasson et al., 1990; Phillips et al., 1994). The most common acidogenic microorganisms include Clostridium thermoaceticum, C. Ijungdahlii, Peptostreptococcus productus, A. woodii, Eubacterium limosum, and B. methylotrophicum. Many of the reported fermentation studies have shown a high acetic acid production compared to the other organic acids. Younesi et al. (2005) reported an acetate con­centration of 1.3 g/L at 1.4 atm pressure using C. ljungdahlii.

Butyrate is synthesized by the chemical intermediate acetyl-CoA reacting with butyryl — CoA (Brown, 2006). Acetic and butyric acid yields are highly dependent on the types of microbe and the substrate. Worden et al. (1989) reported that the production of butyrate was increased by 10-folds at the expense of acetate yield when the pH shift was from 6.8 to 6.0. Recovery of organic acids produced during syngas fermentation may provide oppor­tunity for additional revenue generation from coproduct.

A better approach for biomass conversion is the integrated hydropyrolysis and hydro­conversion of biomass to directly produce fungible gasoline and diesel fuel or blending components is carried out in two integrated stage. The first stage is a medium pressure, catalytically assisted, fast hydropyrolysis step completed in a fluid bed under moderate hydrogen pressure. Vapors from the first stage pass directly to a second-stage hydro­conversion step where a hydrodeoxygenation catalyst removes all remaining oxygen and produces gasoline and diesel boiling range material. All the process steps are completed at essentially the same pressure, so that compression costs are minimized. A unique feature of this process is that all the hydrogen required for this process is produced by reforming the Ci—C3 hydrocarbons, so no additional hydrogen is required. Pyrolysis is carried out in the presence of hydrogen at high pressure. The advantage of hydropyrolysis is the high quality of the products at the maximum liquid yield. The disadvantage is the high hydrogen consumption, which leads to high processing costs, but this is only a short-term economic consideration. If the H2 from a carbon-free source becomes cost competitive, the hydropyrolysis can become commercially exploitable technique for the conversion of ligno — cellulosic biomass with complete utilization of carbon content (Agrawal and Singh, 2009; Marker et al., 2009).

Syngas-fermenting microorganisms such as C. Ijungdahlii (Phillips et al., 1994), C. carboxydivorans, C. autoethanogenum, and B. methylotrophicum (Bredwell et al., 1999) follow the acetyl-CoA pathway (sometimes referred to as Wood-Ljungdahl Pathway) to produce biofuels (Henstra et al., 2007). Microorganisms that produce the intermediate acetyl-CoA from carbonyl or carboxyl precursors are known as acetogens (Brown, 2006). Though many acetogenic microbes produce acetate from alcohols and fatty acids, some are capable of produc­ing organic acids and alcohols using CO2 and H2 (autotrophic acetogens) or CO (unicarbo — notrophic acetogens) as their substrates.

Figure 1 shows the simplified acetyl-CoA pathway leading to the production of bio-based products such as ethanol, butanol, and butyrate and acetic acids from syngas. The essential reducing equivalents (-CO, — CoA, — Co-CH3) are produced from H2 and CO by hydrogenase and CO dehydrogenase (CODH) enzymes, respectively. In addition, the bifunctional CODH enzyme produces a carbonyl group from the reaction of carbon dioxide and water (Henstra et al., 2007). The produced reducing equivalents are then converted to acetyl-CoA by acetyl — CoA synthase (ASC) complex.

During the metabolic pathway, intermediate acetyl-CoA performs two major roles—it acts as a precursor for the cell macromolecule, and it serves as an energy source. It is essential to maintain a strict anaerobic environment during the acetyl-CoA pathway to avoid the con­sumption of reducing equivalents by other metabolic pathways (e. g., aerobic respiration). After several successive reactions, CO2 is reduced to a methyl (-CH3) group with the expense of 6 electrons and adenosine triphosphate (ATP). The produced methyl groups then react with the coenzyme and produce — Co-CH3. During the later stage of the acetyl-CoA pathway,

the produced metabolites (-C0-CH3, — CoA) react with CO to produce acetate. The enzyme complex—acetyl-CoA synthase enhances the reaction rate. This reaction recovers the meta­bolic energy invested during the early stages of the pathway. Acetate is further reduced to produce ethanol.

4.3 Inhibitory Compounds

In general, the syngas mixture contains constituents such as ethylene (C2H4), ethane (C2H6), acetylene (C2H2), tar, ash, char particles, and gases containing sulfur and nitrogen (Ahmed et al., 2006). These impurities impair the fermentation process through scale forma­tion in the pipes/joints, and inhibition of microbial catalysts and enzymes resulting in low cell growth and product yield. Datar et al. (2004) reported a cell dormancy, hydrogen uptake shutdowns, and a shift in metabolic pathways from acidogenesis to solventogenesis and vice versa, when the syngas was used without conditioning. Introduction of a 0.025-p. m filter to remove tar, ash, and other particulate matter from the biomass-derived producer gas was able to overcome cell dormancy (Ahmed and Lewis, 2007). Further, the authors reported that nitrous oxide (NO) was found to be a potential inhibitor of hydrogenase enzyme activity, which reduced the available carbon for product formation. In order to eliminate the inhibitory effects of NO, some studies suggested to improve the gasification efficiency or to scavenge NO by chemicals including sodium hydroxide, potassium permanganate, or sodium hypo­chlorite. In a separate study, Klasson et al. (1993) reported that the growth of C. ljungdahlii was not significantly affected by H2S concentrations as high as 5.2% (v/v).

It is evident that the biomass-generated syngas has inhibitory compounds that have adverse effects on syngas fermentation efficiency. Some of these impurities can be reduced by biomass pretreatment. Turn et al. (2003) reported that the fuel characteristics of sugarcane
bagasse could be improved by pretreatments including milling and leaching. The authors reported a reduction of N, S, and Cl content of the sugarcane bagasse by 13%, 36%, and 62% from their ultimate analysis values (% dry basis) of 0.48%, 0.22%, and 0.65%, respectively. By combining the pretreatments, milling-leaching-milling, the authors reported a further reduction in N, S, and Cl contents by 27%, 82%, and 94%, respectively, from their initial contents. These pretreatments can be implemented to other lignocellulosic biomass feedstocks in order to reduce the production of nitrogen and sulfur compounds during gasi­fication. Takara and Khanal (2011) introduced a new concept of wet or green processing of biomass for upfront juice extraction for coproduct generation, and the utilization of clean fiber for biofuel production. The authors reported the elimination of nitrogen compounds from the biomass to provide clean biomass feedstock for thermochemical conversion.