Bioenergy

Cornstarch is processed to ethanol by either the dry-grind or wet-milling process. The two processes differ in how the kernel is initially treated to access starch for enzymatic hydrolysis. In addition to the use of sugar in ethanol production, wet-milled starch can also be sold as dried or modified corn starch, or converted enzymatically to dextrins and sweeteners, or fermented to any number of products. Alternate fermentation products include amino acids, vitamins, citric acid, and lactic acid. Some wet mills also produce vitamins, enzymes, pharmaceuticals, nutraceuticals, films, solvents, pigments, polyols, or fibers. Wet-milled corn fiber can also be converted to ethanol. As with corn, ethanol is produced from cane in two primary types of facilities, integrated sugar/ethanol production facilities and autonomous distilleries. Integrated sugar/ethanol production facilities are capable of switching cane processing between sugar and ethanol depending on economic driving forces, whereas autonomous plants are designed to process sugarcane solely for the production of ethanol. As with corn-based ethanol production facilities, sugar plants employ conventional distillation to concentrate ethanol to near-azeotropic (96%) concentrations for subsequent dehydration. Future biorefineries that process the whole corn plant (starch, fiber, and stover) or the whole sugarcane plant (sugars and bagasse) could produce liquid fuel, edible oil or sugars, animal feed, power, and polymers or chemical intermediates. Starch and sugars will continue to play an important role in ethanol production, even as lignocellulosic feedstocks come into production.

Traditional Saccharomyces cerevisiae ferments glucose to ethanol rapidly and efficiently, but it is limited in its fermentation of pentose sugars (xylose and arabinose) to ethanol. For future sustainable and cost-efficient lignocellulosic biomass conversion to ethanol, there exist two major challenges: heterogeneous sugar utilization and stress tolerance in engineering microbial catalytic fermentors for bioethanol production. This chapter reviews the current knowledge on the composition and structure of lignocellulosic biomass, its pretreatment and enzymatic saccharification to simple sugars. It also discusses strain development of S. cerevisiae for efficient fermentation of the biomass-derived sugars to ethanol. Endoglucanase from Mucor circinelloides strain was found to have a wide pH stability and activity. There is an increasing demand for the development of thermostable, environmentally compatible products and for substrate-tolerant cellulases with increased specificity and activity for the application of converting cellulose to glucose for the fuel ethanol industry. Well-functioning hexose transporter family members maintain an easy flow of glucose uptake for the yeast and produce ethanol through glycolysis. Significant progress has been made recently for inhibitors generated from biomass pretreatment. The chapter focuses on the representative inhibitors furfural and 5-hydroxymethylfurfural (HMF). Development of yeast strains that can efficiently utilize heterogeneous sugars and withstand stress conditions in the bioethanol process is key for sustainable, economic and cost-competitive industry dealing with lignocellulosic biomass conversion to ethanol. A comprehensive genomic engineering approach will allow to meet the challenges for efficient lignocellulosic biomass conversion to ethanol in the next decade and beyond.

Pichia stipitis is a source of genes for engineering xylose metabolism in Saccharomyces cerevisiaea task undertaken in numerous laboratories around the world. Increasing the capacity of P. stipitis for rapid xylose fermentation can greatly improve its usefulness in commercial xylose fermentations. Genetic transformation with URA3 is more efficient than transformation with the modified Sh ble marker. Cultivation conditions can strongly affect expression of fermentative enzymes in P. stipitis. Unlike S. cerevisiae, which regulates fermentation by sensing the presence of glucose, P. stipitis induces fermentative activity in response to oxygen limitation. The coupling of Xyl1 and Xyl2 activities therefore tends to result in the consumption of NADPH and accumulation of NADH. Preliminary data based on expressed sequence tags indicate that transcripts for fatty acid synthase (FAS2) stearoyl-coenzyme A desaturase (OLE1) are induced under oxygen-limiting conditions. Xylanase production by P. stipitis has been recognized, and the organism has also been transformed with heterologous xylanases to increase xylanase activity. A genetic system has been developed that includes the auxotrophic markers URA3 and LEU3 along with modified forms of the phleomycin D1 resistance marker, Sh ble, and the Cre recombinase. Many genes in P. stipitis are found in functionally related clusters. Further metabolic engineering and strain selection are needed to increase the overall fermentation rate and ethanol tolerance of P. stipitis for the commercial bioconversion of hemicellulose hydrolysates.

A vigorous debate on gradual substitution of petroleum by use of renewable alternatives such as starch or sugar to ethanol or cellulosic biofuels dominates the political and economic agenda worldwide. Ethanol has been produced from sugarcane since the 1990s; an aggressive program has achieved the use of neat ethanol in new cars. There are various reports on lignocellulosic ethanol production using biomass. These indicate that ethanol from lignocellulosic biomass reduces “greenhouse gases” (GHG) emissions by about 80% compared to gasoline, whereas ethanol from corn reduces them by 20 to 30%. The cellulase system in Clostridia thermocellum comprises multiple enzyme complexes. In the case of hemicellulose, enzymatic saccharification by C. thermocellum occurs, generating xylose and xylobiose; both are utilized by Thermoanaerobacterium saccharolyticum for production of ethanol, lactic acid, and acetic acid. The combination of both microorganisms in the production of ethanol has been central in the development of the consolidated bioprocessing concept of researchers. Although the coculture system is very promising, several barriers exist such as end product inhibition by the produced ethanol. This disadvantage can almost be counterbalanced by the process of ethanol distillation from dilute broths. The work by researchers on the structure of the cell surface multisubunit complex called the cellulosome has been very important for understanding the detailed interactions between the organism and its enzymes and the binding affinity to cellulose through the cellulosic binding domain.

This chapter considers use of thermophilic bacteria to produce ethanol in a consolidated bioprocessing (CBP) configuration. It mainly focuses on cellulose conversion, as it represents the largest technical challenge for development of CBP-enabling microorganisms, with an emphasis on recent developments pertaining to processing of cellulose by cellulolytic microorganisms. Thermophilic bacteria was first considered for CBP with respect to diversity and ecology, utilization of nonglucose sugars, and ethanol tolerance. Since some pretreatment processes solubilize essentially all of the hemicellulose, it may or may not be necessary that insoluble hemicellulose be enzymatically hydrolyzed in the context of industrial processing of cellulosic biomass. The major component of angiosperm hemicellulose is xylan that can be esterified or acylated by phenolic residues such as ferulic acid and p-coumaric acid. Therefore, it is not surprising that hemicellulolytic bacteria must express a large set of cooperating enzymes in order to efficiently and completely hydrolyze hemicellulose and assimilate products of this hydrolysis. The authors concluded that a system similar to catabolite repression may be responsible for the apparent inhibition of cellulase synthesis and proposed that over time as cellobiose was metabolized, its concentration would drop below the point necessary to inhibit cellulase transcription. This work contradicted earlier studies which had concluded that the endoglucanase activity in C. thermocellum was constitutive.

The cellulosome was first isolated on the basis of the cellulose-binding function of the anaerobic thermophilic bacterium Clostridium thermocellum. Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose. The cellulose utilization systems in cellulosome-producing bacteria include over 100 different genes that must be orchestrated and timely expressed. The concept of directly converting biomass to ethanol by a mixed clostridial fermentation was fashionable some 30 years ago when it was found that the product pattern of C. thermocellum in favor of ethanol could become almost quantitative in stable coculture with another ethanol-producing anaerobe. The action of the exocellular protuberance-bound cellulosome may serve to delay or limit diffusional loss of the hydrolyzed sugar to the environment and/or competing bacteria. Consolidated bioprocessing (CBP) was recently extended for direct production of ethanol in yeast by cloning an endoglucanase and a ß-glucosidase in Saccharomyces cerevisiae. At present, C. thermocellum, as a very potent cellulolytic, anaerobic thermophile, still seems to be the microorganism of choice for future bioethanol production from biomass. Theoretically, C. thermocellum can be engineered metabolically to produce better yields of ethanol or other products. Eventually, yeast cell surfaces may be modified to contain designer cellulosomes for direct ethanol conversion. The combination of CBP with the designer cellulosome concept may ultimately provide optimized degradation of specific cellulosic feedstocks for bioethanol production.

This chapter discusses fully characterized cellulosomes from mesophilic bacteria consisting of two major components. These components include (i) one or more scaffolding proteins called scaffoldins that contain enzyme binding sites called cohesins and (ii) cellulosomal enzymes containing dockerin domains. The cohesin-dockerin interaction between the scaffolding protein and cellulosomal enzymes allows the assembly of the extracellular multisubunit enzyme complex. The scaffoldins minimally contain a carbohydrate binding module (CBM) and enzyme binding domains. In addition, depending on the scaffoldin, there may be hydrophilic domains, dockerins, even an enzyme function, and other domains of unknown function. The cellulosomes of mesophilic bacteria must play a major role in the turnover of carbon in nature, since most of the anaerobic habitats are in the temperate range of 15 to 45ºC in contrast to thermophilic organisms that require temperatures above 50ºC. An analysis of the expression of cellulosomal genes of Clostridium cellulovorans revealed that many of the cellulase and hemicellulase genes were expressed coordinately when cells were grown on cellulose or cellobiose. Since cellulosomes contain both cellulases and hemicellulases, it was of interest to determine whether synergism existed in the degradation of substrates such as corn cell wall by the enzymes.

Many investigations were conducted to characterize the cohesin-dockerin interactions. These studies include measuring cohesin-dockerin affinity, identifying critical amino acid residues by site-directed mutagenesis, and determining the molecular structures of the dockerin, cohesin, and its complex. Cohesins are highly conserved within the same scaffolding protein, with sequence identities higher than 50%. The interactions between the cohesin of the Clostridium thermocellum CipA scaffolding protein and the dockerin of cellulosomal catalytic components are categorized as type I, and the interactions between the dockerin of CipA and its counterpart are categorized as type II. The type I cohesins and dockerins include those modules from different microorganisms. This classification, however, is based on sequence homology and does not necessarily imply recognition among the same type of modules. Indeed, interspecies specificity was demonstrated using cohesins and dockerins from C. thermocellum and Clostridium cellulolyticum, respectively. In this work, Pages et al. found that C. thermocellum Cel48A (i.e., CelS), with its dockerin, did not recognize cohesin 1 of the C. cellulolyticum scaffolding protein CipC. The extremely high affinity between cohesin and dockerin and their important roles in cellulosome assembly have prompted interest in determining their molecular structures to elucidate the molecular mechanism of the cohesin-dockerin recognition. The first success in X-ray crystallography of a cohesin-dockerin complex was brought about by using a crystal of the dockerin of C. thermocellum Xyn10B and cohesin 2 of CipA, coexpressed in Escherichia coli and purified as a complex, yielding a good crystal suitable for X-ray analysis.

This chapter focuses on the contributions that domain interactions and calcium have on the properties of carbohydrate-active enzymes. It also focuses on the interactions between domains in one of the largest cellulosomal catalytic components, cellobiohydrolase A (CbhA). CbhA is the only Clostridium thermocellum cellulosomal enzyme whose domain interactions and role of calcium have been studied in detail by different techniques including genetic manipulations, crystallography, circular dichroism (CD) spectroscopy, and differential scanning calorimetry (DSC). In the presence of calcium, the stabilities of the domains are relatively independent, while in the absence of Ca2+, domain interactions play a stabilizing role. Thermal unfolding in buffer assumes coexistence of protein molecules (i) with calcium bound to all binding sites, (ii) with partially lost calcium, and (iii) without calcium. This chapter describes modular architectures of carbohydrate-active enzymes and analyzes the role of interdomain interactions in the structure, stability, and functionality of the interesting and important proteins. Even linkers between domains are crucial for the functionality of carbohydrate-active enzyme in that they serve as “molecular springs” allowing catalytic sites to reach and hydrolyze new glycosidic bonds, while the CBM is still bound to the substrate surface. As a general conclusion one may say that domain interactions in modular carbohydrate hydrolytic enzymes enhance the activity of the catalytic domains of the enzymes.

This chapter focuses on the development of two related bacteria that have proven to be effective in a variety of physical and chemical processes: Escherichia coli and Klebsiella oxytoca. Development of recombinant microbes that utilize a variety of sugars for ethanol production in laboratory media under optimum growth conditions has been repeated by several laboratories around the world. The use of dilute acid at temperatures above 140ºC is effective for the hydrolysis of hemicellulose in bagasse without significant loss of sugars or the production of degraded by-products. Dilute acid hydrolysis of hemicellulose has been used in order to produce high concentrations of hemicellulose sugars for fermentation by E. coli strain KO11-RD1. The yield of ethanol from acidic hydrolysis of cellulose is limited due to the poor recovery of glucose during the acid hydrolysis process. The degradation of glucose occurs very rapidly under conditions necessary for cellulose hydrolysis. Therefore, the use of cellulolytic enzymes has been pursued for several decades as a means of increasing the ethanol yield from cellulose. The simultaneous saccharification and fermentation (SSF) model has the following advantages over the sequential hydrolysis and fermentation process model: (i) lower enzyme dosages required for efficient conversion, (ii) compatibility with coproduction of enzymes during ethanol fermentation, and (iii) lower free-sugar concentrations during the SSF process.

The major portion of total secreted protein is represented by cellulases and xylanases. In general, cellulases and xylanases of Chrysosporium lucknowense have a great potential for degradation of lignocellulosic materials. The strategy for isolation of individual cellulases has been described in this chapter and is based on the sequential use of different purification techniques, such as anion-exchange chromatography, hydrophobic chromatography, and gel filtration. The cellulase system of C. lucknowense consists of several endoglucanases, cellobiohydrolases, and β-glucosidase. Cellobiohydrolases are the key components of multienzymatic cellulose complexes which are responsible for conversion of cellulose to soluble sugars. Xylanases are known for the production of fermentable monomeric xylose for production of biofuel and xylooligosaccharides for nutraceutical applications. Xylanases increase the digestibility of feed by lowering viscosity in the intestinal tract and decreasing absorption and water-holding capacity of feeds with significant content of nonstarch polysaccharides (NSP) by destruction of arabinoxylan. The ability of xylanases to efficiently decrease the viscosity of arabinoxylan makes them potential candidates to degrade NSPs and to be used as feed additives, and also to depolymerize pre-treated biomass in the production of cellulosic ethanol. All xylanases produce xylose, xylobiose (major product), and xylotriose as final products of arabinoxylan degradation. Filamentous fungal cell factories are the best hyperproducers of a complex array of glycosyl hydrolases such as cellulases, hemicellulases, and accessory enzymes.

After the oil supply shocks in the 1970s, research on development of alternative sources of liquid fuels was intensified. The initial emphasis was on processes that used coal as a feedstock, but later investigations were on the direct fermentation of lignocellulosic biomass where the biomass is first treated to convert it to fermentable sugars, followed by the familiar alcoholic fermentation, generally by yeast. Synthesis gas can be fermented or otherwise converted to a number of desired products, including ethanol. It can be chemically converted to liquid fuels. Clostridium carboxidivorans has been shown to ferment 100 mmol of fructose to 23 mmol of ethanol, 81 mmol of acetate, and 4 mmol of butanol. Upon fermentation of an equivalent amount of carbon monoxide (600 mmol), the end products produced shifted to 96 mmol of ethanol, 12 mmol of acetate, and 24 mmol of butanol. The presence of reducing agents alone can shift products of fermentation from acetate to ethanol. The ability to produce butanol from synthesis gas appears to be the most attractive feature of Butyribacterium methylotrophicum. C. ljungdahlii was isolated from chicken yard waste based on its ability to ferment synthesis gas to ethanol. The first commercial fermentation of synthesis gas to ethanol will probably use C. ljungdahlii as the microbial catalyst. C. carboxidivorans can directly ferment biomass, at least the cellulose fraction.

This chapter explores the microbiology and biochemistry of acetate conversion to methane, a key component of biomethanation. It provides a fundamental background appropriate for stimulating advances to improve the process that will ensure biomethanation among the competitive alternatives to fossil fuels. Biomethanation of organic matter in nature occurs in diverse habitats such as freshwater sediments, rice paddies, sewage digesters, the rumen, the lower intestinal tract of monogastric animals, landfills, hydrothermal vents, coastal marine sediments, and the subsurface. Methanosarcina species synthesize tetrahydrosarcinapterin (H4SPT) which serves the same function as tetrahydromethanopterin (H4MPT). The aceticlastic and CO2 reduction pathways generate primary sodium and proton gradients that are the only possible driving forces for ATP synthesis. The production of acetate from complex biomass by fermentative and acetogenic anaerobes and the subsequent conversion of acetate to methane by aceticlastic methanogens are of primary importance in the biomethanation process. Aceticlastic methanogenesis is the major factor controlling the rate and reliability of the process; thus, a comprehensive understanding of these methanogens is paramount for developing an efficient process for biomethanation of renewable and waste biomass for use as a biofuel. Although the enzymology of reactions leading from acetate to methane by Methanosarcina species is fairly well understood, there have been only a few investigations reported on the mechanism of energy conservation and regulation of gene expression. Further, global proteomic and microarray analyses have identified a host of proteins and genes in Methanosarcina species, many with unknown functions, that may be important or essential.

The degradation of organic matter by methanogenesis is the most complex and the most efficient way of transforming organic matter into an energetically useful product. This chapter deals with the complex cooperations in methanogenic microbial communities under different treatment regimes and the technological perspectives in the optimization of energy recovery from biomass treatment in the present and in the future. Energy-rich products other than methane can be produced fermentatively from biomass. A real breakthrough in the treatment of high-strength wastewaters was the development of the Upflow Anaerobic Sludge Blanket (UASB) technology developed by G. Lettinga and his coworkers in Wageningen, The Netherlands, in the 1980s. Although the reason for the development of the microbial aggregates remains obscure, the UASB technology has proven to be applicable to many different types of high-load wastewaters and has conquered the market in this field nearly worldwide. All major constituents of living biomass or organic waste materials can be converted to methane plus CO2, with the only exceptions being lignin and lignocellulose. It is the energetic efficiency that makes the overall process possible at all, but it also limits its kinetic and dynamic versatility.

The most efficient systems for biodegradation of polymeric organic compounds are mixed cultures that have evolved in some insect and mammalian guts. The efficiency and economic viability of converting organic wastes to biofuels depends on the characteristics of the waste material, especially the chemical composition and the concentrations of the components that can be converted into products that can be used as fuels. As mixed-culture fermentation involves large microbial communities, only certain compounds can be produced. Some products cannot be generated because they are converted to other compounds by the mixed culture more quickly than they are formed. When glucose-containing waste streams, such as those that are high in starch or cellulose, are used to produce bioenergy, butyrate may be one of the most important organic acid products. The hydrogen yield in mixed-culture bioprocessing can be increased by physically separating the anaerobic oxidation of sugars from hydrogen production by conducting the reactions in the anode and cathode, respectively, of a microbial fuel cell (MFC). Diverse microbial communities with metabolic flexibility should be more resistant to bacteriophage attack because different species or strains with similar metabolic functions can take over. Bioaugmentation can be used when modeling or systems biology analysis shows that a metabolic pathway that is needed to produce a useful energy carrier or its precursor is missing from the community metabolome.

Biogas can be made from most biomass and waste materials regardless of the composition and over a large range of moisture contents, with limited feedstock preparation. Methane-producing communities are very stable and resilient, but they are also complex and largely undefined. The methanogenic Archaebacteria uniquely catabolize acetic acid and one-carbon compounds to methane. The methanogens are obligate anaerobes that can pick up electrons from dead-end fermentations, through interspecies hydrogen transfer, and shuttle these electrons through a unique form of respiration which results in the reduction of carbon dioxide to methane. Codigestion with manure often enhances the conversion of other biomass and waste feedstocks through balancing micronutrients. Organic acids, pH, and alkalinity are related parameters that influence digester performance. The major alkalis contributing to alkalinity are ammonia and bicarbonate. Biowastes and biomass crops can be gasified in a reduced atmosphere combustion process to convert the biomass into a mixture of CH4, CO2, CO, and H2. Any improvement in conversion efficiency that enhances cellulosic ethanol yields is equally applicable for biomass conversion to methane. Methane yields from seaweeds, grasses, and crops all approach theoretical yields, such that as much as 80% of biomass energy content could be recovered in methane. Processing of terrestrial and marine energy crops to biomethane can result in higher energy yields than that of other biofuels.

This chapter provides an introduction to naturally occurring methane hydrates: their physical properties, formation, occurrence, and distribution in the natural environment, current estimates of resources, and potential for exploitation. The hydrate stability zone (HSZ) in marine and subsurface permafrost sediments can be delineated as the overlap of the pressure-temperature region of hydrate thermodynamic stability and the hydrothermal/geo-thermal gradients. In both marine and permafrost sediments, within the region of pressure-temperature stability, gas hydrates may form wherever the concentration of methane exceeds solubility in pore waters. The chapter summarizes the principal metabolic pathways involved in methanogenesis. The most important substrates for bacterial methanogenesis are acetate and H 2/CO2/. Until recently, probably the most widely cited global estimate of hydrate-bound gas was 21 × 1015 m3 of methane (STP) or ~10,000 gigatons of methane carbon. The success of the initial field studies at Mallik well led to a second research program, the Mallik 2002 consortium, this time with the aim of investigating methane production. The forecast for future tight supplies of natural gas, along with increasingly higher prices, point to growing demand for alternative supplies. The exploitation of gas hydrates is seen by many as a means to meet the demand, with some analysts suggesting that marine gas hydrates may begin contributing to natural gas markets in less than 10 years.

Methanol is an easily storable source of hydrogen. As a motor fuel, methanol has a favorable octane rating of 110, and most of today’s production goes directly into motor fuels or becomes a component of methyl tertiary-butyl ether, a fuel additive, or an esterification component of vegetable fatty acids in biodiesel. Today, methanol is produced by three main processes that are differentiated by the raw materials used: coal and oil, natural gas, and carbon dioxide. Catalytic reduction of carbon dioxide with hydrogen to methanol is inherent in every commercial methanol production process from coal, oil, or gas. The residual carbon dioxide formed from burning fossil carbon raw materials can be reduced with hydrogen produced by nonfossil energy sources. It has been shown recently that direct electrolysis of carbon dioxide and water can be applied to produce methanol. A considerable part of the organic component of the biomass is converted into end products, CH4 and CO2. The fermentation pathway from sugars and other substances (such as fats and amino acids) via acetic acid to methane is very straightforward, although there must be strictly anaerobic conditions in the fermentation system. Methane is the end product of this anaerobic conversion pathway. It can be oxidized to methanol and finally to carbon dioxide.

This chapter discusses the potential of methanol as an alternative fuel along with the prospects for its production using biomimetic pathways for efficient conversion of carbon dioxide to methanol based on single-carbon biotransformations. It focuses on methanol production through biocatalysis and is organized in three parts. First, the effects of fuel sources and their influence on the global carbon cycle and atmospheric accumulations of CO2 are discussed. Second, the potential utility of methanol as an alternative fuel and the scope of different methods for its commercial production are outlined. Finally, the use of biological systems in efficient conversion processes leading to methanol is elucidated with specific emphasis on dehydrogenase-catalyzed synthesis of methanol from carbon dioxide. The stabilization of enzymes in sol-gel materials provides a strategy for efficient utilization of enzymes in conversion of carbon dioxide to methanol. Immobilization of these enzymes confers additional thermal and environmental stability to the enzyme structure due to elimination or minimization of protein unfolding pathways. The moles of methanol produced are plotted as a function of the moles of the terminal electron donor (NADH). The enhancement of methanol production in sol-gel was due to confinement and matrix effects. The overall yield of the reaction for methanol production through this pathway depended on several factors. The sequential enzymatic conversion pathway to methanol production from CO2 provides several significant advantages. In the long range, with appropriate resource allocations, enzymatic biomethanol production pathways offer appealing prospects for practical development of new self-sustainable technologies.

This chapter discusses hydrogenase structure-function relationship studies in which new properties of modified enzymes might serve as an inspiration source for rational optimization of hydrogenases for biotechnological processes. A key point is that these reactions that appear as competitors for biotechnological purposes are often essential for cell survival or development. In part, this explains the difficulty and the slow progress in biohydrogen research. Two research directions can be proposed to overcome this kind of limitation: improve the substrate specificity of hydrogenase or, more radically, redirect redox intermediates. The V74M-L122M hydrogenase oxidized by oxygen remained in the same redox state as the native enzyme oxidized anaerobically, as demonstrated by the predominance of an Ni-B EPR signal (while Ni-A is predominant in the oxygen-exposed native enzyme) and by the abundance of a hydroxyl-bridging ligand at the active site in the structure. It was shown that it is possible to improve dioxygen resistance of [NiFe] hydrogenases. The enzyme bias, substrate specificity, and oxygen resistance are the main domains in which some progress has already been made, opening the way towards future applications. But other issues, like heterologous expression of [NiFe] hydrogenases that would facilitate molecular research and organism engineering or deciphering the catalytic mechanism that would allow the development of biomimetic catalysts, are also the subjects of intensive research and will contribute to biohydrogen implementation.

This chapter focuses on a biological process for hydrogen generation that depends on the environmentally benign use of biomass and solar energy. A group of anoxygenic photosynthetic bacteria known as purple nonsulfur bacteria (PNSB) produce large amounts of hydrogen under normal growth conditions by using nitrogenases as opposed to hydrogenases. However, hydrogen production by this route tends to be short-lived because of the extreme oxygen sensitivity of hydrogenases and nitrogenases. The chapter reviews the fundamental biology of nitrogenase-catalyzed hydrogen production by PNSB. Researchers found that photohydrogen production was inhibited by nitrogen gas. There are numerous strategies for strain development that can be expected to lead to improvements and stabilization of the hydrogen production process. In addition to their practical usefulness, the application of such strategies will lead to an improved understanding of the hydrogen production process as it operates in the context of whole cells. The fundamental unit of peripheral light-harvesting systems, also known as light harvesting 2, consists of two other types of α and β polypeptides. Two metabolic processes that consume large quantities of reductant and thus have the potential to divert electrons away from nitrogenase-catalyzed hydrogen production by whole cells of PNSB are poly-hydroxyalkanoate (PHA) synthesis and carbon dioxide fixation. In many ways studies of nitrogenase-catalyzed hydrogen production by anoxygenic phototrophic bacteria are still in their infancy.

This chapter emphasizes photobiological, H2 producing organisms and processes that are able to link photosynthetic water oxidation (reductant-generation) directly to [FeFe]-hydrogenase-catalyzed H2 production function. The biological catalysts involved in H2 metabolism are either nitrogenases or hydrogenases. Interestingly, the [NiFe], [FeFe], and FeS-cluster free types of hydrogenases are almost completely segregated within specific groups of organisms, suggesting convergent evolution. Two distinct H2 photoproduction pathways have been described in green algae, and there is evidence for a third, light-independent, fermentative H2 pathway coupled to starch degradation. A section summarizes the genetics, expression, maturation, structure, and modeling aspects of [FeFe]-hydrogenases, which catalyze H2 production in green algae. The hydrogenase structural genes that have been cloned and sequenced from species of Chlamydomonas, Chlorella, and Scenedesmus are homologues of the [FeFe]-hydrogenases from bacterial organisms. A majority of the [FeFe]-hydrogenase genes and proteins so far isolated exhibit complex structures that are organized into modular domains. Experimental investigations on the molecular engineering of O2 accessibility in [FeFe]-hydrogenase are currently under way. A better understanding of anaerobic metabolism in Chlamydomonas and metabolic fluxes associated with diurnal periods of light and dark will facilitate the development of physiological models able to predict metabolic fluxes under various environmental conditions. Photosynthesis and H2 production in unicellular green algae can in principle operate with a nearly 100% absorbed photon utilization efficiency. The rate of electron transport in the thylakoid membrane of photosynthesis is of importance for defining yield and efficiency of the overall process.

This chapter discusses the microbiology of microbial fuel cells with emphasis on fuel cells powered by electricigens. Electricity production with electricigens is significantly different from that of other types of microorganisms. The ability of electricigens to directly transfer electrons to the anode surface also alleviates the need for unstable, and potentially toxic, mediators. The fact that, as far as is known, there has been no evolutionary pressure on microorganisms to produce electricity suggests that electricigens may not be optimized for electricity production. Introducing genes to increase production of the outer surface cytochrome, OmcS, or pilin did not show increased power production. Geobacter species are capable of accepting electrons from electrodes poised at low potential for the reduction of various electron acceptors. Geobacter-catalyzed reduction of U(VI) to U(IV) at electrode surfaces can precipitate uranium contamination from groundwater, precipitating U(IV) on the electrode. Of the microorganisms known to contribute to electricity production in microbial fuel cells, only electricigens offer the possibility of highly efficient, self-sustaining conversion of waste organic matter and renewal biomass to electricity. However, the study of electricity production with electricigens is clearly in its infancy.

This chapter provides an overview of the microbial communities found in microbial fuel cells (MFCs), the interactions that drive the community structure, the processes performed by the communities, and how engineering affects the microbial resources within MFCs. Microbial electricity generation in MFCs relies on the drive of bacteria to acquire maximum energy. The electrode potential represents an important tool to control and increase the biocatalyst activity in relation to electricity generation. Moreover, the electrode potential will, as the key factor in the energy metabolism, determine the trade-off between fermenting and respiring organisms, thereby influencing the microbial composition. Several studies have described the microbial composition in MFCs. When comparing these data, several conclusions regarding the microbial community composition can be derived. First, various inocula can be used to successfully enrich electron-transferring organisms in an MFC. Second, several authors have concluded that MFCs strongly enrich organisms that utilize the electrode as final electron acceptor, both in a direct and in an indirect way. Third, although the MFC is an appropriate device to enrich electricity-producing communities, a typical electricity-generating microbial community has not been established yet. The majority of reported taxonomic classes are Proteobacteria (64%) followed by Firmicutes (13%) and nonclassified sequences (13%). The large variety of microorganisms found in MFCs suggests that many organisms can interact within the electricity-generating process. The influences of both the electron transfer interactions and the substrate transport within the biofilm on the development of the microbial community are discussed.

This chapter focuses on the regulation of solvent formation in solventogenic clostridia and in particular in Clostridium acetobutylicum ATCC 824, the most widely studied solventogenic Clostridium at a genetic level, and until recently the only sequenced one. C. beijerinckii and other clostridia are also discussed to the extent that relevant molecular details are known and pertinent to the subject matter. When grown at neutral pH under conditions of high NAD(P)H availability, the culture of C. acetobutylicum is termed alcohologenic as only butanol and ethanol are produced. The chapter focuses on these and related questions with emphasis on more-recent and genomically based work that has not been previously reviewed. The five solventogenic genes on pSOL1 are organized into three operons. Solvent and acid tolerance can be classified as a complex genetic trait, and its characterization requires not only the study of the ''key players'' but also the study of the organism response as a whole. Notable among the methodological improvements in this work were the improved performances of gene inserts from serial enrichment as opposed to a single round of enrichment in batch culture. It seems likely that one or more of the 178 pSOL1 genes is responsible for increased tolerance to butanol. Early attempts relied on the first systematic use of metabolic stoichiometry in order to calculate metabolic fluxes and carry out a large-scale metabolic flux analysis on the overall primary metabolism of C. acetobutylicum.

This chapter discusses practical approaches for biobutanol production, including strategies for reducing or eliminating butanol toxicity to the culture, as well as exploiting both physiological and nutritional aspects of the fermenting microorganisms in order to achieve better product specificity and yield. Recent developments in liquid biofuel technology, the uncertainty of petroleum supplies, the finite nature of fossil fuels, and environmental concerns have revived research efforts aimed at obtaining butanol from renewable resources. Solventogenic clostridia and many other anaerobes transport sugars into cell membranes through a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), and this PTS is involved in the transfer of a phosphate group from PEP to the substrate sugar. Generally, process controls during fermentations may be classified as physical (agitation speed, temperature, pressure, and aeration rate), chemical (pH, redox potential, dissolved oxygen, and dissolved CO2), and biological (biomass concentration, oxygen uptake, and production rates of H2, CO2, CH4, etc.) variables. The solventogenic clostridia have the ability to reassimilate the fermentation intermediates (acids) for solvent or butanol production and stabilize the pH in the process. pH measurement during butanol fermentation is important in order to accurately monitor the fermentation progress and in extreme cases prevent "acid crash." Accurate online pH monitoring is important for early detection of poorly buffered pH media.

From ethanol to vegetable oils, solvents have always been extremely important to the chemical industry from its inception. Until the advent of the petrochemical industry, solvents were biologically derived. With the petrochemical revolution, more extensive production, more variety, and more efficient solvents became economically feasible. ABE fermentation by Clostridium acetobutylicum, for instance, allows for mainly four solvents, namely, ethanol, acetone, butanol, and acetic acid (other minor products being acetoin and butyric acid). An alternative for the production of alcohol solvents is the thermochemical platform, which converts syngas (carbon monoxide and hydrogen) produced from the gasification of biomass, into Fischer-Tropsch (F-T) fuels and mixed alcohols through catalysis. Methanogenesis can be inhibited by maintaining a low pH; however, low pH also inhibits the production of the desired carboxylic acids. For the fermentation in the MixAlco process, methane analogs, such as bromoform or iodoform, are preferred as methane inhibitors. The ketones obtained from dry distillation of the calcium carboxylate salts may be sold as such, or they may be further converted by hydrogenation into secondary alcohols. The wide diversity of products that the MixAlco process allows meeting the diverse solvent demand that the petrochemical industry has created. When technology is fully established and large plants are built, it will be possible to pay more for the biomass and grow crops specifically for energy and chemical production, which will be mostly processed into alcohols for the fuel market.

Researched data suggest that membrane-associated proteins in the ethanol-adapted strain are either synthesized in lesser quantities or not properly incorporated into the cell membrane. In this study, 49 different proteins were identified and among them were two highly abundant surface layer proteins, flagellum components, and paralogs of the high-molecular-weight surface layer protein. These identified proteins may represent new virulence factors. The current status for reduction of expression of multiple genes is antisense RNA technology. The introduction of plasmids bearing genes of acid pathways showed only a modest effect on the acid content. This suggests that increasing the levels of certain enzymes may not affect the overall metabolite profile if they are already in adequate amount and the network is limited by other factors. Among the strategies discussed to increase the butanol fraction of solvents in Clostridium acetobutylicum, that is, by manipulating expression levels of genes for acid and solvent production, regulators, heat shock proteins, and sporulation, altering gene expression for cell membrane synthesis would be expected to directly address solvent tolerance as well as production capabilities. The study of the genes encoding the 1,3-PD operon of Clostridium butyricum VPI1718 revealed three genes, dhaB1, dhaB2, and dhaT. Studies detailed in this chapter reflect an increased level of complexity in the process of metabolic engineering and recognize that attempts to improve the desired phenotype must be accompanied by a deeper understanding of the organism as a whole if success in strain engineering is to be achieved.

Much of the world’s oil is produced by water injection. Water injected through injection wells helps to maintain the reservoir pressure required to sweep the oil to the surface through production wells. When injected water breaks through, an oil-water mixture is produced. After separation from produced oil, the produced water can be reinjected, a production strategy referred to as produced water reinjection (PWRI). PWRI is commonly practiced in landlocked reservoirs where access to water may be limited. The fraction of water produced from oil fields subjected to water injection (the water cut) generally increases with time. Oil production by water injection often results in increased sulfide levels (souring), because sulfate-reducing bacteria (SRB) couple the oxidation of degradable oil organics to the reduction of sulfate to sulfide. Nitrate injection also stimulates heterotrophic nitrate-reducing bacteria (hNRB), which couple either incomplete oxidation of degradable oil organics (to acetate and CO2) or complete oxidation of oil organics (to CO2 only) to the reduction of nitrate to nitrite, nitrogen, or ammonia. The need to provide both a hydrocarbon substrate and a fermenting inoculum designed for high yields of biosurfactant adds to the complexity of traditional microbially enhanced oil recovery. Nitrate injection changes the microbial community downhole to one in which the activities of hNRB and in some cases nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) are more prominent.

Given our current dependency on fossil fuels and the dwindling supplies thereof, efforts must be made to ensure availability of fossil fuels and to minimize the energy-associated environmental footprint. This chapter tries to indicate how biotechnology might assist in achieving these goals with specific respect to fossil energy reserves. Methane is now believed to be the primary by-product of anaerobic oil biodegradation in many petroliferous deposits. There are a number of microbial processes that are potentially useful, either to improve oil production rates from individual wells or to increase the ultimate amount of oil recovered from a reservoir. It is important to note that for most microbial oil recovery technologies, multiple products and activities are involved and it is likely that these act synergistically. The incomplete oxidation of hydrocarbons could generate alcohols or fatty acids from the hydrocarbon molecule or stimulate the production of biosurfactants and bioemulsifiers derived from microbial cells. Oil production of carbonaceous sandstone reservoirs in Romania increased by 30 to 40 barrels per day when treated with a mixed microbial population adapted for rapid growth under reservoir conditions with molasses as the substrate. Microbial oil recovery processes will gain more widespread acceptance and application only when quantitative measures of performance can be reliably obtained.

This chapter focuses on the structure and regulation of the pathways utilized by various microbes (bacteria, algae, and yeasts) for the production of fatty acids and triacylglycerols (TAGs). An important observation with regard to the possibility of microbial biodiesel production was made when fatty acyl-ACP thioesterase (FAT) enzymes were overproduced in Escherichia coli. Thus far it represents the most efficient way to uncouple fatty acid formation from phospholipid and membrane biosynthesis in E. coli. Phosphatidic acid (PtdOH) is a key branching point in de novo lipid metabolism, and it is converted either to CDP-diacylglycerol (DAG) or DAG depending on the organism. CDP-DAG and DAG serve as intermediates in membrane phospholipid biosynthesis and, in addition, DAG is converted to TAG. Phosphatidate phosphatases (PAPs) in coordination with phospholipid-producing enzymes are key regulators of the flux of carbon towards TAGs. PAPs catalyze the conversion of PtdOH to DAG; the primary destination of DAG is the synthesis of membrane phospholipids, whereas excess DAG is directed towards TAG. The emerging theme from genome comparisons underlines the evolution of distinct regulatory mechanisms in various phylogenetic groups. All free-living organisms have the machinery to synthesize fatty acids, and conceptually, they could be exploited for biodiesel production. However, the photosynthetic organisms provide the unique opportunity to couple CO2 sequestration to lipid accumulation and subsequent biodiesel production.