To establish an environmentally sustainable biohydrogen technology, multidis­ciplinary research approach is vital. Process engineering and optimization of operational factors govern the performance of any biological system and also have considerable influence on fermentative H2 production. The persistence of an acidic microenvironment due to production of soluble acid metabolites as end-products inhibits the process leading to low substrate conversion efficiency to H2. Apart from lower conversion efficiency, one of the important aspects to be paid signifi­cant attention is the non-utilized organic fraction that usually remains as a soluble fermentation product from acidogenic process. Various routes to utilize residual organic fraction of acidogenic process as substrate can be explored. Integration of multiple processes possible for additional revenue generation in the form of addition energy (H2, bioelectricity, methane, etc.) and wastewater treatment utilizing acido — genic effluents are depicted in Fig 7. Application of genetic engineering aspects to stimulate conversion process efficiency is one potentially promising research area.

Design and development of bioreactor systems for H2 production is one of the areas where considerable focus is required. Scaling up of the process to pilot or large scale to generate baseline engineering data will sustain the technology with respect to commercialization. Interaction between the research community and industry from time to time to understand the requirements and design the technology accord­ingly holds the key to the successful commercialization of this process. Moreover, the process to convert existing/operating anaerobic reactors producing methane to

H2 production will pave the way for large scale implementation of this technology and helps to achieve continuous H2 production.

Acknowledgments I acknowledge Dr. J. S. Yadav, Director, IICT and Dr. P. N. Sarma, Head, BEEC, IICT for their encouragement and inputs of V. Lalit Babu, G. Mohanakrishna, S. Veer Raghuvulu, S. Srikanth, B. Purushotam Reddy, M. V. Reddy, M. Prathima Devi, R. Kannaiah Goud and M. Lenin Babu. Biohydrogen and bioelectricity research in BEEC is supported by Department of Biotechnology (DBT), Government of India in the form of research grants (BT/PR/4405/BCE/08/312/2003 and BT/PR8972/GBD/27/56/2006).

Researchers have performed all three fermentation processes (batch, fed-batch, and continuous) for biomass conversion into ethanol. The most suitable fermen­tation strategy depends upon the growth kinetics of the microorganism, the type of hydrolysate, and the economics of the process. For ethanol production from lig — nocellulosic biomass, batch fermentation has been extensively utilized in the past. The batch process is a multivessel approach that allows flexible operation and easy control in the bioconversion process [33]. In fed-batch fermentation, the micro­bial cells can be acclimatized at low substrate concentrations that later assist in accelerating the rate of ethanol formation during the entire course of the biocon­version process. Fed-batch fermentation processes are ideal to obtain a high cell density, which may help to achieve higher ethanol yields with greater productivity. Higher cell density also helps to reduce the toxicity of lignocellulose hydrolysates, particularly acid hydrolysates, to yeast cells. Continuous fermentation is another state-of-the-art technology in which microorganisms work at a lower substrate con­centration, maintaining higher ethanol concentration during the entire course of the fermentation reaction [34]. Table 3 summarizes the fermentation profiles of different microorganisms utilizing a variety of lignocellulose hydrolysates.

# Soliddiquid (Lignocellulose substrate: dilute acid solution). ## Details are not available.

H2 production by dark fermentation (acidogenic or acetogenesis) processes shares many common features with methanogenic-anaerobic digestion [7, 11, 12]. Anaerobic conversion requires four major steps and five physiologically dis­tinct groups of microorganisms to convert hydrocarbons from complex to simple molecules through H2 and acid as intermediates finally, to carbon dioxide (CO2) and methane (CH4) (Fig. 1a). Fermentative/hydrolytic microorganisms hydrolyze com­plex organic polymers to monomers, and then ferment those monomers to a mixture of low-molecular-weight organic acids and alcohols. Obligatory H2 producing ace — togenic bacteria (AB) oxidize fermentation products to acid intermediates and H2,

which also include acetate production from H2 and CO2 by acetogens and homoace — togens and finally acetoclastic methanogens convert organic acids to CH4 and CO2 [4,11,13]. H2-producing AB grow in syntrophic association with hydrogenotrophic methanogens (H2 consuming), resulting in low H2 partial pressure thus allowing acetogenesis to become thermodynamically favorable by interspecies H2 transfer

[11] .

Although the TGER did not perform to its full potential during the 90 day assess­ment and validation, it did demonstrate its ability to convert waste to energy and reduce diesel fuel consumption in a harsh operating environment. Below is the system level parameters recorded during live testing in Iraq. Due to equipment problems, the TGER was not able to demonstrate its ethanol production capabilities and provide enough data to statistically evaluate the bioreactor performance. The harsher conditions in Iraq also required more maintenance time for the pelletizer, thus reducing their pellet production capabilities. These issues and others con­tributed to the reduced fuel efficiency of the TGER while in operation in Iraq.

Below are specific data taken from various days when the TGER was operating at its best in Iraq. Figure 9 illustrates the ability of the TGER to conserve diesel

fuel when running at high loads. The specifications for the Kohler 60 kW generator used on the TGER rates the engine’s fuel consumption at 4.6 gallons per hour (gph) when less than 100% load. 100% load for the Kohler generator set using a 3-phase, 120/240 V 4P8 alternator at prime rating is 54 kW. The TGER maintained 50 kW of off board power (usable power) for approximately 2 h. During that same time the engine’s diesel fuel consumption was on average 1.5 gph, a diesel fuel savings of 2.76 gph.

Figure 10 illustrates the power efficiency of the TGER. The yellow line rep­resents all the power consumed by the TGER’s subsystems and is referred to as parasitic power. All remaining power generated by the TGER (50 kW) is available for use by the customer, and is represented by the light blue line. To determine the TGER’s power efficiency (pink line), we divided the power available to the customer (light blue line) by the total power generated (dark blue line). The TGER’s average power efficiency was approximately 77.37% during the recorded timeframe.

Figure 11 illustrates the TGER’s ability to continue to conserve diesel fuel in adverse environmental conditions. The generator exceeded the recommended load of 54 kW and generated 55.5 kW of off board power while consuming only 2.5 gph of diesel fuel. The most likely cause of the increase in fuel consumption from 1.5 to 2.5 gph was due to foreign debris (i. e. sand and dust) entering the system and causing the gasifier filters to clog, thereby reducing the amount of syngas supplied to the engine. This forced the engine to compensate by supplying more diesel fuel into the engine in order to maintain 55.5 kW of off board power. Even under these sub-optimal conditions, the TGER was able to conserve 2.23 gph of diesel fuel.

Table 3 shows data taken during field testing on 30 May 08 that was input into the TGER Energy Conversion Model. The model calculates the percent contribution

Fig. 11 Fuel efficiency and power (28 May 08)

that diesel fuel versus biofuels has to generating electrical energy. The model cal­culated that, of the total energy produced, the biofuels contributed 77.26% of the required energy and diesel fuel contributed 22.74%.

Figure 12 illustrates the effect of the introduction of ethanol on fuel consump­tion of the generator. Fuel consumption matches closely with the increase in power

Energy content of feed

Electrical energy production

Total (kWh) 343

Offboard (kWh) 230

Total thermal-to-electrical energy conversion efficiency (% of energy content of feed) 23.0%

Offboard energy conversion efficiency (% of thermal energy content of feed)

15.4%

Diesel fuel savings (gallons)

33

Energy delivery efficiency (% of electrical energy for offboard use)

67.1%

%Contribution to feed energy Diesel 22.74%

Biofuels 77.26% output until 1:30 pm, after which the fuel consumption drops off abruptly while the power output remains relatively steady. At 1:30 pm ethanol was introduced into the engine at rate of 0.5 gph causing the diesel fuel consumption rate to drop by more than 0.25 gph. Ethanol was supplied to the engine for approximately 30 min until mechanical difficulties with the ethanol pump began to occur and forced the operators to turn the pump off. When the ethanol pump is turned off the diesel fuel consumption gradually goes up while the power output remains relatively steady.

Table 4 shows the use of the TGER Energy Conversion Model to analyze the per­formance of the TGER on 1 August 08. Biofuels contributed 92.92% of the required energy to generate electricity and diesel fuel contributed 7.08%. This shows that the TGER can run almost entirely on biofuels, although the increase in biofuel contribu­tion did have a negative affect on the thermal to electrical conversion efficiency. The increase in the contribution of energy from biofuels lowered the thermal to electrical conversion efficiency from 23% on 30 May 08 to 16.8% on 1 August 08, which is attributable to the fact that the Kohler generator was specifically designed to run on diesel, rather than biofuels.

When wastewater is used as a fermentative substrate for H2 production, the extent of substrate degradation is important when process efficiency is considered [71]. There is a trade-off between technical efficiency based on H2 production and sub­strate removal at different feeding pHs. Neutral pH is ideal for wastewater treatment while acidic pH is useful for effective H2 production [21, 26]. Balanced conditions for effective combined performance and process optimization are especially impor­tant to sustain process economic viability. Process performance was evaluated using two diverse mathematical approaches [data enveloping analysis (DEA) and design of experimental (DOE) methodology] [71]. The role of some important factors such as type and origin of inoculum, pre-treatment procedure, inlet pH, co-substrate addi­tion and feed composition were evaluated for combined process efficiency by the DEA methodology. DEA analysis showed that the untreated anaerobic inoculum under acidic conditions using simple wastewater as fermentative substrate showed combined process efficiency. Taguchi’s DOE methodology was used to enumerate the role of selected factors on H2 production and substrate degradation with the final aim of optimizing the process [71]. This helped to identify the influence and con­tribution of individual selected factors on the process and to derive the relationship between variables and operational conditions. By adopting the derived optimum conditions, the performance with respect to H2 production and substrate degradation could be improved significantly.

The commercial installation of AD technologies is facilitated or obstructed by multi­ple interactive factors, respectively termed drivers or barriers. Drivers are factors that stimulate, enable, or facilitate implementation of a technology or project, whereas barriers are the factors that function in the opposite direction. Both drivers and barriers may be technological, economic/financial, environmental, or sociopoliti­cal, and may also include subjective psychological components such as uncertainty, perception, or fear. Despite their importance, the drivers and barriers for commer­cial implementation of AD by companies or farms are rarely disclosed or detailed in the literature because the information is typically related to business operations and confidential. However, experience from on-site and cooperative studies of AD for candidate factories or farms have shown that the satisfaction and resolution of multi­ple drivers and barriers, respectively, is crucial in the decision making to implement a specific AD project. Experience with pilot-scale AD studies conducted by anaer­obic digester vendors has shown that both drivers and barriers are multi-faceted and interdependent and vary in importance depending on a host of factors associated with candidate factories and their biomass wastes. An AD project is unlikely to proceed unless the full range of drivers and barriers are considered and the drivers outweigh the barriers. The following section will discuss the drivers and barriers in general, and how the advancement of AD technologies can contribute to tipping the balance towards the drivers by mitigating many of the barriers, including those of economic and political nature.

Keiko Watanabe, Norio Nagao, Tatsuki Toda, and Norio Kurosawa

Abstract Composting is an efficient and cost-effective process for organic waste treatment. In order to expand our knowledge regarding microorganisms in the com­posting reactor, bacterial community structures in a variety of composting processes were examined by 16S rRNA gene (rDNA) clone analysis including denaturing gradient gel electrophoresis (DGGE), as a case study. As previously reported, the dominant bacteria consist of members of the order Bacillales in a typical compost­ing condition with woodchips as the bulking agent. However, these aerobic bacteria decreased to 14%, and anaerobes or facultative anaerobes arose when the decom­position rate of organic compounds dropped following aggregation of the contents. In the composting reactor operated with plastic bottle flakes as bulking agent, the order Lactobacillales co-dominated with the Bacillales, regardless of reactor size, accounting for about 70% of the detected organisms during first week of the opera­tion, gradually decreasing to about 30% with maturation of the composting process. Most species detected by clone analysis have not been cultivated, and may be VBNC (viable but non-culturable) species, implying symbiotic interactions among the microorganisms. In addition, the 16S rDNA-clone and DGGE methods are also introduced in this chapter.

Keywords Aggregate ■ Bacterial community ■ Bulking agent ■ Clone analysis ■ Compost ■ Denaturing gradient gel electrophoresis (DGGE) ■ Large-scale fed-batch composting reactor ■ Plastic bottle ■ Polyethylene terephthalate ■ 16S rRNA gene (16S rDNA)

K. Watanabe (B)

Faculty of Engineering, Department of Environmental Engineering for Symbiosis, Soka University, 1-236, Hachioji, Tokyo, Japan e-mail: kewatana@soka. ac. jp

O. V. Singh, S. P. Harvey (eds.), Sustainable Biotechnology,

DOI 10.1007/978-90-481-3295-9_8, © Springer Science+Business Media B. V. 2010

1 Introduction

Composting is one of the efficient and cost-effective biological processes to treat organic waste. However, some trouble may occur in a composting reactor, for exam­ple, aggregation of contents, decreasing pH and decreasing rate of decomposition. In composting processes, thermophilic and mesophilic microorganisms have important respective functions in terms of nutrient recycling and decomposition of complex organic substrates [1]. Therefore, an understanding of the microbial community and its succession is important to effectively manage the composting process. Thus, in this chapter, bacterial communities in the various composting reactors are revealed by using molecular biological methods, as a case study. These methods are simi­larly applicable to environmental samples, anaerobic digestion treatment reactors, and industrial reactors.

Xylitol is a naturally-occurring sugar with a wide spectrum of potential applica­tions. It has a sweetening power matching that of sucrose (table sugar), and is used as a sugar substitute in the food processing industry [43]. Xylitol produces a perceived sensation of coolness in the mouth as it comes in contact with saliva because of its negative heat of solution [43]. Xylitol can be produced through microbial transformation reactions by yeast from D-xylose, or by both yeast and bacteria from D-glucose [44]; D-xylose can also be directly converted into xylitol by NADPH-dependent xylose reductase [45].

Fermentation is the process of deriving energy from the oxidation of organic com­pounds using an endogenous electron (e-) acceptor, which is usually an organic compound [14]. This is in contrast to cellular respiration, where e — are donated to an exogenous e- acceptor, such as oxygen, via an electron transport chain (ETC). Considering glucose as substrate, fermentative H2 production starts with the con­version of glucose to pyruvate through glycolysis by both obligate and facultative anaerobic bacteria. In facultative anaerobes, pyruvate is converted to acetyl-CoA and formate, which is catalysed by pyruvate formate lyase (PFL) [3] and H2 is produced from formate by the formate hydrogen lyase (FHL) complex. In obligate anaerobes, pyruvate is converted to acetyl-CoA and CO2 through pyruvate ferre — doxin oxidoreductase (PFOR) and this oxidation requires reduction of ferredoxin (Fd) [3, 15]. The fate of pyruvate in the case of anaerobic operation depends on the operating pH. Under acidic condition pyruvate is converted into volatile fatty acids along with H2 by acidogenic bacteria. Neutral operation leads to the formation of CH4 and CO2 by methanogenic bacteria. Under basic pH, anaerobic digestion leads to solventogenesis. At all the pH conditions, H+ shuttling takes place between metabolic intermediates with the help of various redox mediators under anaerobic operation. The H+ from the redox mediator is detached by a specific dehydrogenase (NADH-dehydrogenase) and combined with the e- from oxidized ferredoxin to gen­erate H2 in presence of the hydrogenase enzyme (Fig. 1b). Hydrogenase activity is higher at acidic pH but with increase in pH, metabolic pathway might proceed to the next step of anaerobic digestion where H+ get reduced to CH4 (methanogenesis) or ethanol (solventogenesis).

Biodegradation of substrate is always accompanied by the release of protons (H+) and electrons (e-) associated with various redox reactions and enzymes. Dehydrogenase is one of the important enzymes involved in the inter-conversion of metabolites and the transfer of protons (H+) between metabolic intermediates through redox reactions using several mediators (NAD+, FAD+, etc.). Redox medi­ators are capable of carrying H+ and e-, otherwise known as energy carriers as they are involved in biological energy (ATP) generation [16]. Generally, in the anaerobic microenvironment, inter-conversion of substrates takes place through degradation that increases the availability of H+ in the cell. The protons associated with redox mediators are the main source of fermentative H2 production. The protons from redox mediators are detached in presence of NADH-dehydrogenase and reduced to H2 in presence of the hydrogenase enzyme with the help of e — donated by oxidized ferredoxin (co-factor) [3]. Hydrogenases are complex metalloenzymes that can be classified into three groups based on the number and identity of the metals in their active sites: [NiFe]-, [FeFe]- and [Fe]-hydrogenases [17]. These enzymes are also responsible for the reversible conversion of molecular H2 into two H+ and two e — [H2 ^ 2H+ + 2e-] [3]. The dehydrogenase activity is crucial along with the hydro­genase activity as it maintains H+ equilibrium in the cell through redox reactions and inter-conversion of metabolic intermediates. Nitrogenase enzymes are also involved in H2 production along with nitrogen-fixation. Nitrogenases irreversibly catalyze
the reduction of molecular nitrogen to ammonium by consuming reducing power (e- mediated by ferredoxin, NAD+ etc.) and ATP. Nitrogenase catalyzes H+ reduc­tion in the absence of nitrogen gas. Even in nitrogen atmosphere, H2 production is catalyzed by nitrogenase as a side reaction at a rate of one-third to one-fourth that of nitrogen-fixation. Nitrogen-fixing cyanobacteria are potential candidates for H2 production by nitrogenase but it is an energy-consuming process due to breakdown of many ATP molecules.