A very complex community of bacteria and archaeal methanogens drives the entire AD process [36, 65]. Fungi and protozoa are also found in anaerobic digesters [60] although their functions and contributions to the AD process are not known. The cell densities of microbes in anaerobic digesters are among the highest in managed envi­ronments, with bacteria being the most predominant (up to 1010 cells/mL of digester content) followed by methanogens. The entire AD process can be described as a synergistic process of four sequential phases: hydrolysis, acidogenesis, syntrophic acetogenesis, and methanogenesis (Fig. 1). Each phase is mediated by a distinct functional group, or guild, of microbes [36, 91]. During the first phase, some fac­ultative or strictly anaerobic bacteria (e. g., Clostridium spp.) hydrolyze the biomass polymers (e. g., polysaccharides, proteins, and lipids) present in the feedstocks, giv­ing rise to monomers or oligomers (e. g., glucose, cellobiose, amino acids, peptides, fatty acids, and glycerol). This hydrolysis step is catalyzed by the extracellular hydrolytic enzymes such as amylases, cellulases, xylanases, proteases, and lipases secreted by the hydrolytic bacteria. Kinetically, the hydrolysis step can proceed rapidly for soluble feedstocks such as starch. However, for insoluble lignocellulosic feedstocks that contain recalcitrant embedded lignin, the hydrolysis phase is rather slow and often becomes a major rate-limiting step of the entire AD process [2].

The resulting hydrolytic products are immediately fermented to short chain fatty acids (SCFA), CO2, and H2 during the subsequent fermentative acido — genesis by another guild of facultative or strictly anaerobic bacteria (e. g., Bacteroides, Clostridium, Butyribacterium, Propionibacterium, Pseudomonas, and Ruminococcus). The major SCFA formed include acetate, propionate, butyrate, formate, lactate, isobutyrate, and succinate, with acetate predominating. Small quantities of alcohols (e. g., ethanol and glycerol) are also produced. The fermen­tative acidogenesis typically proceeds rather rapidly [10]. In fact, when feedstocks

containing large amounts of readily fermentable carbohydrates (e. g., sugars and starch) are digested at high organic loading rates, the production of SCFA can exceed their consumption, leading to SCFA accumulation and consequential AD upset or even failure [10].

The final phase of AD involves methanogens of the Archaea domain. Methanogens are strict anaerobes and produce CH4 as the major end-product of their catabolism. Most methanogens are fastidious microbes and only grow on a few substrates within a narrow spectrum of environmental conditions (neutral pH, Eh <­300 mV, etc.). Methanogens use a unique methanogenesis pathway to produce CH4 [36]. Hydrogenotrophic methanogens produce CH4 via the reduction of CO2 by H2 or by the conversion of other C1 substrates (e. g., methanol and methylamines), while acetoclastic methanogens convert acetate to CH4. It should be noted that the former accounts for approximately one third while the latter accounts for two thirds of the CH4 produced in anaerobic digesters. This is because acetate is the major end product of the acidogenesis step in all anaerobic digesters [86]. In spite of this, only a few species of acetoclastic methanogens have been known and they are within genera Methanosaeta (formerly Methanothrix) and Methanosarcina. Methanosaeta spp. are obligate acetoclastic methanogens, while species of Methanosarcina also use C1 substrates. Hydrogenotrophic methanogen species are found in gen­era Methanobacterium, Methanospirillum, Methanobrevibactor, Methanococcus,

Methanomicrobium, Methanoculleus, Methanogenium, and Methanothermobacter. All methanogens contain a unique cofactor, F420, that is autofluorescent at a wavelength of 420 nm [38]. Some methanogens, especially hydrogenotrophic methanogens, contain so much of it that they appear blue when viewed under a microscope. Several trace elements, especially nickel and cobalt, are required by methanogens for methanogenesis and growth. For some feedstocks, supplementa­tion with trace elements can significantly enhance methane biogas production and process stability [48]. Because of the low energy yield from the methanogenesis pathway, most methanogens grow slowly, especially acetoclastic methanogens (e. g., Methanosaeta spp. have a generation time of 3.5-9 days) [36]. However, methano — genesis is typically not a rate-limiting step of the entire AD process because the low-energy yield of the methanogenesis pathway forces it to run rather rapidly. Additionally, methanogens are susceptible to a host of factors (e. g., pH, ammonia, and metals) so they are often implicated in instability or sub-optimal performance of AD [17].

The small amounts of SCFA with three or more carbons (e. g., propionate, butyrate, isobutyrate, valerate) and the ethanol produced during the fermentative acidogenesis as well as the long chain fatty acids derived from lipid hydrolysis can not be used directly by any known methanogens. A unique guild of strictly anaer­obic bacteria (referred to as syntrophic acetogens) can oxidize these intermediates to acetate, H2, and CO2 so that they can serve as the substrates of methanogenesis [75, 91]. However, the oxidation of these fatty acids and ethanol under fermentative conditions (referred to as syntrophic acetogenesis) is thermodynamically unfavor­able; and hydrogenotrophic methanogens are needed to reside in close proximity to rapidly consume the H2 produced by the syntrophic acetogens through interspecies hydrogen transfer [23]. Syntrophomonas wolfei and Syntrophobacter wolinii are thought to be important syntrophic acetogens in anaerobic digesters, with the former primarily oxidizing butyrate and the latter oxidizing propionate. With a generation time of greater than one week, syntrophic acetogens grow extremely slowly [24]. As a result, the solid retention time (SRT) in digesters has to be long (15 days or longer) to retain enough syntrophic acetogens. Hence, syntrophic acetogenesis can be a rate — limiting step during AD, and failure or suboptimal performance encountered during AD operation often involves this guild of bacteria, which is exemplified by AD fail­ure when the organic loading rate was too high and the production of non-acetic SCFA exceeded that of their utilization [47]. Thus, syntrophic acetogens are impor­tant members of the microbial community of stable AD processes even though the carbon flux through them is relatively small, and it is critical to maintain a balanced production and consumption of these non-acetic SCFA by avoiding organic over­loading. It should be noted that because they cannot be cultured as single cultures, syntrophic acetogens are not well studied. The recent advancement of genomics and metagenomics offers new opportunities to better understand this important guild of bacteria in anaerobic digesters (see [55] for a recent review).

Several features of feedstocks can have profound effects on AD, such as the con­tent of readily fermentable carbohydrates, particle sizes of insoluble feedstocks (the hydrolysis step is especially affected by particle sizes), nutrient content and balance, and presence and concentrations of inhibitory compounds. Feedstocks rich in starch and/or proteins are easier to digest than lignocellulosic feedstocks. Reduction of par­ticle size of insoluble feedstocks can significantly speed up AD and increase CH4 yields. Microbes need numerous nutrients to grow, with nitrogen and phosphorous being the most important. The optimal carbon (expressed as chemical oxidation demand, COD) to N to P ratios (COD:N:P) for efficient AD differ with different feedstocks and the AD technologies used. For most feedstocks, a C:N ratio of 25-32 is suitable for most AD processes [8].