Alcoholic fermentation involves the production of alcohols such as ethanol and butanol from biomass. Fermentation has been used since ages to produce alcohol from carbohydrate substrates. The feedstock for this fermentation has been agri­cultural commodities such as sugarcane, beet sugar and corn starch—the more common one being starch. The microorganism used for this fermentation has been mainly, yeast. The technology for such fermentations is very well established and has been commercially used for production of ethanol, with state-of-the art fer­mentation plants, the world over. However, the food versus fuel controversy has prompted the search for other feedstocks such as cellulosic/lignocellulosic biomass for use as substrate for fermentation to ethanol. The bioethanol so produced can be used as a fuel as such or, more appropriately, as an admixture in diesel, as gasohol. The use of such substrates, especially lignocellulosic substrates, for successful commercial fermentation, is still a challenging task mainly due to the highly refractory nature of lignocellulose. In addition to physicochemical methods for efficient hydrolysis of lignocellulose, enzymatic hydrolysis methods are also being researched the latter having an advantage of being carried out in situ in the fer­menter. Persistent research efforts in this area have resulted in the development of efficient and economic fermentation processes and technologies even for such a refractory material. The world’s first cellulosic ethanol demonstration plant has been set up at Yonroe-Tennesee and has begun operations since January 2010.

The biochemistry of lignocellulose/cellulose conversion into easily fermentable sugars has been studied exhaustively and a multitude of reviews and treatises on the subject is available in the literature. This section will discuss the recent advances that have taken place in the area. Lignocellulose consists of cellulose which is a glucose polymer; hemicellulose, which consists of mixed hexoses and pentoses; and xylan which is a xylose polymer. All these substances need to be essentially converted into monosaccharides before they can be fermented by the usual fermentation process. This process of breaking down the complex poly­saccharides into simple soluble sugars is called saccharification. Saccharomyces cerevisiae was the predominantly used species for this conversion. However, this microorganism, which is conventionally used for conversion of starch into ethanol,

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— First type of anaerobic digestion reactor developed; still in

use

— Good mass transfer

— Cannot be operated at high hydraulic loading rates

— Not suitable for biomass with low concentration of readily

biodegradable substances

— Can handle high-strength feedstocks with large amounts

of suspended solids

— High loading rates possible

— Active microbial mass retained in reactor

Applications

Batch CSTR produces 4,000 m3 of biogas per day in biomethanation plant at Karlsruhe, Germany Centralized biogas plant operating three thermophilic CSTRs with a total volume of 7,000 m3 in Lemvig, Denmark

Type of reactor/technology	Salient features Applications
Anaerobic Filter Reactor (AFR)	Comprises a tank filled with rocks, gravel, or plastic Suitable for feedstocks rich in carbohydrates granules which act as a filter medium as well as a Not suitable for feedstock with high separated substrate on which most microbes adhere and grow as a solids(SS) such as manure slurry unless SS is removed biofilm
• Anaerobic expanded bed reactor(AEBR)	— Some microbes grow as clusters/granules within the void spaces — Hydraulic residence time(HRT) and solids retention time (SRT) are separated — Suitable for feedstocks where phases 3 and 4 are prolonged, requiring long (>20 days) SRT — High possibility of clogging of filter — Design is a variant of AFR, similar in all aspects except that the filter medium is fluidized instead of

Biomass Conversion to Energy

immobilized • Anaerobic fluidized bed reactor — Large surface area is provided for digestion reaction (AFBR) — Friction between fluidized particles promotes transfer of substrates, nutrients, and metabolic products across biofilms, simultaneously preventing excessive build-up of bio-film — Effluent is re-circulated — High organic loading rates possible — Reduced reactor clogging — High SS (up to 10%) can be treated — Energy consumption is higher compared to AFR — Scale-up is more difficult — Requires longer start-up time and uniform distribution of influent (continued)

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Type of reactor/technology	Salient features Applications
Expanded Granular Sludge Blanket Reactor (EGSB)	— Latest development in anaerobic digesters Suitable for high strength feedstocks — It is a USAB reactor with a greater height to diameter ratio, enabling greater up-flow velocity (>4 m/h) — Part of effluent is re-circulated
Internal Circulation Reactor (IC)	— Increase in up-flow velocity expands the sludge bed and eliminates dead zones, thus improving mass transfer and digestion rates — High loading rates possible (HRT < 2 h) — It is a combination of USAB and EGSB reactors—design Suitable for treatment of a variety of industrial waste similar to two USAB reactors stacked one on top on the waters, including food processing and manure waste other waters — Lower portion has an expanded granular sludge bed — Influent enters at the bottom through a distribution system and is mixed with the effluent which is recirculated from the top to the bottom through a down pipe — Due to the presence of the sludge bed at the bottom, most of the microbiological reactions of anaerobic digestion processes occur in this lower compartment — The reactor has a down pipe (from bottom to top of the reactor) and a rise pipe (in the upper compartment of the reactor), which cause internal circulation of the water and sludge in the reactor — the rising gas causes a gas-lift, carrying water and sludge upward to the gas-liquid separator, through the rise pipe, consequently causing some water and sludge to drain downward through the down pipe
	(continued)

Biomass Conversion to Energy

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is not suitable for lignocellulosic-based ethanol production because Saccharomy — ces is capable of fermenting glucose alone to ethanol, whereas lignocellulosic biomass hydrolysate usually consists of a mixture of oligosaccharides. A number of novel enzymes and improved microbial strains, which have been specifically engineered to convert such a recalcitrant substance like lignocellulose into easily fermentable sugars, have been successfully developed. Some of the microbial strains developed are Scheffersomyces stiptis, Candida shehatae, Kluyveromyces marxianus, Escherichia coli and Zymonas mobilis [25]. The enzymes responsible for breaking down cellulose from lignocellulosic biomass comprise a multitude of enzymes which fall into the category of glycosyl hydrolases which exist in the form of a complex assembly of enzymes called “cellulosome”. These glycosyl hydrolases include both cellulases and non-cellulosic structural polysaccharidases. The details of the different modules of enzymes comprising the cellulosome are discussed in detail by Berg Miller et al. [26]. The true cellulases, present in the glycosyl hydrolases, cleave the p-1,4-glucosidic bonds of cellulose, resulting in the production of cellobiose. A number of other enzymes, having varying degrees of specificities, are responsible for hydrolyzing different forms of cellulose present in the cellulosic plant material. Non-cellulosic structural polysaccharidases are a diverse group of enzymes that are capable of cleaving the different types of bonds present in the main chain backbone (xylanases and mannanases) and the side chain constituents (arabinofuranosidases, glucuronidases, acetyl esterases, xylosidases, and mannosidases) of the substrate. Ladisch et al. [27] provide an elaborate description of the cellulose enzyme system and the mode of action of fungal cel — lulases. Current research efforts in the field of enzymatic hydrolysis of cellulosic plant material are going increasingly toward identifying newer glycosyl hydrolases. The currently followed approach for accomplishing this is by attempting to genetically modify the most efficient existing microbial systems such as those found in the grass eating ruminant animals, e. g., Fibronobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens into making newer and more efficient glycosyl hydrolases by using molecular engineering concepts. The research progress in the field is comprehensively outlined by Berg Miller et al. [26].

An alternative to the above-mentioned enzymatic hydrolysis of lignocellulosic biomass is acid hydrolysis, which has a few advantages over enzymatic hydrolysis in that, it is quick, no dedicated support of enzyme production system is required, and high temperatures can be used, allowing lower acid concentrations. However, the major drawback of acid hydrolysis process is the degradation of the hexoses and pentoses to acids such as hydroxymethyl furfural (HMF) from glucose and furfural from xylose, in addition to some other acids produced. These acids reduce the activity of the ethanol producing microorganisms. HMF further breaks down to formic acid which may lead to a total inhibition of ethanol formation. Figure 1.20 shows a schematic of the various acids produced during pretreatment/acid hydrolysis of lignocellulosic biomass.

In addition to these acids, metals leached out from the hydrolysis equipments and other SO2 inhibitors released from additives also retard microbial growth and other metabolic activity. More than a 100 such inhibitors have been detected. Liu

Fig. 1.20 Fermentation inhibitors produced during degradation of lignocellulosic biomass (Adapted from [28])

et al. [28] have classified such inhibitory compounds on the basis of the functional group present on the inhibitor agent. These degradation products have an inhibi­tory effect on the ethanol producing organisms, reducing the yield of ethanol from the process. An obvious solution to this problem lies in the removal of the alde­hyde and/or other inhibitory agents at regular intervals. This can be carried out by physicochemical processes such as vacuum evaporation to reduce the volatile inhibitors; alkali treatment, using Ca(OH)2 or NaOH to precipitate out substances having aldehyde and ketone functional groups; and adding activated charcoal or diatomaceous earth to physically adsorb the inhibitory agents, thus improving the yield of ethanol. Use of anion — and cation — exchange resins has also been inves­tigated with results more favorable than all the other methods mentioned above. A combination of two or more methods is preferred depending on the nature of inhibitors present. Enzymatic treatment using peroxidases and laccases obtained from the lignolytic fungus Trametes versicolor has been found to improve the yield of ethanol by removing the phenolic inhibitors from the substrate. Alterna­tively, in situ ‘detoxification’ solutions to this problem are being explored. One such solution comprises development of inhibitor-tolerant strains of yeast or bacteria that can withstand the presence of the inhibitors. The inhibitor conversion pathways and mechanisms of in situ detoxification have been reviewed by Liu et al. [28].

Using recombinant yeast for improved ethanol production is yet another area in which ongoing research efforts are likely give good returns. The improvement of

cellulase expression in S. cerevisiae has also been exploited. The contribution of a number of researchers in developing strains with improved cellulose expression has been compiled by Liu et al. [28]. Similarly, hemicellulase expression in S. cerevisiae has also been studied.

The above developments, along with simultaneous advances in fermentation technologies, are certainly expected to take ethanol production from the more economical lignocellulosic biomass to new heights.

The fermentation technologies used for fermentations have also advanced rapidly, with many progressive modifications in the conventional batch and con­tinuous fermentation processes. Combination of both, the batch and continuous process, called the fed-batch processes have also been developed. Fermentations using novel immobilized cell systems have been used to enhance the efficiency and productivity of the fermentation processes. Methods such as ‘‘growth arrested process’’ have been developed, where a high productivity of intermediate meta­bolic products such as lactate and succinate, as well as other organic acids, has been achieved by arresting the growth of the microorganisms at the particular stage at which the target products are produced, by maintaining the conditions in the reactor which stop further growth of the organisms [29].