Production of Methane Biogas as Fuel Through Anaerobic Digestion

Abstract Anaerobic digestion (AD) is a biotechnology by which biomass is converted by microbes to methane (CH4 ) biogas, which can then be utilized as a renewable fuel to generate heat and electricity. A genetically and metabolically diverse community of microbes (mainly bacteria and methanogens) drives the AD process through a series of complex microbiological processes in the absence of oxygen. During AD, bacteria hydrolyze the polymeric components (e.g., polysaccharides, proteins, and lipids) present in the feedstock and further ferment the resulting hydrolysis products to short chain fatty acids (SCFA), H2 and CO2 , which are ultimately converted to methane biogas (a mixture of CH4 and CO2 ) by archaeal methanogens.Various biomass wastes (e.g., livestock manure, crop residues, food wastes, food-processing wastes, municipal sludge, and municipal solid wastes) are especially suitable for AD. As one of the few technologies that can both costeffectively generate bioenergy and reduce environmental pollution, AD has been increasingly implemented in different sectors to convert otherwise wasted biomass to bioenergy. AD technologies can be categorized in many different ways. Each AD technology has its own advantages and disadvantages that make it suitable for particular feedstocks or objectives (i.e., production of energy or stabilization and treatment of wastewaters). Both drivers and barriers exist for commercial implementation of AD projects, with the former stimulating, enabling, or facilitating AD implementation, while the latter function in opposite direction. This chapter will provide an overview of the microbiology underpinning the AD process, and discuss the characteristics of the biomass wastes suitable for AD and the AD technologies appropriate for each type of these feedstocks. The drivers and barriers for AD as well as the AD technology gaps and future research needs will also be discussed.

Z. Yu (B) Department of Animal Sciences and Environmental Science Graduate Program, The Ohio Agricultural Research and Development Center, The Ohio State University, Columbus, OH 43210, USA e-mail: yu.226@osu.edu

O.V. Singh, S.P. Harvey (eds.), Sustainable Biotechnology, DOI 10.1007/978-90-481-3295-9_6, C Springer Science+Business Media B.V. 2010


Z. Yu and F.L.Schanbacher

Keywords Anaerobic digestion · Biomethanation · Methanogens · Methane biogas · Digesters · Biomass wastes · Feedstocks Abbreviations AD BMP BOD CAFO CMCR COD CSTR DRANCO EGSB HRT MPFLR MSW OFMSW OLR RDP SCFA SRT SS TPAD TS UASB VS anaerobic digestion biochemical methane potential biological oxygen demand confined animal feeding operation completely mixed contact reactor chemical oxygen demand continuously stirred tank reactor dry anaerobic combustion expanded granular sludge bed hydraulic retention time mixed plug-flow loop reactor municipal solid wastes organic fraction of municipal solid wastes organic loading rate ribosomal database project short chain fatty acids solid retention time suspended solid temperature phased anaerobic digestion total solid upflow anaerobic sludge blanket volatile solid

1 Introduction
Anaerobic digestion (AD) is underpinned by a series of bioconversion processes that transform organic compounds, especially biomass wastes, to methane biogas (a mixture of approx. 60% CH4 and 40% CO2 ). Although it has been used for more than a century in treatment of municipal sludge and high-strength organic wastewaters from industries, the main objectives have been to stabilize and sanitize the sludge and to remove the organic pollutants from the influents, with relatively little focus on biogas production. Recently, AD received tremendous renewed interest as the demand for and price of fuels continue to rise. AD is looked upon to be an important biotechnology to help build a sustainable society by simultaneously producing

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renewable bioenergy and protecting the environment. Indeed, a diverse range of feedstocks (e.g., municipal sludge, food-processing wastes and wastewaters, livestock manures, the organic fraction of municipal solid wastes (OFMSW), crop residues, and some energy crops) are being diverted to AD for increasing biogas production [4]. Although AD is a relatively slow process and its operation and performance are sometimes unstable, the methane biogas derived from biomass wastes has become competitive, in both efficiency and cost, with heat (via burning), steam, and ethanol production [31]. In this chapter, the microbiological underpinning of the AD process as well as the recent understanding of the microbial communities driving AD will be discussed from a biotechnological perspective. This chapter will also provide an overview of the common characteristics of feedstocks that have great biogas potentials and the AD technologies suitable for each of these types of feedstocks. The drivers and barriers for commercial AD implementation as well as the AD technology gaps and the research needs will also be discussed.

2 The Microbiology Underpinning Anaerobic Digestion
A very complex community of bacteria and archaeal methanogens drives the entire AD process [36, 65]. Fungi and protozoa are also found in anaerobicdigesters [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 acidogenesis 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




Digestate CH4, CO2

Hydrogenotrophic methanogenesis H2, CO2, or HCOO-

Aceticlastic methanogenesis CH3-COO-

Syntrophic acetogenesis Non-acetic SCFA (propionate, butyrate, lactate, etc) Fermentative acidogenesis

Fermentative acidogenesis

Fermentative acidogenesis

Monomers and oligomers (sugars, AA, LCFA, peptide) Hydrolysis Feedstock Polymeric feedstocks

Fig. 1 The four phases of anaerobic digestion process

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 conversionof 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 ageneration time of 3.5–9 days) [36]. However, methanogenesis 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 belong (15 days or longer) to retain enough syntrophic acetogens. Hence, syntrophic acetogenesis can be a ratelimiting 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, withnitrogen 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].

3 Methane Biogas Production from Different Feedstocks
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, ]). 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.

3.1 Anaerobic Digestion of Municipal Sludge (Biosolids
Municipal sludge includes primary sludge and waste activated sludge derived from centralized wastewater treatmentplants 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 inorganic 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 37a—Š 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.

3.2 Anaerobic Digestion of Animal Manures
Animal manures represent a huge methane biogas potential. As estimated, 106 million dry tons of animal manures are produced each year in the USA, with approximately 87 million dry tons being available for methane biogas production [69]. Given a BMP of 200–400 m3 CH4 /dry ton [8], the amount of animal manures available for AD provides a potential of 17–35 billion m3 of CH4 per year in the USA. The animal manures produced from confined animal feeding operations (CAFOs) offer one of the most abundant single feedstocks available for large-scale methane biogas productions. The composition and physical features (e.g., water contents) of animal manures vary widely from species to species and from operation to operation [58]. In general, animals manures have relatively high water contents, ranging from 75% (poultry manure) to 92% (beef cattle manure). Most of the animal manure is organic matter, with VS contents ranging from 72% (poultry manure) to 93% (beef cattle manure) of TS. Inorganic nutrients, including N


P and K, are rich in animal manures, especially poultry manure. Because most of the readily degradable substances, especially carbohydrates, have been digested and absorbed by the animals, animal manures have very little readily fermentable substrates. Additionally, animal manures have high concentrations of aminonitrogen such as urea and ammonia and a large pH buffering capacity against acids. Thus, the fermentative acidogenesis during AD of animal manures typically does not result in significant pH decline, but high concentrations of ammonia can result, causing toxicity to methanogens, especially in thermophilic digesters where methanogens are very susceptible [43]. Furthermore, animal manures contain large amounts of microbial biomass, including bacteria and methanogens. Consequently, AD reactors digesting animal manures, especially livestock manures, can be started without the addition of external digested sludge as a start culture or inoculum. Because of the relatively low contents of readily degradable substances, the methane biogas production from animal manures is generally slow. Thus, when digested alone a long retention time is needed. Co-digestion with nitrogen-poor yet carbohydrate-rich feedstocks, such as food-processing wastes and OFMSW, can substantially enhance CH4 production and stabilize the AD process of animal manures [59, 94]. Some animal manures, especially dairy cattle manure, contain sand from the sand bedding [42], which settles in AD reactors and can cause operational problems if not dealt with properly. Due to the large differences in many physicochemical characteristics and degradability, different manures may require different AD technologies for efficient and cost-effective AD. Here the AD technologies suitable for beef manure, diary manure, swine manure, and poultry litter will be discussed. 3.2.1 Animal Manure Dung and Poultry Litter The manuresfrom 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 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 ADefficiency. 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 (https://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 (55a—Š C) are responsible for the improvement observed in TPAD systems [91].

3.3 Anaerobic Digestion of Solid Food and Food-Processing Wastes, Organic Fraction of Municipal Solid Wastes (OFMSW), and Crop Residues
These wastes are characterized by varying water contents, but high VS contents (>95%). However, these parameters vary considerably. Most food wastes have balanced nutrients and large amounts of readily fermentable carbohydrates and thus are among the most suitable feedstocks for AD. According to a recent study, 348 m3 of CH4 can be produced per dry ton of food wastes within only 10 days of AD [93]. Food wastes amount to approximately 43.6 million dry tons each year in the USA [81]. This represents a potential of 15.2 billion m3 of CH4 per year. During food processing, a significant portion of foodstuffs also ends up in wastes or wastewaters. For example, 20–40% of potatoes are discarded as wastes during processing. National data on the amount of food-processing wastes are not available.


Production of Methane Biogas as Fuel Through Anaerobic Digestion115

The state of California generates more than 4 million dry tons of food-processing wastes each year [54], potentially producing 1,200 million m3 of CH4 . This translates into an annual potential of several billions m3 of CH4 in the USA. Except for the wastes from animal meat processors, most food-processing streams are relatively poor in nitrogen, but rich in readily fermentable carbohydrates. As such, food-processing wastes can be co-digested with other nitrogen-rich feedstocks (e.g., municipal sludge or animal manures) to enhance AD system stability and CH4 production [46]. Approximately 250 million dry tons of MSW are produced annually in the USA. The organic fraction, such as paper, yard trimmings, and food scraps, is biodegradable and can be converted to methane biogas. Although the composition of MSW varies dramatically depending on society, season, collection, and sorting, OFMSW accounts for more than 50% of the MSW in most societies. Most OFMSW has little moisture or readily fermentable carbohydrates and is relatively deficient in N or P, but has a relatively large BMP (300–550 m3 CH4 /ton) if digested adequately [25]. The OFMSW generated annually in the USA has a CH4 potential of 37.5 billion m3 . Crop residues amount to an estimated 428 million dry tons each year in the USA. Although the majority of crop residues is typically left in the field, approximately 113 million dry tons are recoverable and available for conversion to methane biogas [69]. Crop residues typically have relatively low water contents, high VS contents, and variable contents of readilyfermentable carbohydrates. Most crop residues are non-leguminous and are poor in available nitrogen. The BMP of crop residues varies from crop to crop (from 161 to 241 m3 CH4 /ton) (124). If subjected to proper AD, at least 20 billion m3 of CH4 can be produced annually from the crop residues available for biogas production in the USA. Similarly for other nitrogen-poor biomass, codigestion of crop residues with animal manures or municipal sludge substantially improves CH4 yield [50]. In the EU, 1,500 million dry tons of biomass are available each year for biomethanation within the agricultural sector, with half of this being crops intended for bioenergy production [5]. It should be noted that production of bioethanol and biodiesel from energy crops only utilizes a fraction of the biomass, and implementation of AD by the bioethanol industry can generate substantially more energy (up to 30% of the total energy of the initial biomass) [3, 74]. This also holds true for many other biomass-based processes producing non-food products. All these types of feedstocks likely contain bulky materials, such as peeling, papers, stems and leaves. Pretreatment, especially reduction of particle size by grinding or milling, is typically required to enhance AD [40]. Other pretreatments such as alkaline pretreatment [53] have also been evaluated to further enhance the hydrolysis step in laboratories, but few of them have been implemented in fullscale AD plants. As mentioned earlier for the AD of livestock manures, co-digestion with other nitrogen-rich biomass (e.g., municipal sludge oranimal manure) can also substantially stabilize the AD process and increase CH4 production [50, 94]. The above mentioned wastes have relatively low water contents. They can be digested using some wet AD processes (e.g., CSTR and CMCR) after dilution. The Lemvig Biogas plant in Denmark is one example of such wet AD. It is a centralized biogas plant consisting of three thermophilic CSTR with a total volume of 7,000 m3


that digests various types of organic industrial wastes, source-sorted MSW, and manures [7]. The biogas produced is used to generate electricity and heat. Apparently, dry AD is advantageous for these low-moisture feedstocks because it eliminates the need to dilute the feedstocks to a fluid state and produces a lowmoisture digestate, which is easier to transport and disperse [90]. The DRANCO technology is a dry AD technology successfully used to convert low-moisture organic wastes (e.g., OFMSW and crop residues) to methane biogas [21]. The DRANCO technology requires the feedstock to be shredded and milled first to reduce particle sizes (15 kGy 130 MPa 75–94 MPa 50 MPa 80 MPa 28 MPa 50–70 MPa 30 MPa

[61] [25] [9] [32] [10] [72] [50] [71] [8, 21] [90, 91] [50, 80] [70, 93] [41] [41, 67] [88] [45] [65]

Thermophilicpizophiles

51 MPa 75 MPa 45 MPa 20 MPa 40 MPa


282 Table 1 (continued) Environmental Parameters Types Mesophilicpeizophiles Desiccation Xerophiles Definition 15 MPa 60 MPa Anhydrobiotic Examples Desulfovibrio profundus

R. Kumar et al.

References [4] [51] [84] [79] [61]

Pseudomonas sp. Ms300 Artemiasalina; Nematodes Microbes, Fungi, Lichens Haloarcula, Halobacterium, Haloferax, Halorubrum, Dunaliella salina

Salinity

Halophiles

Salt loving (2–5 M Nacl)

pH

Alkaliphiles

pH>9

Natronobacterium, [61] Natronococcus, Bacillus firmus OF4, Spirulina sp. (all pH 10.5) Ascidianus, Desulfurolobus, [61] Sulfolobus, Thiobacillus, Cyanidium caldarium, Ferroplasma sp. [41] [67] [61]

Acidophiles

pH80a—Š C) to psychrophilic (maximum growth 1 M and temperatures > 80a—Š C. Organisms exposed to osmolaritic environments can develop osmolyte strategies that have been referred to as extremolytes. Extremolytes provide protection to globular proteins, nucleic acids and whole cells. These protective effects may


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partially be secondary effects of protein stabilization (e.g., stabilization of membrane proteins) in microorganisms. Extremolytes provide protection to this cells against drying environment probably by replacing of water molecules by hydroxyl group of Ectoine [59] and thus stabilize membrane fluidity. Hydroxyectoine (4S-2methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) was originally discovered from an extremely halophilic phototrophic eubacterium Halorhodospira halochloris, isolated from Wadi Natrun, Egypt by Galinski et al. [33]. Later, hydroxyectoine substance was also found in a wide varieties of halophilic and halotolerant bacteria. The ability to accumulate ectoine was observed among the organisms such as E. coli, B. subtilis, C. glutamicum, and S. melilotii [46, 86]. The carbohydrate extremolytes such asmannosylglycerate (Firoin) and mannosylglyceramide (Firoin-A) are among top value-added extremolytes. Firoin accumulates in the cells in response to heat stress in thermophilic microbes. The chemically reactive end of the sugar of firoin forms a glycosidic bond with a hydroxyl group of glyceric acid or glyceramide. Rhodothermus marinus synthesizes both mannosylglycerate and mannosylglyceramide. Anionic mannosylglycerate is accumulated in the cells in response to heat stress. Whereas, uncharged mannosylglyceramide increase in the cells with elevated NaCl levels. Mannosylglycerate was also found in eukaryotic mesosphilic red algae [49]. Archaeabacteria are well known as extremophilic candidates among the microorganisms. Typical halotolerant and hyperthermophilic archeabacteria Pyrococcus furiosus and Thermotoga maritima accumulate negatively charged derivatives of inositol and glycerol at extreme temperature and high salt concentration [22, 82]. Di-myoinositol-1 -phosphate (DIP), a phosphodiester derivative of the uncharged osmolyte myoinositol found in eukaryotes, which provides tolerance against salinity. γ-Diglycerol phosphate (DGP) was identified as a new extremolyte in Archaeoglobus fulgidus and shown to be an effective protein stabilizer in vitro [56, 73]. DGP is also known to accumulate in response to elevated external NaCl concentrations, while temperature increases lead to enhanced DIP accumulation [55]. Similarly, cyclic 2 -diphosphoglycerate (cDPG) and cyclic trianionic pyrophosphate were found to accumulate in archaea Methanothermobacter thermoautotrophicus. The primaryrole of 2 -diphosphoglycerate (cDPG), in Methanothermobacter may be as a phosphate storage compound which may provide protection to glyceraldehyde-3-phosphate dehydrogenases at high temperature [42, 66]. Other novel extremolytes and their applications are being summarized in the Table 2. Besides conventional means, non conventional sources such as marine macro and micro flora have been explored to isolate the novel drug candidates to inhibit or activate the vital signalling pathways lead to cure or prevent the particular diseases or disorder. Bryostatins, isolated from bryozoan, Bugula neritina, one of the novel protein kinase C (PKC) inhibitors is being evaluated for cancer cure. The marine microalgae, cyanobacteria, and heterotrophic bacteria, living in association with invertebrates (e.g. sponges, tunicates, and soft corals etc.) have been identified, as the original sources of many bioactive compounds (Kahalalide F, E7389, Curacin A, Salinosporamide A and Eleutherobin) and may be used as important


Extremophiles: Sustainable Resource of Natural Compounds-Extremolytes Table 2 Extremolytes and their specific applications Compounds Hydroxyectoine Extremophilic organisms Streptomyces strain Applications Protection of oxidative protein damage (LDH) Reduction of VLS in immunotoxin therapy Stabilization of retroviral vaccines Induction of thermotolerance in E. Coli Protection of P. putida against anhydrobiotic stress Enzyme stabilization against heating, freezing, and drying Protection of LDH against heat and freeze-thawing Inhibition of insulin amyloid formationStabilization of tobacco cells against hyperosmotic stress Block of UVA-induced ceramide release in human keratinocytes Protection of the skin barrier against water loss and drying out Protection of skin immune cells against UV radiation Reduction of UV-induced SBCs Prevention of UVA-induced photoaging Cytoprotection of keratinocytes Stabilization of enzymes against thermal stress and freeze drying Stabilization of recombinant nuclease Thermostabilization of proteins and rubredoxin Treatment of patients with severe psoriasis.


References [1] [6] [24] [63] [64] [59] [36] [2] [69] [37] [18] [13] [19] [18] [20] [16, 81] [31] [55, 56] [40]

Ectoine

Halorhodospira halochloris

Mannosylglycerate Rhodothermus marinus DGP Kahalalide F Archaeoglobus fulgidus Elysia rufescens/ Bryopsis sp. (mollusc/green alga) Halichondria okadai (sponge, synthetic) Lyngbya majuscule (cyanobacterium)

E7389 (halichondrin B derivative) Curacin A

Treatment for breast cancer

[3]

Potent inhibitor of cell growth and mitosis

[85]




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drug candidate in human interest [3, 38, 40, 85]. The therapeutic agent Scytonemin isolated from an extremophilic marine cyanobacterium Stigonema sp. collected from Waldo Lake, Oregon and characterized as a protein serine/threonine kinase inhibitor [87]. Various other molecules have been screened in the hope of human interest to cure the cancer and related diseases. A battery of such compounds has been explored in the extremophilic marine organisms [29, 34, 43, 58, 60, 68, ].

4 Future Implications of Extremolytes
Multipleirradiations such as X-rays, gamma rays, UV rays, and other electromagnetic radiations have shown tremendous application in human life. Also, various radioisotopes are being used in agriculture, medicine, diagnostic and therapeutic purposes. Apart from that, various nuclear installation sites or reactors are always prone to accidents. Besides the planed radiation exposure for human benefit, the unplanned radiation catastrophe cannot be ruled out. Under this scenario, development of an effective radioprotector is important. The extremophilic microbes and their niches are the best models having abilities to provide molecules of human interest for radioprotection. These sustainable resources of microorganisms have been explored in the past to find the lethal radiation environment [39, 48, ]. We at Institute of Nuclear Medicine and Allied Sciences (INMAS) are involved in exploring the functional properties of radioresistant bacteria, isolated from anoxic rock samples, potentially leading to the development of an effective radioprotective biomolecule in future. Biomolecules extracted from the radioresistant bacteria has been tested for their antioxidant activities, in vitro DNA and protein protection properties and radioprotective efficacy in vitro and in vivo models. Studies performed at INMAS revealed potential support in lower animals, exposed to lethal doses of gamma radiation. The bio-molecule designed from radioresistant bacteria was also found to be capable enough to protect the radiosensitive organs (such hemopoitic, gastrointestinal and reproductive system) in micemodel system. A future implication of mode of bio-molecule administration can be predicted to enhance the immunomodulatory activity with less post irradiation infection. The chances of survivability in irradiated mice, pre-treated with drug, are expected to increase compared to the control mice.

5 Expert Commentary and 5 Year View
In the past two decades thousands of molecules and drug candidates have been screened from different mesophilic microorganisms and evaluated for their possible therapeutic human applications. Many microbial bioproducts are being used as life saving drugs. However, comparatively insignificant efforts were focused on extremophilic microbes as the potential drug reservoirs of the future. In the recent years, much attention has given to the areas of astrobiology, oceanology, nuclear




energy, food production under extreme conditions. It is now being accepted that extremophiles have tremendous potential and can be sustainable resource for novel bioproducts. On futuristic approach, the radioresistant bacteria can be explored for innovation of radioprotective biomolecules that can be used in nuclear catastrophe, and may utilize to protect space travelers. The isolation and maintenance of radioresistant bacteria remains a challenging issue due to their specific nutritional requirement, fear of pathogenesis, and unsecured genomic integrity, but the value-added bioproducts from extremophiles are of great potential utility. The antifreeze extremolytes are always in demandfor people living at subzero temperature and many mountaineers facing frostbite disorder. Other than maintenance of extremophiles, the high throughput screening (HTS) and reference chemical libraries are limited that can screen a wider range of molecular specificity for strategic application of biomolecules. However, the mass spectrometry methods in analytical chemistry such as Electro Spray Ionization Ion Cyclotron Resonance Fourier Transform Mass (ESI-ICR FTMS), Fluorescence Activated Cell Sorter Multi SETTM System (FACS-MS) and Nuclear magnetic resonance (NMR) methods such as Nuclear Magnetic Resonance-Structure Activity Relationship (NMR-SAR) and Saturation Transfer Difference- Nuclear Magnetic Resonance (STD-NMR) are being effectively utilized to screen chemical libraries. Not surprisingly, the sophisticated analytical instrumentation and logistic support may be a limiting factor until proven effective analysis of specified biomolecule [52]. Apart from exploring extremolytes, the evaluation of biological effectiveness, safety and toxicity of extremolyte is one of the major concerns routing drug development. The availability of animal models to generate effective data of specified biomolecule is still limited. The approaches and resources of extremophiles are broad, and have clear potential to generate value-added products for human society.

References
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