Temperature affects H2 production, metabolite product distribution, substrate degra­dation and bacterial growth. Most studies on H2 production have been conducted under ambient (15-27°C), mesophilic (30-45°C), and moderate thermophilic (50-60°C) temperatures, with a few studies of mixed cultures under extreme thermophilic conditions, over 60°C [88, 89]. The optimal temperature for H2 production via dark fermentation varies widely based on the type of biocatalyst and the carbon substrate used. For pure cultures, the optimal temperatures are reported to be in the range of 37-45°C, whereas for mixed microflora diverse optimum tem­peratures were reported [62]. Both mesophilic and thermophilic temperatures were observed to be optimal for fermentative H2 production processes. Thermophilic conditions were reportedly advantageous due to its thermodynamics [15, 62] which gives higher reaction rates with better process performance and decreased problems with contaminating H2-consuming microorganisms. Although higher temperatures allow more favorable reaction kinetics, rapid changes in system pH may inhibit H2 producing bacteria [90]. The changes in soluble metabolite composition were also observed with changes in operating temperature, resulting in metabolic pathway shifts correlated to bacterial functions dominant at that particular temperature [91]. Temperature control might not be a feasible option for process control.

The manures from beef cattle feedlots (or barns that do not use water to flush the animal manure) and poultry barns have relatively low water contents. They are often applied to farmland as fertilizer and thus have not been commonly subjected to AD. However, these two types of manures can be digested using dry AD pro­cesses [37] such as the dry anaerobic combustion (DRANCO) process, ECOCORP process, BEKON process, Kompogas process, and Linde process. These dry AD processes have several advantages over wet AD technologies and are described later in this chapter. Although not demonstrated on either type of manures [27], anaero­bic leaching bed reactors may be suitable for the AD of these manures without any dilution.

Both types of manures can be diluted to slurry and digested in conventional wet AD reactors. For beef cattle manure, a slurry containing 12% TS can be digested, but for poultry litter, a higher dilution (TS <3%) is needed to minimize inhibition by ammonia [44]. Inevitably, such dilutions create the need for large reactor volumes and high capital and operating cost. Pretreatment may be needed to remove the un­ingested diet (e. g., hay in beef cattle manure) or course materials (e. g., bedding materials and feather in poultry litter) prior to AD. Because of the presence of high solid contents, only CSTR, CMCR, and mixed plug-flow loop reactor (MPFLR) are suitable for AD of diluted beef cattle manure and poultry litter [20]. However, in a pilot study [9], upflow anaerobic sludge blanket (UASB) reactors were shown to be suitable and more efficient for the AD of diluted poultry feces. Co-digestion and thermophilic AD are also shown to improve digestion of poultry litter [16]. Thermophilic AD of poultry litter can be difficult due to the resultant high ammonia concentrations that render the AD process unstable [16]. The future will probably see more application of AD to both beef cattle manure and poultry litter, either alone or in co-digestion with other feedstocks, to both harvest bioenergy and produce fertilizer.

Some trace metals, organic compounds, nutrients and H+ concentration generally have a stimulating effect on the enzymatic activity pertaining to biochemical pro­cesses and might enhance process efficiency if added at optmial concentrations (Table 5). Hydrogenases that are able to catalyze the oxidation of H2 or the reduc­tion of H+ are classified into two major families: the [Ni-Fe] hydrogenases and the [Fe-Fe] hydrogenases, according to the metal content at their active site [130].

[Ni-Fe] hydrogenases have a higher substrate affinity [131]. During H2 production process catalyzed by the [Ni-Fe] hydrogenases, electrons are transported through an intra-molecular electron transfer chain from the redox partner such as NADH or NADPH to the active site, meanwhile, H+ are also transferred to the active site, and gets reduced by the e — to produce H2 [132, 133]. Since nickel is a fundamental com­ponent in [Ni-Fe] hydrogenases, it may influence the fermentative H2 production by influencing the activity of [Ni-Fe] hydrogenases and thus plays an important role in fermentative H2 production [91, 129]. A trace level of nickel is required for acti­vation or function of [Ni-Fe] hydrogenases and thus is conducive to fermentative H2 production [134]. Enhanced H2 production potential was observed with increas­ing Ni2+ concentration from 0 to 0.1 mg/l [91]. Trace metals such as magnesium, sodium, zinc and iron showed considerable affect on the fermentative H2 produc­tion with magnesium being the most significant one [134]. A nutrient formulation containing these four trace metals has shown a 66% enhanced H2 production rate as compared to the control. Iron is an important element which helps to mediate between hydrogenase and nicotinamide adenine dinucleotide (NADH)-ferredoxin reductase [135, 136]. Low iron concentration limits hydrogenase activity to effi­ciently mediate a reversible reaction between H2 and an electron donor such as reduced ferredoxin, thereby limiting H2 production [2]. About a 1.59-fold increase in H2 production and six-fold increase in hydrogenase activity was observed by increasing the FeSO4 concentration from 2.7 to 10.9 mg/l [137]. The role of metal ions (Mn+, Mg+2, Fe+3, etc.) as well as primary and secondary metabolites (adeno­sine mono phosphate, phosphoenolpyruvate, etc.) which have stimulation effects on the enzymatic activity pertaining to fermentative H2 production need to be studied to enumurate their specific function.

Hemicellulases, which catalyze the hydrolysis of plant cell polysaccharides, are multi-domain proteins generally containing structurally discrete catalytic and non­catalytic modules [27]. The most important non-catalytic modules consist of carbohydrate binding domains (CBD), which facilitate the targeting of the enzyme to the polysaccharide, interdomain linkers, and dockerin modules. The dockerin modules mediate the binding of the catalytic domain via cohesion-dockerin inter­actions, either to the microbial cell surface or to enzymatic complexes such as the cellulosome [27, 28].

The coordinated action of hemicellulases is necessary to obtain a satisfactory yield of pentose sugars from lignocellulosic as summarized in Fig. 2. Therefore, the development of low-cost and commercial hemicellulases is expected to be a limelight research area for cellulosic ethanol production. Table 2 shows the hemicel- lulase titers from different microorganisms and their mechanistic applications [29].

The microbial community residing in digesters largely remains a black box [65]. This is largely attributed to the difficulties and inability to grow these microbes in laboratory media. The use of cultivation-independent DNA-based molecular biology and metagenomic techniques makes it possible to define the membership and functionality of this complex microbial community (e. g., reviewed in [41]). As indicated by the more than 5,265 bacterial and 839 archaeal 16S rRNA gene sequences of anaerobic digester origin archived in the Ribosomal Database Project (RDP) (as of this writing, unpublished data), our knowledge on this microbial community has expanded tremendously [61,65]. These sequences represent approx­imately 2,500 species of bacteria (based on 97% 16S rRNA gene sequence identity) and 160 species of archaea (unpublished data). Our statistical prediction suggests that AD reactors can have at least 3,500 species of bacteria and 170 species of archaea. The continued studies using both molecular biology and metagenomic techniques should provide a better knowledge on the microbial community struc­ture, population dynamics, adaptation, granulogenesis, and metabolic kinetics in digesters. Eventually, this knowledge will help develop more efficient and stable AD technologies.

Both TGER prototypes underwent a third party assessment conducted by the US Army Aberdeen Test Center. Three high risk and five medium risk hazards were identified on the TGERs. All risks were mitigated with minor hardware modifica­tions, and sufficient safety devices and equipment were supplied as part of the basic issue items (BII). 007-DT-ATC-REFXX-D5104

Given that the mission of the Rapid Equipping Force is to quickly respond to field commanders’ requests by accelerating new technologies, the two first stage TGER prototypes were deployed by intent at what was considered to be the mini­mum technical readiness level for field evaluation. TGER assessment during the 90 day deployment to Victory Base Camp, Iraq met its objectives by identifying the key engineering challenges needed to advance from a first stage scientific prototype to an acquisition candidate system (Fig. 8).

The Iraq deployment validated the utility of the TGER system as an efficient means to address a complex, mixed, wet and dry waste stream while producing power. The science and technology underlying the hybrid design of the TGER is unique and has considerable advantages over other unitary approaches. The engi­neering of the TGER system and, in particular, the difficulties which arose in having to modify third-party commercial off the shelf equipment to TGER purposes, were an expected and commensurate problem.

Overall, the TGER performed well as a system for the first month of deployment. During the second month, unanticipated problems with the downdraft gasifier arose

Fig. 8 Deployed TGER which required considerable remedial attention by the technicians. With remote coordination with the manufacturer, many of these problems were quickly resolved, but the overall reliability and performance of the downdraft gasifier was in gen­eral decline over the three months, resulting in considerable down-time during the deployment.

Despite some initial tankage limitations (due to a delay in site prep by the Victory Base Camp DPW) and intermittent performance of the chiller system due to extremely high (120°F) ambient temperatures, the bioreactor performed well during the first month. The chiller was eventually upgraded with one of greater capacity, but during the final month the system encountered a compro­mised heat exchanger, some pumping problems, and apparent loss of biocatalyst efficacy due to heat exposure. The technicians were able to bypass the failed heat exchanger, modify pump elevations and add fresh biocatalysts to recover system performance.

About halfway through deployment, one of the two laboratory pelletizers became inoperative and could not be recovered. This resulted in a shift from a daily to an intermittent duty cycle (every other day) as the operators could not produce suf­ficient waste fuel pellets to keep the downdraft gasifier running continuously. The downdraft gasifier requires 60 lb/pellets/h and both pelletizers were needed to meet that throughput.

Alternatively, the biggest issues anticipated prior to deployment, i. e., the viability of the waste processing equipment involving the shredder, material transport/feeding and generator flex-fuel control performed reliably and were generally trouble free. Our pre-deployment effort on these critical system tasks ensured the system per­formed reasonably well during the first month, and allowed the other engineering issues to emerge from the background for proper identification and characterization for remedy.

Despite the mechanical issues, when the various elements of the TGER system were pulled together (routinely during the first month, then intermittently during the last two months) the system performed remarkably well. Field data demon­strated operations at or near 90% efficiency, with excellent throughput of both liquid and dry waste. The system generally conserved water at steady state and no environmental or safety problems emerged.

Various reactor configurations, viz., suspended growth, biofilm/packed-bed/fixed bed, fluidized bed, expanded bed, upflow anaerobic sludge blanket (UASB), gran­ular sludge, membrane based systems, immobilized systems, etc., have been used successfully to produce H2 by fermentation processes. Biofilm/attached-growth sys­tems are generally robust to shock-loads compared to the corresponding suspended growth systems, with the biofilms acting as a buffer to reduce the effective concen­tration of toxic chemicals to which the organisms are exposed, protect the culture from predation, provide improved reaction potential due to the presence of high cell densities and provide resilience and resistance to change in the process parameters [26, 92-95]. Generally bacteria achieve maximum growth rates in biofilm resulting in improved reaction potential finally leading to stable and robust system which are well suited for treating highly variable wastewater. Cell-immobilization approaches and granular processes also showed good H2 production efficiency.

Various modes of reactor operation viz., batch, fed-batch, semi-batch/continuous, periodic discontinuous batch (sequencing batch operation) and continuous have been used to produce H2. About a 25% improvement in H2 production and sub­strate degradation efficiency was reported with batch mode operation compared to the corresponding continuous mode operation [92]. The efficacy observed in fed — batch mode operation might be attributed to the reduced accumulation of soluble metabolic intermediates formed during acidogenic fermentation due to fill-draw mode operation [24, 26, 31, 38, 92]. A fed-batch mode of operation with acidic pH showed highest H2 production [92]. Poor biomass retention/cell washout encoun­tered during continuous mode operation can be prevented to some extent with a batch mode operation [92, 96, 97]. Batch mode operation coupled with a biofilm configuration combines the operational advantages of both systems and helps to maintain stable and robust cultures suitable for treating highly variable wastewater [21-25, 98-100].

Morphologically similar bacteria were observed in the scanning electron microscopy (SEM) image [26] of the biofilm formed on the fixed-bed of bioreac­tor producing H2 from the treatment of chemical wastewater (Fig. 4a). The biofilm reactor was inoculated with selectively enriched H2-producing consortia and oper­ated under an acidic microenvironment for more than 300 days. SEM imaging visualized slightly bent, scattered and short chain rods (predominant) along with a relatively low frequency of cocci shaped bacteria of approximate length of 10 p, m. SEM images of isolated bacteria strains from a biofilm reactor (acidogenic mixed culture) (Fig. 4b, c) visualized slightly bent, rod shaped, thick fluorescent capsid bacteria with (~10 ^m in length). Images of both the isolated strain and mixed consortia showed comparatively similar morphology demonstrating the presence of related groups of bacteria proliferated in the bioreactor producing H2. Transmission electron microscopy (TEM) image showing sub-cellular structures of the isolated bacteria from an acidogenic mixed culture [26] (Fig. 4d). TEM image showing oval centralized spore formation with sub-terminal endospore development in rod shaped bacteria (1-7 ^m in length). Terminal bulging with granulose accumulation was not observed. Flagellum attached subapically to the bacterium (two times length of the cell body) was observed. Vegetative cell surrounded by thick membrane (peptido — glycan layer) with two layers (inner and outer forming fibrillar capsule structure) on the cell surface was also visualized (Fig. 4e).

Some small dairy and swine farms do not use water to flush their barns so they produce manure with low water contents. These manures can be digested using dry AD [27]. Large dairy and swine farms, however, use water to flush the manure out of the barns and hog houses, respectively, generating manure slurries of mod­erate solid contents (>8%). Traditionally, both types of slurries are stored in waste lagoons built on the farms. By installing a flexible or floating gas-impermeable plas­tic cover, such lagoons can be easily converted to a unique type of digesters, covered lagoon digesters [68]. Covered lagoons typically have a long retention time (several months or longer) and high dilution rates [11]. Because of impracticality in temper­ature control, covered lagoons are left to operate at ambient temperatures and can produce biogas efficiently only in areas with moderate and elevated year round tem­peratures. Covered lagoons are simple and cheap to construct, operate, and maintain, which justifies their low AD efficiency. Another disadvantage is the slow but contin­uous accumulation of undigested solids at the bottom of the lagoons, which is costly to remove. One example of covered lagoons is located at Royal Farms in Tulare, California. It has three cells with a surface area of nearly 2,800 m2. Supported by the US EPA AgSTAR Program (http://www. epa. gov/agstar/index. html), it was started in 1982 and has been in operation ever since. The biogas produced has been enough to fuel two Waukesha engine-generators to generate electricity to meet all of the farm’s electricity needs with excess being sold to the local utility. The heat recovered from the generators is used as supplemental heat in the nursery barns, and the stabilized effluent is used as fertilizer. Barham Farm in North Carolina also operates a covered lagoon that has an effective volume of 24,500 m3. It digests the manure slurry generated from 4,000 sows. Baumgartner Environics, Inc. and MPC Containment Systems, LLC are two providers and installers of anaerobic lagoon covers.

Another type of digester that has been successfully and commonly used in AD of dairy manure slurry is non-mixing plug-flow reactors [15], which can successfully digest manure slurries with high solid contents (up to 11-14%). With a HRT of 21 to 40 days, methane biogas containing more than 60% CH4 can be produced at rates from 0.37 to 0.79 m3/m3 reactor volume/d. As estimated from the bio­gas yields of three such digesters, the daily biogas production ranged from 1.16 to 2.41 m3 per cow per day [88]. Although non-mixing plug-flow reactors are nearly maintenance-free, the gas production is rather slow due to poor mass transfer. Recently, MPFLR has been built at several dairy farms in the USA by GDH, Inc. Herrema Dairy located in Fair Oaks, Indiana operates a MPFLR, which receives more than 400 m3 of manure slurry of 8% solids that is generated by 3,800 heads of cattle daily. Operated mesophilically with a HRT of 17 days, this reactor pro­duces enough biogas to steadily fuel two Hess engine-generators of 375 kWh each. The separated solids from the effluent are dried and reused for bedding in the barns, while the heat recovered from the engine-generators is used to heat the digester, barns, and alleyways.

Both CSTR and CMCR have been used in AD of dairy manure slurry. The con­tinuous mixing significantly enhances biogas production and reduces HRT (from months to 10-20 days) [11, 15]. Thus, implementation of CSTR and CMCR signif­icantly reduces the digester volumes required to digest the manure derived from a given number of cows or hogs. Although these two types of digesters cost more to build and operate, the increased costs may be offset by the increased biogas produc­tion and TS reduction. Other types of reactors that have been tried on AD of manure slurries include hybrid reactors [26] and anaerobic filter reactors [87, 88]. However, to prevent clogging of the filter media of these two types of reactors, the SS has to be separated prior to feeding to these biofilm-based digesters, resulting in reduced biogas production [88]. The superiority of these digesters remains to be determined.

Recent studies have focused on improvement of VS degradation and concomitant increase in biogas production. Co-digestion with food wastes or crop residues was found to dramatically increase (by 2-3 folds) biogas production [51, 59]. This is attributed to the increased input of readily degradable substrate from these wastes. Temperature-phased AD (TPAD) also substantially improves AD [78], and TPAD of dairy manure slurry can be completed within a short HRT. The increased conversion rates at elevated temperature (55°C) are responsible for the improvement observed in TPAD systems [91].

Metabolic engineering is one of the promising areas which can be advanta­geously used to enhance H2 production rate in dark fermentation processes. By the use of recombinant DNA technology one can try to restructure metabolic network to improve the production of H2. Microbial metabolic manipulation by gene over expression, mutation and gene knocking out techniques were used for this purpose. H2 molar yields can be increased significantly through metabolic engi­neering efforts [109]. Table 6 documents some of the work carried out in this area pertaining to fermentative H2 production. By engineering the genetic expression of microorganisms the H2 production rate can be influenced directly or indirectly.

1.1.1 Ethanol

Bioethanol is a clean-burning (emits less CO2 and other green house gasses due to availability of free O2), non-petroleum liquid fuel that is considered to be a safe sup­plement to gasoline for transportation. The production and combustion of ethanol do not contribute to the total amount of carbon dioxide in the atmosphere [3, 21]. Ethanol can be mixed with gasoline in 10% (E10), 20% (E20), and 22% (E22) blends without engine modifications, but higher-level blends (such as 85% or 95%) require some engine modification. As a fuel additive, ethanol provides oxygen to the fuel, thus improving fuel combustion and reducing tailpipe emissions of carbon dioxide and unburned hydrocarbons.