Chapter 2 Literature review
2.1 Anaerobic processes: multi-step metabolism processes
2.1.1 Microorganisms and metabolism
2.1.1.2 Methanogens
We all know anaerobic processes are accomplished by consortia, which relate to hydrolytic and fermentative bacteria, acetogens and methanogens. Methanogens play a central role in the whole anaerobic processes, and there have three reasons: first, methanogens belong to the Archaea, and their physiologies and structures are distinct from bacteria. Archaea are unlike true bacteria due to presence of membrane lipids, absence of basic cellular characteristics (e.g., peptidoglycan), and distinctive ribosomal RNA (Rittmann and McCarty, 2001; Khanal, 2008). Second, compared with other anaerobes, methanogens grow slowly. If existence of a large amount of inhibitors in anaerobic processes, it will harm methanogens and reduce the processing performance of AD. And third, various modes of operation will affect the community composition of methanogens, for example, hydrogenotrophic methanogens are dominant in thermophilic AD, but the major methanogens in the
mesophilic AD are acetotrophic methanogens. In addition, unsuitable retention time will resulting in methanogens too late to grow due to washing out. Although both hydrogenotrophic and acetotrophic methanogens are responsible for producing methane, there are still many differences between them.
Methanogens that are responsible for producing methane are usually classified acetotrophic and hydrogenotrophic methanogens according to their biomethanation precursors. Moreover, the hydrogenotrophic conversion contributes up to 28% of the methane production, on the other hand, the acetotrophic conversion is responsible for surplus 72% of the methane production (McCarty, 1964a; Khanal, 2008). Tab.
2-1 is a classification of methanogens, we can find three classes methanogens including methanobacteria, methanococci and methanomicrobia, respectively. H2 and CO2, acetate and formate are important substrates for methanogenic bacteria to produce methane. Formate concentration is low than other substrates due to it is rapidly produced and consumed. All species, especially hydrogenotrophic methanogens, can use H2 as an elector acceptor to reduce CO2 to methane, these bacteria can synthesize methane by formate as well as H2 and CO2. But H2 and CO2
aren’t only approach to accomplish biomethanation, acetate cleaves methane and bicarbonate is common reaction in AD. Although methanogens using acetate as the substrate are few, they still play a key role in anaerobic reactor since major biomethanation occurs via this way. Methanosaricina species are known to use acetate as the substrate, they often exist in a reactor which has high acetate concentration (Bryant, 1979; Demirel and Scherer, 2008; Khanal, 2008).
In regard to hydrogenotrophic methanogens, these bacteria utilize not only H2 as elector donors reducing CO2, buy also formate to produce methane, besides, these processes carry out either directly or indirectly. Although acetate, formate and H2
Tab. 2-1 Classification of methanogenic bacteria (adapt from Demirel and Scherer, 2008)
Class I. Methanobacteria (substrate: H
2/CO
2, carbon source: formate)
Order I. MethanobacterialesFamily I. Methanobacteriaceae
Class II. Methanococci (substrate: H
2/CO
2, carbon source: formate)
Order I. MethanococcalesClass III. Methanomicrobia (substrate: H
2/CO
2, carbon source: formate)
Order I. MethanomirobialesFamily III. Methanospirillaceae (known to be hydrogenotrophic) Genus I. Methanospirillum
Order II. Methanosarcinales (known to be acetate- and methylotrophic) Family I. Methanosarcinaceae
Genus I. Methanosarcina Genus II. Methanococcoides
Tab. 2-1 Continued on next page
Tab. 2-1 Continued
Family III. Methanospirillaceae (known to be hydrogenotrophic) Genus I. Methanospirillum
Order II. Methanosarcinales (known to be acetate- and methylotrophic) Family I. Methanosarcinaceae
compounds that contain methyl groups, mono-, di-, and trimethylamine, and dimethyl sulfide in terms of literatures (賀延齡, 1998; Khanal, 2008).
Different operation of AD can obviously change the community that is a composition of methanogens as well as acetogenic bacteria. Thermophilic condition or operating at short retention time can favor rod like or coccoid hydrogenotrophic methanogens. In addition, Methanosaeta spp. are the dominant aceticlastic methanogen at low acetate concentration, but they decrease fast when the acetate concentration increases. Contrarily, high acetate concentration is accompanied an increase in Methanosarcina spp. (Demirel and Scherer, 2008).
Mesophilic operation is a more common method then thermophilic operation in AD
recently due to their resistance to extreme environment and potentialities on Environmental Engineering. Tab. 2-2 is Suryawanshi et al. (2010) listed new species thermophilic methanogens found in recent years from many previous studies, these archaea were found in wildly varying habitats.
Operation modes certainly change the both community of fermentative anaerobes and methanogens. Liu et al. (2002) examined the start-up of two acidogenic reactors under mesophilic (37℃) and thermophilic (55℃) conditions carried out with methanogenic granular sludge as an inoculum and dairy wastewater as feed. From the DGGE results, due to pH drop to 5.5, the domains Bacteria and
Archaea populations showed significant shifts after 13 days operation accompanied
with an increase in VFAs production, a decrease in methane formation, and rapid sludge disintegration. Methanosaeta were abundant at the interior of the seed sludge and many other biogranules, but the dominant population changed fromMethanosaeta to Methanomicrobiales, Methanobacteriales and Methanococcales
when reactors were operated at a high VFAs concentration and a low pH condition, besides, the microbial community change was more significant and rapid in the thermophilic reactor. Although obvious community changes took place at the first 13 days for both reactors, a longer period up to 71 days was required to make the microbial community more stable.The methanogens population change in the study of Liu et al. (2009) was found
Methanoculleus (hydrogenotrophic) and Methanosarcina (acetotrophic) were
responsible for methane generation in thermophilic upflow anaerobic filter reactor.Sasaki et al. (2011) carried out their experiment with thermophilic (55℃) AD using artificial garbage slurry as feed. In addition, they also took a tracer experiment using
13C-labeled acetate and found approximately 80% of the acetate was decomposed via a non-aceticlastic oxidative pathway (Eq. 2.14), whereas the remainder was converted
to methane via an aceticlastic pathway (Eq. 2.13). The Archaea 16S rRNA analyses demonstrated the hydrogenotrophic methanogens Methanoculleus spp. accounted for
>90% of detected methanogens, and the acetotrophic methanogens Methanosarcina spp. were minor.
Aceticlastic cleavage
CH
3COO
-+ H
2O → CH
4+ HCO
3(Eq. 2.13) Non-aceticlastic cleavage
CH
3COO
-+ 4H
2O → HCO
3-+ HCO
3+ 4H
2+ H
+HCO
3-or (HCO
3-) + 4H
2+ H
+→ CH
4or (CH
4) + 3H
2O (Eq. 2.14)
From their thermophilic (55℃) co-digestion of OFMSW and lipid-rich wastes, Martín-González et al. (2011) found Methanobacterium, Methanoculleus and
Methanosarcina were detected. Methanobacterium and Methanoculleu belong to
hydrogenotrophic methanogens, however Methanosarcina belongs to acetotrophic methanogens. No another acetotrophic methanogen, Methanosaeta, were detected in their result indicating Methanosaeta were unfavorable in thermophilic condition.Tab. 2-2 Profiles of thermophilic methanogens (adapted from Suryawanshi et al., 2010)
No. Methanogen Site of occurrence Cell
morphology
40-75 Blotevogel and Fischer, 1985
Genus: Methanocaldococcus
2 M. jannaschii Submarine hydrothermal vent, East Pacific Rise, (2600 m depth) 3 M. infernus Deep sea hydrothermal vent
chimney, Mid-Atlantic Ridge
4 M. fervens Deep sea hydrothermal vent core, Guaymas Basin, California
Tab. 2-2 Continued 2
No. Methanogen Site of occurrence Cell
morphology
7 M. thermophilus Sediment, Crystal River, Nuclear power plant, Florida
10 M. thermophila UASB bioreactor, paper-mill wastewater, The Netherlands
Irregular cocci －ve 0.1-0.3 Ma, Met 5.0-7.5
42-58 Jiang et al., 2005
Genus: Methanopyrus
11 M. kandleri Hydrothermal Rod heated deep sea sediment, California
Tab. 2-2 Continued 3
No. Methanogen Site of occurrence Cell
morphology
13 M. thermophila Anaerobic digester (55℃), New York, USA 15 M. wolfeii Mixture of sewage sludge and river
sediment, USA 19 M. marburgensis Mesophilic sewage sludge Cylindrical
irregularly rod
＋ve
NS HC5.0-8.0
45-70 Wasserfallen et al., 2000
Tab. 2-2 Continued on next page
Tab. 2-2 Continued 4
No. Methanogen Site of occurrence Cell
morphology
Biogas plant, Germany Regular and irregular cocci
－ve 0.3-2.0 F, HC
6.5-7.5
30-70 Huber et al., 1982;
Whitman, 2002b 21 M. okinawensis Western Pacific deep sea, Okinawa
Trough, Japan
24 M. igneus Submarine vent, Kolbeinsey ridge, Iceland (106 m depth)
NS NS NS NS NS 45-91 Burggraf et al., 1990;
Whitman, 2002c 25 M. formicicus Black smoker chimney, Kairei field,
Central Indian Ridge
Irregular cocci NS 0.1-1.5 F, HC 6.5-8.5
53-83 Takai et al., 2004