Methane Fermentation Process As Anaerobic Digestion of Biomass: Transformations, Stages and Microorganisms

Review

Methane fermentation process as anaerobic digestion of biomass: Transformations, stages and microorganisms

Krzysztof Ziemiński1*, and Magdalena Frąc2

1Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Poland. 2Department of Plant and Soil System, Institute of Agrophysics, Polish Academy of Sciences, ul.Doswiadczalna 4,. 20-290 Lublin, Poland.

Accepted 3 February, 2012

Anaerobic decomposition of organic compounds is conducted in close cooperation of specialized bacteria of different types, including mostly hydrolyzing, digestive, acetogenic, homoacetogenic, sulfate-reducing (VI) and methanogenic bacteria. A great interest in the anaerobic digestion process results mainly from its advantages, as compared to aerobic methods. The main advantages of the methane digestion process are: Production of an insignificant amount of biomass and lower energy input, as compared to degradation conducted under aerobic conditions. This paper reviews the transformations, stages and microorganisms that participate in methane fermentation process.

Key words: Biogas, methane fermentation, methanogens, microorganisms, anaerobic digestion.

INTRODUCTION

The demand of the world economy for electrical and 1 million equivalent hectares for energy crops may thermal energy in over 88% is covered from non- provide approximately 65 TWh energy in the primary fuel. renewable resources, mainly petroleum and natural gas The use of biogas, acquired in this way, for production of (IEA, 2006). The extraction, processing and combustion electrical energy and heat would allow the reduction of of these raw materials adversely affect the natural the annual emission of CO2 by about 57 million tons. environment, besides they are gradually exhausted The European Parliament Committee on Industry, (Energy, 1997; EC, 2007). According to forecasts, the External Trade, Research and Energy calls on to demand for fuels used for energy production will be increase efforts within the research on new technologies further increased. of using biogas as a biofuel. Studies are carried on to use The European Union’s energy policy aims at using biogas not only for the production of electrical energy and more and more unconventional sources of energy. It was heat but also to introduce it in the natural gas supplied assumed that up to 2010 in the European Union the network, which would allow to reduce Europe’s electrical energy produced in the environmentally friendly dependence on the import of natural gas from the third way would constitute 22.1% of the total amount of countries. In the European Union in 2007 the production produced energy. On the one hand, this is connected of biogas amounted to over 6 million tons. Since then, a with energy safety; on the other hand it contributes to 20% increase annually has been noted in the amount of improvement of the environment. Until 2050 the use of produced biogas (EuroObserver, 2008; Fachverband, modern technologies of energy acquisition should cause 2009). Biogas production from large quantities of 50% reduction of CO2 emission (IPCC, 2000). The use of agricultural residues, animal wastes, municipal and industrial wastes (water) appears to have potential as an alternative renewable energy for many African countries if relevant and appropriate research is carried out to adopt *Corresponding author. E-mail: [email protected]. Tel: +48 42 the biogas technology to the local conditions in African 636-36-39. countries (Mshandete and Parawira, 2009). Biogas is 4128 Afr. J. Biotechnol.

presently obtained not only from landfill degasification, such as: Sewage sludge, manure, industrial wastewater anaerobic sludge digestion in wastewater treatment and organic fraction of municipal wastes (Classen et al., plants, but more and more often also from industrial 1999; Finstein, 2010; Verstraete et al, 2002). In the biogas-plants where the combined processing of organic process of biochemical degradation, the complex organic municipal and industrial wastes as well as sewage sludge com-pounds become decomposed to simple organic and is conducted (Mata-Alvarez et al., 2000; Weiland, 2010). inorganic compounds (Bryant, 1979). During the methane Such procedure is connected with drastic reduction of the digestion process, microbiological reduction of sulfates possibilities of landfilling organic wastes; in 2010 the (VI) to sulfides and hydrogen sulfide occurs along with mass of biodegradable wastes was reduced to 75% as anaerobic ammonification and reduction of nitrates (V) to compared to the wastes disposed in 1995. ammonia. Apart from assimilative reduction of nitrates (V), denitrification may occur (Scherer et al., 2000). Initial stages of anaerobic metabolism are similar to PROCESS OF ANAEROBIC DIGESTION those of aerobic processes. When dissolved oxygen is

Methane digestion is a complex, reduction process of a missing, some chemolithotrophic organisms make use of number of biochemical reactions occurring under oxidized mineral compounds (sulfates (VI) and nitrates anaerobic conditions. Under symbiotic effects of various (V)) as the final acceptor of hydrogen. Oxidation anaerobic and relatively anaerobic bacteria, multi mole- proceeds then, as under aerobic conditions, through the cular organic substances are decomposed into simple, respiratory chain, but the final products are respectively chemically stabilized compounds – mainly of methane hydrogen or molecular nitrogen and energy (Santosh et and carbon dioxide (Naik et al., 2010). Generally, this al., 2004). The process of digestion releases into the process consists of liquefaction and hydrolysis of environment the high energy final products, such as insoluble compounds and gasification of intermediate alcohol or methane. Methane digestion is a complex products. This is accompanied by a partial or complete process which undergoes four phases: Hydrolysis, mineralization and humification of organic substance acidogenesis – acidification phase, acetogenesis, (Lyberatos and Skiadas, 1999). An advantage of the methanogenesis (Figure 1). Involved in biochemical process of anaerobic digestion is production of biogas, a conversions of H2 and CO2 to methane and acetate to high energy fuel which may be used to produce environ- methane and CO2 are various enzymes and prosthetic mentally-friendly energy. It is basically for this reason that groups which occur only in methanogenes. The basic scientists and power industry companies have been structures of these compounds are presented in Figure 2 interested in anaerobic digestion for almost 140 years. and comprise: Deazariboflavine derivative F420, Biotechnology of biogas production usually refers to methanopterin, methanofurane, nickel-tetrapyrol factor digestion of various types of organic wastes, food F430 and coenzyme M (mercaptan sulfonate). Autotrophic industry wastewater, sewage sludge, animal excrements binding of CO2 by methanogenes occurs without a share or organic fraction of municipal wastes etc. In some of the reaction of ribulose-bisphosphatic cycle. Synthesis countries subjected to anaerobic digestion are plants of cellular material with CO2 occurs through the reductive deliberately grown for this purpose, e.g. maize (Boone et pathway of aceto-CoA with pyruvate (Mashaphu, 2005; al., 1993; Mata-Alvarez et al., 2000). Currently in many Saxena et al., 2009). European countries, the production of biomass as a At the first stage of the process, CO2 is bound by substrate for the biogas plants has developed. In the methanofurane (MFR) which is then reduced to methenyl, most extreme European case, the government of methylene, methyl and at the final stage – methane, Germany has taken steps in 2011 to reduce even mono- which is bound in turn by coenzymes: Tetrahy- culture maize production for energy purposes (Graaf and dromethanopterin, 2-methylthioethanesulfonic acid and 2- Fendler, 2010; Weiland, 2010). mercaptoethanesulfonic acid (Medigan et al., 2000). Digestion connected with biogas production may play a Hydrogenase accounts for assimilation of H2. As a result triple part. First, it is a method of converting the energy of activation of hydrogen by hydrogenases which react contained in biomass into a useful fuel (biogas) which with factor F420, a reducing force is provided. Most may be stored and transported. Second, it is a method of methanogenes use H2 as a source of electrons, which is recycling of organic wastes into stable soil additives, that connected with the occurrence of hydrogenases. is, valuable liquid fertilizer and energy. Third, it is a Methanopterin accounts for the stage of the reduction method of wastes treatment aimed at a reduction of their of CO2 to methyl groups of pyruvate. Methyl groups in the hazardous effects on the environment. carbonylation process are converted into carbonyl groups with a share of carbon monoxide dehydrogenase enzyme (Mashaphu, 2005; Saxena et al., 2009). Involved in TRANSFORMATIONS OF METHANE FERMENTATION methanogenesis pathway are many coenzymes which do PROCESS not have any flavinic or quinonic groups (Figure 2). Metabolism of methanogenesis is unique, because it runs Methane digestion is used for stabilization of wastes, along the pathway which requires coenzymes which do Zieminski and Frac 4129

Figure 1. Stages of methane fermentation process

Coenzyme F Methanopterin – 420 Coenzyme – F430

Figure 2. Coenzymes and prosthetic groups of methanogenes

not occur in any live organisms, except for metha- monocarbon compounds. These compounds appear in nogenes (Smith, 1966; Zeikus, 1977). the environment as a result of the digestion and decay of Methanogenes C1 participated in the metabolic products of vegetable and animal origin and also pathway of methanofurane, methanopterin and pesticides. Methane is produced by methanogenic coenzyme M, whereas coenzymes F420 and B act as archaeons using carbon dioxide as electron acceptor electron donors. C1 compounds do not contain any (Medigan et al., 2000; Mashaphu, 2005). carbon-carbon bonds. They contain monocarbon Deazariboflavine – derivative F420 is a coenzyme of compounds, such as methane (CH4), methanol (CH3OH), electron transfer used by many enzymes, such as dimethyl carbonate (CH3OCOOCH3) and other hydrogenase, formate dehydrogenase, methylene 4130 Afr. J. Biotechnol.

dehydrogenase of tetrahydromethanopterin (H4MPT), phasing. Each of them accounts for degradation of a methylene reductase H4MPT and heterodihydrogen different type of compounds. sulfide reductase. As earlier mentioned, MFR participated in the stage of methanogenese initiation only when CO2 Hydrolysis: During hydrolysis of the polymerized, mostly is bound with furane. In successive stages, it is reduced insoluble organic compounds, that is, carbohydrates, to the level of formyl and transformed to the next proteins, fats are decomposed to soluble monomers and coenzyme of tetrahydromethanopterin. There are four dimers, that is, monosugars, amino acids and fatty acids. types of tetrahydromethanopterin which may occur in This stage of the methane digestion process passes three different degrees of oxidation (Mashaphu, 2005). through extracellular enzymes from the group of hydrolases (amylases, proteases, lipases) produced by appropriate strains of hydrolyzing bacteria. Hydrolysis of BIOGAS hardly decomposable polymers, that is, cellulose and cellucottons is considered to be a stage which limits the Biogas is a digester gas arising from the activity of rate of wastes digestion. During solid wastes digestion, methanogenic anaerobic bacteria which decompose only 50% of organic compounds undergo biodegradation. organic matter. Its composition depends on the type of The remaining part of the compounds remains in their raw material subjected to the digestion process and on primary state because of the lack of enzymes the method of conducting this process and is as follows: participating in their degradation (Conrad, 1999; Parawira Methane CH4 (50–75%), carbon dioxide CO2 (25–45%), et al., 2008). hydrogen sulfide H2S (0–1%), hydrogen H2 (0–1%), The rate of hydrolysis process depends on such carbon monoxide CO (0–2%), nitrogen N2 (0–2%), parameters as: Size of particles, pH, production of ammonia NH3 (0–1%), oxygen O2 (0–2%) and water – enzymes, diffusion and adsorption of enzymes on the H2O (2–7%) (Graaf and Fendler, 2010). The obtained particles of wastes subjected to the digestion process. biogas may be used in various fields of economy Hydrolysis is carried out by bacteria from the group of (Classen et al., 1999; Naik et al., 2010; Verstraete et al., relative anaerobes of genera: Streptococcus, 2002), mainly in technological processes and for power Enterobacterium (Bryant, 1979; Smith, 1966). engineering purposes, including the following: Acidogenesis (acidification phase): During this stage, 1. Production of thermal energy in gas boilers and the acidifying bacteria convert water-soluble chemical production of thermal and electrical energy in associated substances, including hydrolysis products to short-chain units – (from 1 m3 of biogas – in associated production of organic acids (formic, acetic, propionic, butyric, energy 2.1 kWh of electrical energy and 2.9 kWh of heat pentanoic), alcohols (methanol, ethanol), aldehydes, is obtained); carbon dioxide and hydrogen. From decomposition of 2. Production of electrical energy in spark - ignition or proteins, amino acids and peptides arise, which may be a turbine engines; source of energy for anaerobic microorganisms. 3. Using the obtained gas as a fuel in motor-car engines; Acidogenesis may be two-directional due to the effects of 4. Using the obtained gas in various technological various populations of microorganisms. This process may processes, e.g. in the production of methanol. be divided into two types: Hydrogenation and dehydrogenation. The basic pathway of transformations The average efficiency of methane digestion reaches passes through acetates, CO2 and H2, whereas other approximately 0.24 m3 of methane from 1 kg of dry acidogenesis products play an insignificant role. As a organic matter. 1 m3 of biogas of 26 MJ m-3 calorific value result of these transformations, methanogenes may may replace 0.77 m3 of natural gas of 33.5 MJ calorific directly use the new products as substrates and energy value, 1.1 kg of hard coal of 23.4 MJ calorific value or 2 source. Accumulation of electrons by compounds such as kg of firewood of 13.3 MJ calorific value (Arbon, 2002). lactate, ethanol, propionate, butyrate, higher volatile fatty acids is the bacteria’s response to an increase in hydrogen concentration in the solution. The new products STAGES OF ANAEROBIC DEGRADATION OF may not be used directly by methanogenic bacteria and ORGANIC WASTES must be converted by obligatory bacteria producing hydrogen in the process called acetogenesis. Among the Microbiology of anaerobic transformation of organic products of acidogenesis, ammonia and hydrogen sulfide wastes is a process which involves many different groups which give an intense unpleasant smell to this phase of of bacteria, such as hydrolysing, acidifying, acetogenic the process should also be mentioned (Ntaikou et al., and methanogenic bacteria which in the final stage 2010; Classen et al, 1999; Conrad, 1999). The acid produce CO2 and CH4, that is, the main products of the phase bacteria belonging to facultative anaerobes use digestion process (Demirel and Scherer, 2008; Nealson, oxygen accidentally introduced into the process, creating 1997). A specific characteristic of methane digestion is its favourable conditions for the development of obligatory

Zieminski and Frac 4131

Electron Microorganisms Electron donor Product Reaction type acceptor

Fermentative organic carbon organic carbon CO Fermentation bacteria 2

Syntrophic organic carbon organic carbon H Acetogenesis bacteria 2

Acetogenic organic carbon/ CO2 CH 3 COOH Acetogenesis bacteria H2

Methanogenic organic carbon/ CO2 CH 4 Methanogenesis bacteria H2

Figure 3. Microorganisms cooperation in organic matter degradation.

anaerobes of the following genera: Pseudomonas, are capable to produce methane from acetic acid, a vast Bacillus, Clostridium, Micrococcus or Flavobacterium. majority of CH4 arising in the methane digestion process results from acetic acid conversions by heterotrophic Acetogenesis: In this process, the acetate bacteria methane bacteria (Demirel and Scherer, 2008). Only 30% including those of the genera of Syntrophomonas and of methane produced in this process comes from CO2 Syntrophobacter convert the acid phase products into reduction carried out by autotrophic methane bacteria. acetates and hydrogen which may be used by During this process H2 is used up, which creates good methanogenic bacteria (Schink, 1997). Bacteria conditions for the development of acid bacteria which Methanobacterium suboxydans account for give rise to short-chain organic acids in acidification decomposition of pentanoic acid to propionic acid, phase and consequently – too low production of H2 in whereas Methanobacterium propionicum accounts for acetogenic phase. A consequence of such conversions decomposition of propionic acid to acetic acid. As a result may be gas rich in CO2, because only its insignificant part of acetogenesis, hydrogen is released, which exhibits will be converted into methane (Griffin et al., 2000; toxic effects on the microorganisms which carry out this Karakashev et al., 2005). process. Therefore, a symbiosis is necessary for acetogenic bacteria with autotrophic methane bacteria using hydrogen, hereinafter referred to as syntrophy COOPERATION OF MICROORGANISMS OF (Schink, 1997; de Bok et al., 2005). Acetogenesis is a METHANE FERMENTATION PROCESS phase which depicts the efficiency of biogas production, because approximately 70% of methane arises in the Conversions of complex organic compounds to CH4 and process of acetates reduction. Consequently, acetates CO2 are possible owing to the cooperation of four are a key intermediate product of the process of methane different groups of microorganisms (Figure 3). These digestion. In acetogenesis phase approximately 25% of microorganisms may be counted among: primary acetates are formed and approximately 11% of hydrogen, fermentation bacteria, secondary fermentation bacteria produced in the wastes degradation process. (syntrophic and acetogenic bacteria) and two types of methanogenes belonging to domain Archaea. These Methanogenesis: This phase consists in the production microorganisms occur in natural environment and fulfill of methane by methanogenic bacetria. Methane in this various roles during the process of anaerobic degradation phase of the process is produced from substrates which of wastes (Conrad, 1999; Mashaphu, 2005). Syntrophy is are the products of previous phases, that is, acetic acid, a form of symbiosis of two metabolically different groups H2, CO2 and formate and methanol, methylamine or of bacteria, which enables a degradation of various dimethyl sulfide. Despite the fact that only few bacteria substrates (de Bok et al., 2005; Demirel and Scherer,

4132 Afr. J. Biotechnol.

CH3 CH 2 CH 2 COOH butyric acid

Syntrophomonas CH3 COOH + H 2+CO 2

CH3 (CH 2 ) 4 COOH caproic acid

CH4 Methanogenes

Syntrophobacter

CH 3CH 2 COOH propionic acid CH3 COOH + H 2

Figure 4. Metabolism of fatty acids during syntrophy.

2008). Cooperation of the population of microorganisms acid and caproic acid to CH3COOH and H2. Species enables a synthesis of certain products which are then belonging to this genus oxidise also pentanoic and used by another group of bacteria. The bacteria enanthic acids to CH3COOH, CO2 and H2. Oxidation of belonging to domain Archaea, which are involved in the propionate is also an important stage of methanogenesis production of methane exhibit synergistic relationships process in which syntrophic consortia of acetogenic with other populations of microorganisms. They may bacteria and methanogenic archeons obligatorily develop only when hydrogen is used by hydro- participated (Demirel and Scherer, 2008). Most of the genotrophs. Such cooperation between microorganisms propionate-oxidising syntrophic bacteria, so far isolated producing hydrogen and using hydrogen was defined as from different environments, belonged to interspecific transfer of hydrogen (Conrad, 1999; Syntrophobacter genus from δ-proteobacteria group. Mashaphu, 2005). Syntrophy between microorganisms CH3COOH and H2 produced by the mentioned bacteria producing and using hydrogen allows for the growth and are used by methanogenic archeons to produce methane activity of these microorganisms. (Nealson, 1997). Species of Syntrophobacter genus are The phenomenon of interspecific transfer of hydrogen capable to use sulfates as electron acceptor in the enables the growth of the so called syntrophic bacteria process of propionate oxidation. Syntrophic oxidation of (Syntrophomonas, Syntrophospora, Syntrophobacter) propionate is not limited to gram-negative bacteria but which oxidise such compounds as propionate and also to gram-positive bacteria in which a similar butyrate and thereby obligatorily use hydrogen as phenomenon was observed. Two thermophilic, gram- electron acceptor. The reaction of butyrate oxidation, positive species of syntrophic bacteria oxidising carried out by these microorganisms, may occur only propionate have been described: Desulfotomaculum when low pressure of H2 is maintained (Demirel and thermobenzoicum subsp. thermosyntrophicum and Scherer, 2008; McMahon et al., 2004). Syntrophic Desulfovibrio (de Bok et al., 2005, Mashaphu, 2005). bacteria cannot grow in the form of pure cultures, but only Desulfotomaculum, similarly to Syntrophobacter may use when accompanied by microorganisms using hydrogen sulfates as electron acceptors. Desulfovibrio may produced by them, e.g. mutanogenic archeons. So participate, along with Metanobacterium genus, in syntrophy is a process in which the compound’s decom- forming CH3COOH and H2 in the process of anaerobic position occurs, with participation of two or more respiration and digestion, using sulfate and lactate to microorganisms and none of them can use this make these products. Metabolism of fatty acid during compound separately. Syntrophic bacteria produce syntrophy was presented in figure 4. adenosine triphosphate (ATP) as a result of substrate phosphorylation or oxidation, used in their metabolism of not only fatty acids but also alcohols produced by other METHANOGENES – KEY MICROORGANISMS OF THE microorganisms as a result of digestion. These METHANE FERMENTATION PROCESS compounds are converted to acetate and hydrogen which are used by methanogenic archeons (de Bok et al., Methanogenes are strict anaerobes; the presence of 2005). Bacteria of Syntrophomonas genus oxidise butyric oxygen is lethal for them. These microorganisms contain Zieminski and Frac 4133

neither catalase nor superoxide dismutase. Due to methanogenic bacteria were classified to domain extraordinary sensitivity of these microorganisms to Archaea. Among the microorganisms within domain oxygen, their biochemistry, physiology and ecology are Archaea, four groups are distinguished, the most visible less known. being Crenarchaeota and Euryarchaeota. In taxonomic Methanogenes as absolutely anaerobic micro- and phenotypic respect, methanogenes belong to organisms inhabit anaerobic environment ecosystems, Euryarchaeota group (Vignais et al., 2001). such as tundras, marshlands, rice fields, bottom deposit, Methanogenic bacteria are divided into 4 classes, 5 swamps, sandy lagoons, tanks where wastewater is orders, 9 families and 26 genera (Demirel and Scherer, decomposed, sewage sludge, solid wastes landfills, 2008). Phylogenetic classification of methanogenes is ruminants’ stomach (Bryant, 1979; Smith, 1966; Zeikus, presented in Figure 5. 1977). These microorganisms are particularly sensitive to As earlier mentioned, according to assumed changes in temperature and pH, their development being classification of live organisms, methanogenes are inhibited by a high level of volatile fatty acids and other archeons. Unlike bacteria, methanogenes do not have a compounds, that is, hydrogen, ammonia, sulphur typical peptidoglycan (mureinic) skeleton and are hydrogen in the environment (Zeikus, 1977). characterized by a different metabolism. Besides, Among methanogenic microorganisms, we can methanogenes’ cytoplasmatic membrane consists of distinguish psychro-, meso- and thermophilic micro- lipids composed of isoprenoid hydrocarbons glycerol organisms. Mesophilic and thermophilic bacteria lipids. Methanogene ribosomes exhibit a similar size to described in literature exhibit high activity within that of eubacteria ribosome, but the sequence of temperature, respectively 28 to 42ºC and 55 to 72ºC. So principles in ribosome RNA, especially 16S rRNA, is far, no anaerobic psychrophilic bacterium has been found completely different (Watanabe et al., 2004). which would exhibit activity at a temperature lower than Methanogenes are largely differentiated 25ºC. Temperature is very important for methanogenic morphologically. Methanogenes exhibit almost all shapes bacteria, due to a limited temperature resistance of their occurring in bacteria: Cocci (Methanococcus), rods enzymatic structures. (Methanobacterium), short rods (Methanobrevibacter), Methanogenic bacteria usually develop in inert Spirillaceae (Methanospirillum), sarcina conditions, with environmental pH from 6.8 to 7.2. This, (Methanosarcina), filiforms (Methanothrix). The size of however, does not mean that methanogenesis does not these microorganisms ranges from 0.3 to 7.4 µm occur in environments of acid or alkaline reaction. (Karakashev et al., 2005). The properties of selected Methanogenes which decompose acetates methanogenes are presented in Table 1. Typical (Methanosarcinabarkeri and Methanosarcina sp.) were reactions carried out by methanogenes during anaerobic isolated from environments of approximately pH 5, while processes are shown in Table 2. methylotrophic and hydrogen-oxidising methanogenes were found in strongly alkaline ecosystems (Smith, 1966). Methanogenic bacteria belong to chemoli- Substrates used by methanogenic bacteria thotrophs, because they are capable to use CO2 as a source of carbon (Smith, 1966; Zeikus, 1977). Methanogenes process a limited quantity of simple Methanogenes are an important group of micro- organic substrates, the most important of which are: organisms for several reasons, the most important being CH3COOH and H2-CO2 (Conrad, 1999). Most their ability to process organic matter to methane. methanogenic bacteria are capable to use H2 and CO2 for Methanogenic bacteria are used in anaerobic their growth (Table 2), although certain species process decomposition of wastewater, as a part of the wastes CH3COOH, CH3NH2 and HCOOH (Classen et al., 1999). treatment system. Sedimentation processes are also Owing to used substrates, methanogenes are divided used in stabilization of primary and secondary sludge into two groups (Demirel and Scherer, 2008): arising in the process of aerobic wastewater treatment. These microorganisms arouse also some interest in 1. Hydrogenotrophic – which use only H2 and CO2. Partial pharmaceutical industry, because they may constitute a pressure of hydrogen is an important parameter which source of vitamin B12 (Yanga et al., 2004). defines stability and disturbances in the anaerobic

digestion process. Therefore the activity of hydrotrophic Taxonomy and morphology of methanogenes methanogenes is essential for stability and efficiency of the digestion process. Efficiency and activity of All alive organisms, based on analysis and comparison of hydrogentrophic methanogenes are important both in conservative philogenetic features, through analysis of anaerobic processing of simple, soluble types of 16S and 18S rRNA, were classified to three main substrates (such as acetate, ethanol, dextrose, taxonomic units of the living world. Three phylogenetic propionate), and various types of wastes (e.g. oil). The domains may be distinguished: Archaea, Bacteria and role of this group of microorganisms in anaerobic Eukarya. According to analysis of sequence 16S rRNA, processing of biomass of complex organic compounds 4134 Afr. J. Biotechnol.

Archaea

Crenarcheota Korarchaeota Euryarchaeota Nanoarchaeota

Methanobacteria Methanococci Methanomicrobia Methanopyri

Methanobacteriales Methanococcales Methanomicrobiales Methanosarcinales Methanopyrales

Methano - Methano- Methano- Methano- Methano- Methano- Methano- Methanomicrobiaceae bacteriaceae thermaceae coccaceae caldococcaceae sarcinaceae saetaceae pyraceae

Methano- Methano- Methano Methano- Methano- Methano- Methano- Methano- Methanosaeta Methanopyrus bacterium thermus -coccus caldoccocus microbium corpusculaceae sarcina spirillacea e Methano- Methano- Methano- Methano- Methano- Methano- brevibacter thermococcus torris culleus corpusculum coccoides

Methano- Methano- Methano- Methano- sphaera follis halobium spirillum

Methano- Methano- Methano- thermobacter genium halophilus

Methano Methano- -lacinia lobus

Methano- Methano- planus salsum

Methano- methylovorans

Methani- micrococcus

Figure 5. Philogenetic hierarchy of methanogenes. Source: Demirel and Scherer 2008.

is not well known yet. simple compounds for their growth; that is, of acetic acid. Acetotrophic methanogenes are acetate. Acetate is one of the most important obligatory anaerobes which process acetate to 2. Acetotrophic – reduce methyl groups, such as: substrates for methanogenic bacteria, because methane and carbon dioxide. The activity and Species of Methanosarcinales genus which use over 70% of biomethane comes from processing efficiency of this group of microorganisms are Zieminski and Frac 4135

Table 1. Characteristics of selected methanogenic bacteria

Optimum Species Morphology Width/length of cell (µm) Substrate Optimum pH temperature (°C)

Methanobacterium Brytanii Longrods 0.5-1.0/1.5 H2/CO2 37 6.9-7.2

Methanobacterium formicicum Longrods 0.4-0.8/2-15 H2/CO2. formate 37-45 6.6-7.8

Methanobacterium thermoalcaliphilum Rods 0.3-0.4/3-4 H2/CO2 58-62 8.0-8.5

Methanothermobacter thermoautotrophicum Longrods 0.3-0.6/2-7 H2/CO2 65-70 7.0-8.0

Methanothermobacter wolfii Rods 0.4/2.4-2.7 H2/CO2 55-65 7.0-7.5

Methanobrevibacter smithii Shortrods and chains 0.6-0.7/1.0-1.5 H2/CO2. formate 37-39 -

Methanobrevibacter ruminatium Shortrods and chains 0.7/0.8-1.7 H2/CO2. formate 37-39 -

Methanotherums fervidus Shortrods 0.3-0.4/1.3 H2/CO2. formate 83 <7.0

Methanothermococcus thermolithotrophicus Cocci - H2/CO2. formate 65 -

Methanococcus voltaei Cocci 1.5 (diameter) H2/CO2. formate 35-40 6.0-7.0

Methanococcus vannielii Cocci 1.3 (diameter) H2/CO2. formate 65 7-9

Methanomicrobium mobile Shortrods 0.7/1.5-2.0 H2/CO2. formate 40 6.1-6.9

Methanolacinia paynteri Shortrods 0.6/1.5-2.5 H2/CO2 40 7.0

Methanospirillum hungatei Shortrods 0.5/7.4 H2/CO2. formate 30-40 - Methanosarcinaacetivorans Irregularcocci - Methanol. acetate 35-40 6.5

Methanosarcina barkeri Irregularcocci, - H2/CO2. methanol. 35-40 5-7 irregularpackets acetate

Methanosarcina mazeii Irregularcocci, - Methanol. acetate 30-40 6-7 irregularpackets

Methanosarcina thermophila Irregularcocci - H2/CO2. methanol. 50 6-7 acetate Methanococcoides methylutens Irregularcocci 0.8-1.2 (diameter) Methanol 42 7.0-7.5 Methanosaeta Consilii Rods 0.8x2.5-6.0 (dimensions) Acetate 35-40 7.0-7.5 Methanosaeta thermophila Rods 0.8-1.3x6.0 (dimensions) Acetate 55-60 7

Source: Demirel and Scherer (2008).

important parameters in the process of anaerobic mophilic con-ditions (up to 70°C) (Scherer et al., anaerobic manure processing indicate that hydro- conversion of acetate. 2000). Hydrogenotrophic methanogenes were genotrophic methanogenes are a group which In the studies of hydrogenotrophic, methano- also found under the conditions of digestion of maintains a high specific methanogenic activity genes participated in degradation of organic sewage sludge, manure and compost (Jackel et (SMA) and is characterized by invariable number fraction of municipal wastes. The studies were al., 2005). The studies on the effects of at 65°C. The activity of other methanogenes at a conducted under thermophilic and hyperther- temperature on populations of microorganisms in high temperature was considerably reduced 4136 Afr. J. Biotechnol.

Tabela 2.Typical reactions carried out by methanogenes during anaerobic process

Reaction carried out by methanogenes

Hydrogen: 4 H2 + CO2 CH4 + 2 H2O

Acetate : CH3COOH CH4 + CO2

Formate: 4HCOOH CH4 +CO2 + 2 H2O

Methanol 4 CH3OH 3 CH4 +CO2 + 2 H2O

Carbon monoxide: 4 CO + 2 H2O CH4 + 3 H2CO3

Trimethylamine: 4(CH3)3N + 6 H2O 9 CH4 + 3 CO2 + 4 NH3

Dimethylamine: 2(CH3)2NH + 2 H2O 3 CH4 + CO2 + 2 NH3

Methylamine: 4(CH3)NH2 + 2 H2O 3 CH4 + CO2 + 4 NH3

Methylmercaptans: 2(CH3)2S + 3 H2O 3 CH4 + CO2 + H2S 0 + ++ Metals: 4Me + 8 H + CO2 4 Me + CH4 + 2 H2O

Source: Demirel and Scherer (2008).

(Ahring et al., 2001). Methanogenic bacteria binding IDENTIFICATION OF ANAEROBIC ENVIRONMENTAL hydrogen were found to belong to family MICROORGANISMS Methanobacteriaceae (Boone et al., 1993). Manure is a complex type of substrate, composed of The studies on microorganisms’ ecology require hydrocarbons, proteins and acids. Characteristics of the identification of microorganisms based on universal population of bacteria and archeanes in anaerobic system of classification which reflects microorganisms’ thermophilic processing of manure indicated a evolutional relationship (Hofman-Bang et al., 2003). dominance of two species: Methanoculleus Three basic aims have to be achieved in all studies on thermophilicus (hydrogenotrophic) and Methanosarcina microorganisms’ ecology: thermophila (acetotrophic). The main hydrogentrophic microorganisms, participating in anaerobic processing of 1. Identification and classification of microorganisms; fruit and vegetable wastes comprise 2. Determination of the quantity of microorganisms; Methanosphaerastadtmanii and Methanobrevibacterwolinii 3. Determination of microorganisms’ activity. (Bouallagui et al., 2004). Counted among acetotrophic methanogenes should be the species belonging to genus Methanosarcina. It was Traditional methods of culture and identification found out that during anaerobic processing of sewage sludge and manure, the number of microorganisms of Traditional methods of investigating the microorganisms’ Methanosaeta genus decreased with increasing acetate quantity and identification are based on microorganisms’ in the environment, with simultaneous intensive growth of morphology and phenotypic features (Smith, 1966; the bacteria belonging to Methanosarcina genus which Zeikus, 1977). The studies of methanogenic micro- are acetotrophic methanogenes (Griffin et al., 2000). The organisms carried out by Grothenhius et al. (1991) were studies on the dynamics of the population of anaerobic based on microscopic techniques and consisted in microorganisms participating in degradation of municipal identification of acetotrophic methanogenes based on wastes and sewage sludge indicated that their morphology. Hydrogenotrophic methanogenes were Methanosaetaconcilii was a dominant species among demonstrated by autofluorescence, at the wavelength of acetotrophic methanogenes (McMahon et al., 2004). 420 nm. The rate of the development of digestive bacteria The culture of methanogenes is difficult because of a depends on the type of applied substrate. If the sub- low rate of the growth of those microorganisms, specific strates are carbohydrates, the rate of bacteria generation nutritional requirements and restrictive environmental in acid phase amounts to 5 h, whereas in the case when conditions. fats are the substrate, the time is prolonged to Phylogenetic analysis allows the identification of approximately 72 h. The generation time, in acetogenesis microorganisms according to molecular techniques. This phase, for bacteria using propionic and fatty diagnostic method eliminates the need for the culture of acidsreaches respectively 84 and 131 h. In the these microorganisms. This means that sequences of methanogenesis phase, the generation time ranges nucleic acids may be isolated from environmental sam- between 15 and 85 h (Ilyin et al., 2005; Santosh et al., ples, sequenced and compared to the known sequences 2004). appropriate for identified related microorganisms Zieminski and Frac 4137

(Hofman-Bang et al., 2003). 3. Cloning the products of reaction; In the past, many procedures were published, which 4. Electrophoretic separation using the techniques of enabled calculation and isolation of methanogenic DGGE, TGGE or T-RFLP; bacteria (Ferry et al., 1974; Smith, 1966; Zeikus, 1977). 5. Sequencing the products or clones (Hofman-Bang et One of the possible methods of isolation and culture of al., 2003). methanogenes is the use of LPBM substratum of the following composition: KH2PO4 – 0.75 g, KH2PO4 · 3 H2O Another technique used in the research on – 1.45 g, NH4Cl – 0.9 g, MgCl2 · H2O – 0.2 g, Na2S · 9 methanogenes population is fluorescent in situ H2O – 0.5 g, solution of trace elements Fe, Mn, Co, Zn, hybridization (FISH). Hybridization occurs at the cellular Ca, B – 9 ml, solution of vitamins – 5 ml, distilled water – level and is conducted in situ. It enables determination of 1000 ml. the distribution of microorganisms in the environment. For The substratum is prepared in anaerobic atmosphere labelling of oligonucleotide probes, the fluorescently with 95% share of nitrogen and 5% share of carbon labelled probes are used. This method is based on dioxide (Zeikus, 1977). In conventional studies, the stabilization and permeabilization, hybridization, removal identification of methanogenes is mainly based on of non specifically bounded probes and microscopic analysis of morphological forms. We can distinguish four analysis of dyed cells in fluorescent or scanning electron basic morphological forms of methanogenes, that is, microscope or flow cytometer (Demirel and Scherer, sarcina, rods, spirocheta and spherical forms. The 2008; Tabatabaei et al., 2009). The FISH method was mentioned forms may create various types of aggregates used in identification of dominant methanogenic archeons and clusters, or they may occur in the form of single cells participating in anaerobic degradation of manure and in the environment. Their shapes may be more or less sludge (Karakashev et al., 2005). regular, depending on the species, rods may be shorter The use of advanced techniques of molecular biology is or longer, straight or curved (Zeikus, 1977). essential in the studies aimed at the comprehension and systematization of complex reactions going on during the digestion process (Demirel and Scherer, 2008). Molecular techniques in ecology of anaerobic microorganisms CONCLUSION Contemporary studies on the diversity and function of microorganisms are more and more often based on the A great interest in the anaerobic digestion process results techniques of molecular biology. This is connected with mainly from its advantages, as compared to aerobic the fact that only a part of microorganisms can be methods. The main advantages of the methane digestion cultured in vitro. Molecular techniques enable the process are: Production of an insignificant amount of description of environmental microorganisms’ populations biomass and lower energy input, as compared to according to analysis of appropriate molecules, e.g. degradation conducted under aerobic conditions. Another rRNA. Isolation of the sequence of nucleic acids occurs advantage is the production of biogas which may be used without the culture of microorganisms and isolation of as a source of energy. Furthermore, the anaerobic pure cultures (Nealson, 1997). process does not need energy for aeration, which results In recent years, the research on molecular diversity of in high energy saving. It is characterized by a lower microorganisms developed extensively. Therefore demand for nutritive substances and higher resistance, various techniques are being developed, based on as compared to aerobic processes, with high organic extraction of nucleic acids and adapted to environmental loads. Disadvantages of the process are difficulties studies. These techniques comprise among others: DNA connected with maintaining appropriate concentration of separation, hybridization of DNA-DNA and mRNA-DNA, microorganisms in bioreactor, higher sensitivity to cloning of DNA, sequencing and other techniques based changes in reaction and temperature. Besides, methane on the polymerase chain reaction (PCR), such as digestion process do not always causes a complete Denaturing gradient gel electrophoresis (DGGE), tem- degradation of organic impurities. Besides, it requires perature gradient gel electrophoresis (TGGE), fluorescent heating of wastes to carry out the digestion under in situ hybridization (FISH) (Iqbal et al., 2009; Keyser et mesophilic or thermophilic conditions. In addition, odour al., 2006). nuisance is observed in the case of ineffective Typical studies on methanogenes using molecular hermetization of reactors and containers designed for biology techniques comprise: storage of digested substrates. In accordance with the assumption of the European 1. Extraction of fragments of DNA, rRNA from the Union development of fuel III deficiency has gradually environmental sample; complement conventional fuels. These fuels also reduced 2. Amplification of respective fragments of genes, using CO2 emissions to the atmosphere. One of the main the PCR; research topics is the use of methane fermentation, not 4138 Afr. J. Biotechnol.

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Ilyin VKM, Korniushenkova IN, Starkova LV, Lauriniavichius KS (2005).

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