1.1 Biocatalyst

Biocatalyst (inoculum) selection and its pre-treatment plays a vital role in select­ing requisite microflora for efficient H2 production [4, 7, 15, 30, 71, 72, 73]. Inoculum preparation affects both start up and the overall efficiency of H2 pro­duction. Typical anaerobic mixed cultures cannot produce H2 as it is rapidly consumed by H2-consuming or CH4-producing bacteria (MB) [74]. The most effec­tive way to enhance H2 production from anaerobic culture is to restrict or terminate methanogenesis by allowing H2 to become a metabolic end product. Physiological differences between H2-producing bacteria (AB) and H2-consuming bacteria (MB) forms the main basis for the preparation of the inoculum to start up the acido — genic H2-producing process [72]. Spore-forming H2-producing bacteria can form spores which protect them when they are in an adverse environment (high tempera­ture, extreme acidity and alkalinity), but methanogens have no such capability [72]. Some of the pretreatment methods normally used for selective enrichment of an H2-producing inoculum are listed in Table 3. Methanogenesis could also be elim­inated by maintaining short retention times (2-10 h) during reactor operation [75, 76] as H2-producing bacteria grow faster than the methanogens [72]. Combining different pre-treatment methods also showed a positive effect on the H2 production process [4, 21, 30, 38, 71]. In spite of good enhancement in H2 production, marked reduction in substrate degradation efficiency was observed after applying pretreat­ment methods [4, 30, 71], which can be attributed to the inhibition of MB. The methanogenesis function is required to metabolize intermediates generated from the acidogenic process. Untreated anaerobic inocula showed low H2 yield in spite of effective substrate removal leading to CH4 formation due to the presence of MB.

The influence of various pre-treatment methods applied individually and in combi­nation on H2 production and substrate degradation patterns from the treatment of diary based wastewater under acidic conditions is illustrated in Fig. 2

Any biomass can be used as feedstocks for AD. However, biomass wastes, espe­cially those with a relatively high water content (>50%), are the most common feedstocks suitable for AD. In fact, methane biogas has been produced from mil­lions of tons of biomass wastes arising from municipal, industrial and agricultural sources [91]. The characteristics of biomass wastes vary widely. The common feed­stocks suitable for AD have been discussed with respect to features pertinent to AD and biogas potentials by Yu et al. [91]. The AD of several types of feedstocks has also been reviewed recently (e. g., [15, 66, 85]). Anaerobic digesters can be cat­egorized in many different ways (see [80, 91] for an overview). No AD reactor is universally ideal or superior because each type of reactor has certain advantages and disadvantages that make it appropriate for particular type(s) of feedstocks. In this chapter, the features of individual feedstocks that have substantial methane biogas potentials and the AD technologies that are suitable for their AD will be discussed.

H2 can also be viewed as an energy source and an intermediate towards the production of VFA which can be further transformed to polyhydoxyalkanoates (PHAs), or can be used for biohydrogenation of fatty acids into alcohols [120]. Polyhydroxyalkanoates (PHAs) are a group of biologically derived polyesters that represent a potentially sustainable replacement for fossil-fuel based conventional thermoplastics due to their biodegradability and capability of being produced from renewable resources. During growth-limiting conditions, bacteria produce PHAs as energy and carbon storage molecules. So far, many efforts have been made to pro­duce PHAs from commercial grade VFAs using pure cultures. However, the PHAs produced in this manner are more expensive than polyethylene due to their high production costs [121, 122]. Almost 30% of total PHA production cost is attributed to the carbon source [123]. VFA bound acid-rich waste generated from acidogenic process of H2 production can be used to produce PHAs using PHA accumulating organisms and is a promising approach to decrease the production cost. Production of PHA by mixed microbial cultures using wastes seems to have many advantages when compared to the existing well-known process where pure cultures and single defined substrates are used. Recently, the production of PHAs from the fermenta­tion of syngas was also reported which is economically viable than that from sugar fermentation [124].

Dilute sulfuric acid hydrolysis is a favorable method for pretreatment before enzy­matic hydrolysis and also for the conversion of lignocellulose to sugars [22].

Compared to other pretreatment methods, it is especially useful for the conversion of hemicellulose into xylose, which can be fermented into ethanol by specialized microorganisms [3, 4]. Most dilute acid processes are limited to a sugar recovery efficiency of around 50%. It has been reported that the cell wall structure and com­ponents may be significantly different in different plants, which may influence the digestibility of the biomass [23]. A broad dilute acidic hydrolysis on a variety of lignocellulosic materials with respective ethanol production has been reviewed by Chandel et al. [3].

Methane biogas production is rather slow [89], and large digesters are often required to produce enough biogas to be recovered cost-effectively as energy. AD processes are also susceptible to a host of factors, which can render AD suboptimal or some­times lead to unpredictable upset or total failure of AD [17]. The re-startup and recovery process after failure are often slow [17, 64, 84]. These limitations and other barriers can severely undermine the economic viability of AD processes and make many industries and farmers reluctant to implement this biotechnology. These issues and the research required to improve AD for bioenergy production are briefly discussed below.

1.2 Operation of the Reactors

Small — and large-scale, complete-mixing, composting reactors were used in this study. Three composting reactors for household use (“Namagomi-eater” TK400-H, Matsushita Electric Works, Japan) were used as the small-scale, FBC reactors. The working volume was 15 L. The biomass carrier (or bulking agent) comprised about 5 L of wood chips with a size range of 0.5-2.0 mm, or plastic bottle flakes with a size range of 2.0-10.0 mm. An artificial organic waste sample, made up of 500 g wet wt dog food (VITA-ONE, Nihon Pet Food, Japan) containing about 90% water, was loaded daily into each reactor. The contents in the reactors were gently mixed by automated paddles for 1 min each hour. Mechanical heating was used to maintain the temperature in all the reactors above 35°C to accelerate biodegradation. In the small-scale reactor, three experimental conditions (reactor A, B, and C) were used. In reactor A and C, a high decomposition rate of organic materials was maintained by “partial washing” [14, 16] as follows. Approximately 10% of the contents (0.75 L) were taken out every three days, mixed with 10 L of water, and then filtered on a 35 ^m mesh filter. Upon filtration, the solid part retained on the mesh filter was dried in an oven at 60°C for 48 h, and then re-loaded into the reactor. This process prevents not only a decrease in decomposition rate but also aggregation of the contents in the FBC reactor [14]. In reactor B, there was no maintenance, except for moisture content, where spontaneous aggregation was allowed in the decomposition process. The moisture content in each reactor was kept at 40-50% by the addition of distilled water. Samples were obtained from each small-scale reactor after 60 days of operation.

To compare the difference of the scale of the reactor, large-scale composting reactor was also used (O-1, which we constructed). The working volume was 4 m3. The bulking agent comprised about 2 m3 of plastic bottle flakes with a size range of 2-10 mm. 600 kg wet weight food waste derived from a school cafeteria with 73% moisture content, was loaded into the reactor at the start of the experiment. This is termed batch operating. The contents of the reactor were gently mixed by automated paddles at 1.5 r. p.m. for 30 min once a day. Temperature was not regulated. Samples were obtained from the large-scale reactor, once a day, for 25 days. In the small-scale reactor (reactors A, B, and C), 16S rDNA-clone analysis was performed to compare difference in the bacterial communities under each set of conditions, and in the large-scale reactor, both 16S rDNA-DGGE and clone analysis were performed to analyze how bacterial community succession changes day by day.

The TGER prototypes were fabricated and commissioned at Purdue University and conformed to the following selection criterion:

a. Approach the problem as a “dual optimization” to develop a system which will simultaneously eliminate as much waste as possible while producing as much useful energy as possible.

b. Design of the TGER must be “tuned” to the operational context to ensure an easily available and reliable volume of military waste.

c. The TGER should be designed to be contiguous with both the input source of wastes and the end user for the output energy product, avoiding any reprocessing or transport costs.

d. The TGER must be operationally and tactically deployable via military airframe and able to be transported on the ground via standard military trailer.

e. The TGER should not need additional manpower or machinery costs for waste separation.

f. The process must minimize parasitic costs such as manpower, water, external energy, etc.

g. The refining process should have minimal residual waste.

h. Additional concerns of hazardous waste, safety, and troop use must be consid­ered, and operation should be amenable to unskilled labor.

The selection of gasification and biocatalytic fermentation has strategic value in that both methods are well-demonstrated technologies supported by high levels of research by the Department of Energy and, in the long course, are very likely to improve as new advances are achieved.

Significant new advances in gasification include the introduction of integrated sensors and automated computerized control systems for the process. These recent advances have resulted in gasification technologies with reliable and efficient con­version of waste to energy. Significant recent advances in biocatalytic fermentation include advances in genetically modified or modified via directed evolution enzymes and micro-organisms. Using methods developed at the Laboratory of Renewable Resources Energy at Purdue University, several commercial entities have broken new thresholds in domestic ethanol production techniques by applying new biocat­alysts and processes, the result being the economically viable production of ethanol for fuel [8]. Current advances in enzymatic design and development bode well for further methods to reduce what would normally be considered unusable biomass waste (e. g. paper fines from shredded cardboard and other cellulosic wastes) into usable energy, allowing more energy to be harnessed from the same waste stream.

During the commissioning phase of the TGER, the system was able to deliver reliable power with very low parasitic costs required to operate the system internally. The core processes, gasification and fermentation for conversion of waste to energy, worked very well and the unique hybrid combination of thermochemical and bio­catalytic technologies proved itself to be of considerable merit. These technologies could easily scale up to support military installations such as hospitals and major troop areas by converting waste into power, hot water, and usable fuel while elim­inating costly waste removal expenses. Installation biorefineries could provide cost savings for US and overseas bases, reduce dependence on petroleum-based energy and support environmentally responsible initiatives, highlighting DoD’s support of renewable energy resource technologies.

Depending on organisms and growth conditions, changes in external pH can bring about subsequent alterations in several primary physiological parameters, includ­ing internal pH, concentration of other ions, membrane potential and proton-motive force [77]. pH also influences the efficiency of substrate metabolism, protein syn­thesis, synthesis of storage material and metabolic by-product release. This is especially important for fermentative H2 production where the activity of acidogenic bacteria is considered to be crucial and rate limiting [24, 58]. The restricted nature of specific groups of bacteria at particular pH values helps to maintain the bioreactor in an acidogenic microenvironment. Maintaining pH in the acidic range (5.5—6.0) is ideal for effective H2 production due to repression of MB, thus indirectly pro­moting H2 producers within the system [21, 30, 72]. The activity of hydrogenase is observed to be inhibited by maintaining low or high pH in fermentation [58]. Most methanogens are limited to a narrow pH range (6.8-7.2), while most H2-producing acidogenic bacteria can grow over a broader pH range. AB function well below pH 6, while for MB optimum range is between 6.0 and 7.5 [78, 79]. The pH range of

5.5-6.0 is reported to be ideal to avoid both methanogenesis and solventogenesis [21, 79], which is important for good H2 production. Effective H2 production was observed by maintaining operating pH in and around 6 compared to near neutral pH [21, 75]. Increase in initial/feeding pH (from acidic to neutral) has resulted in suppressed H2 production [21, 26, 31, 32]. However, highly acidic pH (<4.5) is detrimental to H2 production as it inactivates H2 producing bacteria [72, 80].

Cyclic voltammograms (CV) obtained at acidic and neutral pH conditions visu­alized well — defined redox pairs both in forward and reverse scans and the signal corresponded to intracellular electron carriers, NADH/NAD+ (Eo, -0.32 V) [24] (Fig. 3). Shuttling of H+ between metabolic intermediates can be correlated to the e — discharge observed in CV. At acidic pH, the e — discharge was almost similar at

12, 20 and 24 h suggesting the effective H+ shuttling throughout the cycle oper­ation. This helps to maintain the system under acidogenic conditions for longer periods leading to higher H2 production. At neutral operation, the e — discharge var­ied with time and approached maximum at 12 h prior to decrease suggesting the neutralization/reduction behaviour of H+ by MB.

Acidic pH (below 6) showed less substrate degradation efficiency than the corre­sponding neutral operation due to reduced methanogenic activity [24]. Neutral pH illustrated effective substrate removal efficiency over the corresponding acidic oper­ation. Maintenance of acidic conditions in association with pre-treatment has also been observed to be effective in H2 production during treatment of various types of wastewater [30, 31, 38].

VFA (soluble acid metabolites generated from acidogenic fermentation) and pH are integral expressions of acid-base conditions of anaerobic microenvironments which provides information pertaining to the balance between two of the most important microbial groups (AB and MB). Production of acids gradually reduces the buffering capacity of system, which, in turn, results in a decline in the system pH due to accumulation of organic acids leading to process inhibition [23, 81]. If pH is not maintained in the optimum range, cessation of H2 production will result along with a marked shift in microbial population [75]. Relatively higher levels of soluble metabolite production were observed under acidic operation over the cor­responding neutral microenvironment, which corroborated well with H2 production data [26, 31, 38, 82]. Therefore, pH can be considered as a manipulable variable for process control. Among the two process variables viz., influent pH and reactor pH, the later is more difficult to control. Bicarbonate-alkalinity is an important pro­cess parameter which indicates the system buffering capacity in association with pH microenvironment and VFA concentrations.

Sulfate, if present in wastes will be converted into hydrogen sulfide by sulfate — reducing bacteria (SRB) in the anaerobic microenvironment, resulting in toxicity to other anaerobes [83]. SRB are reported to have H2 utilization hydrogenase and can readily use H2 as the electron donor [84]. pH of the system microenvironment has a direct influence on the sulfate reduction linked to H2 production. At acid pH, the SRB activity gets inhibited wherein H2 production is unaffected. H2 production has markedly recovered and increased when pH was reduced to 5.5, even in the presence of higher sulfate concentration (3 g SO42- /l) [85].

Municipal sludge includes primary sludge and waste activated sludge derived from centralized wastewater treatment plants that employ biological treatment of sewage. It is probably the first type of feedstock subjected to AD. It has very high contents (95-99%) of water, low contents (15-20%) of volatile solid (VS, representing the biodegradable portion of total solid, TS), and low contents of readily fermentable carbohydrates [8, 94]. However, most municipal sludge has rich and balanced nutri­ents (nitrogen: 3-6%; phosphorus: 1.0-1.2%; of TS). The biochemical methane potential (BMP) of municipal sludge is relatively small, ranging from 85 to 390 m3 CH4/dry ton. Municipal sludge contains a high density of bacterial cells (mostly aerobic and facultative anaerobic bacteria), some of which may be pathogenic to humans and/or animals. Toxic compounds may also be present in some munici­pal sludge, especially those derived from large metropolitan areas. Approximately

6.2 million dry tons of municipal sludge are produced annually in the USA (based on 1999 data [39]), representing an annual potential of at least 6 billion m3 of methane biogas. At present, however, only a portion of the municipal sludge is digested and the methane biogas yields are relatively low. This is largely attributable to the relatively small net amounts of energy that can be produced. However, when municipal sludge is co-digested with carbohydrate-rich yet nitrogen-poor biomass wastes (e. g., OFMSW and food-processing wastes), the energy yields can increase substantially [4]. For example, in a full-scale two-staged AD system, a 25% increase in organic load rate (OLR) with OFMSW resulted in an increase in biogas yield by 80% and overall degradation efficiency by 10%, which resulted in an increase in electrical energy production by 130% and heat production by 55% [94]. Additionally, when co-digested with carbohydrate-rich yet nitrogen-poor biomass wastes, municipal sludge can stabilize the AD process of the former [46].

Municipal sludge is among the most studied feedstocks in AD. Numerous books and reviews have been published on AD of municipal sludge (e. g. [79]). In gen­eral, because of the presence of high levels of suspended solid (SS), most AD technologies are not suitable for the AD of municipal sludge. Continuously stirred tank reactors (CSTR) and completely mixed contact reactors (CMCR) are most commonly used in AD of municipal sludge [79]. For example, the CSTR with a total volume of 1,350 m3 in Karlsruhe, Germany digests municipal sludge at 37°C and produces approximately 3,800 m3 of biogas of 62-70% methane daily [33]. More recent research efforts have been directed at pretreatment to enhance degra­dation of the solid found in municipal sludge and production of methane biogas (see [28, 45] for reviews). Thermophilic AD, in single — or two-staged systems, is also being increasingly used to enhance biogas production and sanitation [92]. Additionally, because of the low solid contents (1-5%) and low BMP, large digesters are required for the conventional “wet” AD. Currently, “dry” AD technology is being evaluated to produce methane biogas from dewatered biosolids, which have significantly reduced water contents (70-85%) and thus reduced digester volumes [64]. Dewatered biosolids are also ideal feedstocks to be co-digested with other solid feedstocks, such as OFMSW and crop residues.

Shifting the anaerobic reactor from methanogenesis (producing CH4) to acidoge — nesis (to produce H2) is important to make the process more feasible with wider application potential. Bioaugmentation is generally applied to improve the start up of a reactor, to enhance performance efficiency, to protect the existing micro­bial community against adverse effects or to compensate for organic or hydraulic overloading [99, 125]. In this way, the bioaugmentation strategy was applied to an operating anaerobic bioreactor (producing CH4) to shift to acidogenic pro­cess so as to produce H2 [25]. For this purpose selectively enriched H2-producing mixed consortia (in immobilized form) was used as augmenting inoculum. After augmenting, the H2 production rate almost doubled (Fig. 6). Bioaugmentation with co-cultures Clostridium acetobutylicum X9 and Ethanoigenens harbinense B49 showed to improve cellulose hydrolysis and subsequent H2 production rates from carboxymethyl cellulose [67].