Green House Gas Emissions from Termite Ecosystem 2011 Gomati Et Al

Green house gas emissions from termite ecosystem Methanogenic archaea (methanogens) that inhabit the gut of termites generate enormous amount of methane that adds to the global atmospheric methane (CH4). Methane is an important trace gas in the atmosphere, contributing significantly to long wave absorption and bringing in variations into the chemistries of both the troposphere and the stratosphere. In the troposphere, methane acts as a sink for hydroxide (OH) and as a source for carbon monoxide (CO). While in the stratosphere, methane is a sink for chlorine (Cl) molecules and a source of water vapor, which is a dominant greenhouse gas. Analysis has shown that atmospheric concentrations of methane have increased by about 30% over the last 40 years. Such an increase may greatly affect future levels of stratospheric ozone and hence, the climate of the earth. Recent estimates of the total annual source strength of CH4 vary from 400 to 1200 Tg. Activities such as rice cultivation, cattle production, mining, use of fossil fuels and biomass burning is believed to be the cause of increasing methane levels in the atmosphere. To add to this list is the source from termites, which contributes measurable quantities of CH4 ranging from 2 to 150 Tg per year. However, data indicate that while there are large variations in the amount of CH4 produced by different species, the total methane addition due to termites is probably less than 15 Tg per year, thus making a contribution of less than 5% to global CH4 emissions. Furthermore, the review addresses questions related to the biological aspects of termite harboring groups of bacteria that participate in methanogenesis and various other biotechnological potential of unique microbiota as well as possible strategies to mitigate methanogenesis by termite. Key words: Macrotermes, methane, carbondioxide, GHG, methanobacteria, methanosarcina. INTRODUCTION Termites are eusocial insects belonging to the order isoptera that play a major role in tropical ecosystem. One of the most fascinating nutritional symbioses exists between termites and their intestinal microflora: a symbiosis that permits termites to live by xylophagy (Breznak, 1982, 1984). For the microbial ecologist, termite gut represents an excellent model of highly structured micro environments (Cruden and Markovetz et al., 1984). Apart from its natural role of conversion of *** and cellulosic substances into useful products of termite gut, microbiota contributes significantly to greenhouse gas effect through methane generation. *Corresponding author. kvgmathi@yahoo.co.in. However, the total annual source strength of CH4 varies from 400 to 1200 Tg (Brockber 1996). In their report, Zimmerman et al. (1982) estimated the average CH4 production rates of 0.425 mg CH4/termite/day for the lower termite species and 0.397 mg CH4/termite/day for the higher termite families. Though rough, estimates are suggesting an annual methane emission ranging from 2 to 150 Tg per year, in which the total methane source due to termites is probably less than 15 Tg per year, thus making a contribution of less than 5% to global CH4 emissions. The recent reports confirm the figures above keeping the emission levels between 2 and 22 Tg per year (EPA, 2010). However, microbial ecology of the gut of termites has been well documented earlier (Brune and Friedrich, 2000; Erhart 1981; Schultz and Breznak, 1978; Bignell, 1984). In this review, the nature and role of gut Velu et al. 57 Termi t idae Termi t inae Macrotermi t ina e Rhinotermi t i dae Hodotermi t i dae Termopsidae Nasut i termi t inae Apicotermi t inae Serri termi t i dae Kal otermi t i dae Mastotermi t i dae Higher Termites Lower Termites Order: Isoptera Termi te Hodotermopsis sjoestedti (Damp wood) Reticulitermes speratus Coptotermes formosanus Odontotermes formosanus (Fungus growing) Pericapritermes nitobei (Soil feeding) Nasutitermes takasagoensis (Wood feeding) Neotermes koshunensis Cryptotermes domesticus Glyptotermes fuscus (Dry wood) Cockroach and mant i d Figure 1. Flow diagram of phylogeny of termite family (Ohkuma et al., 2001). microbiota of termites in the atmospheric methane contribution is analyzed in detail. Higher and lower termites Termites are divided into two groups, which are lower and higher termites. Lower termites is a group of six evolutionary distinct termite families (the microbial community in the gut of phylogenetically lower termites) comprising both flagellated protists and prokaryotes (Ohkuma, 2007); whereas higher termites comprise only one family. It includes approximately 85% of all termite species that also harbor a dense and diverse population of gut prokaryotes that typically lack eukaryotic flagellated protists. Higher termites secrete their own digestive enzymes and are independent of gut microorganisms in their nutrition. The lower termites also possess this ability, but their production of cellulolytic enzymes is apparently inadequate (Brune et al., 1995; Brune, 2006). Hence, lower termites mostly depend on the activity of gut microorganisms for their nutrition, which are present in the hind gut region (Figure 1). Life cycle of termites Termites' wings of isoptera are essentially similar in size, form (shape) and venation (Richards and Davies 1977), while the length and span of wings are different according to species. Other special characteristics that belong to this order are: social insect with caste differentiation, moniliform antennae, tarsi four segmented, the mouthparts, which are formed for biting; moreover the metamorphosis is incomplete (Comstock and Comstock, 1890; Borror and White, 1998). The termites form colonies of a few hundred to a maximum of seven million individuals. The termites present in a colony consist of several castes, which are morphologically and functionally distinct (O'Brien and Slaytor, 1982). The caste may be divided into two broad groups, reproductive and sterile. Queen termite is the reproductive termite. However, most 58 Afr. J. Environ. Sci. Technol. of the sterile castes are the soldiers and the workers. They subsist on a diet rich in cellulose, which may be in the form of living or dead wood, *** tissues of plants or dung. Some even feed on soil, whereas others have evolved the intriguing habit of cultivating fungus garden as a nutrient resource (Sands et al., 1970). Gut microflora of termites The termite gut consists of fore gut (which includes the crop and muscular gizzard), the tubular mid gut (which as in other insects is a key site for secretion of digestive enzymes and for absorption of soluble nutrients) and relatively, a voluminous hindgut (which is also a major site for digestion and for absorption of nutrients). The morphological diversity of the termite gut microbiota is remarkable and has been documented in recent years for both lower (Brian, 1978) and higher (Eutick et al., 1978) termites. Although some bacteria colonize the foregut and midgut, bulk of intestinal microbiota is found in the hindgut, especially in the paunch, that is, the region immediately posterior to the enteric valve. Bignell (1984) reported that arthropod gut provided a suitable niche for microbial activity, but the nature of microflora and their distribution depended on the physicochemical conditions like pH, redox potential and temperature of that region. Brune (1995) reported that the presence of large number of aerobic, facultative and anaerobic microflora showed that hindguts are a purely anoxic environment together with steep axial pH gradients in higher termites. Among the different physiochemical conditions, pH and redox potential are the important factors which determine the type of microflora in the gut, while the pH of the foregut and midgut is around neutrality, whereas the paunch, colon and *** appear to be slightly acidic. However, the foregut and midgut of termites were aerobic with E0' in excess of +100 mv. The paunch and colon were anaerobic with E0' at about --230 to 270 mv, whereas the hindgut of termites showed --120 to 270 mv, which proved the anaerobic conditions of the gut. In essence, termite gut harbors different kinds of bacteria, fungi and protozoa. Termites are good sources of wood degrading enzymes such as cellulase-free xylanases (Faulet et al., 2006; Matoub and Rouland, 1995), laccases that are potentially involved in phenolic compounds degradation suitable for paper and pulp industry and glucosidases (Yavapa et al., 2005). The meatgenomic analysis of hindgut microbiota of higher termite showed the presence of diverse endoxylanases, endoglucanases, GH94 phosphorylases, glucosidases, nitrogenases, enzymes for carbon dioxide reduction and enzymes used in new ways for producing lignocelluloses based biofuels production (Warneck et al., 2007) and acetate production (Schmidt et al., 1999). Daily hydrogen turnover rates were 9 -33 m 3 H2 per m 3 hindgut volume, corresponding with the 22 - 26% respiratory activity of the termites. This makes H2 the central free intermediate during lignocellulose degradation and the termite gut, with its high rates of reductive acetogenesis, the smallest and most efficient natural bioreactor currently known. The different groups of microorganisms present in the gut of termites are given in Table 1. Acid forming bacteria Anaerobic bacteria present in the gut of termites arecellulolytic, CO2 reducing acetogenic and methanogenic bacteria. Volatile fatty acids are present throughout the intestine and each segment contained a constant volatile fatty acid concentration. Organic acids are produced in the hindgut in the order of acetate > formate > propionate. Mannesmann (1972) reported that acetate is a dominant volatile fatty acid in the hindgut of Reticulitermes flavipes. The acetate, which occurs in the termite hindgut at a concentration of about 80 mm, which can constitute over 90-mol% of VFA, is taken up from the termite tissue for its nutrition (Ohkuma et al., 2001). It is also reported that acetate, propionate and other organic acids produced during microbial fermentation of carbohydrates in the hindgut are important oxidizable energy sources for termites. Schultz and Breznak (1978) reported that the Bacterioides in the guts are capable of fermenting lactate to propionate and acetate by interspecies lactate transfer that occur between Streptococci and Bacterioides. A classical model of the major metabolic reactions occurring in termite hindguts is presented in Figures 2, 2A and 2B. Homoacetogenic bacteria Homoacetogenic bacteria are present in the highly compartmentalized hindgut of soil feeding higher termites (Thayer et al., 1976). The homoacetogenic population is supported by either substrates other than H2 or by a cross epithelial H2 transfer from the anterior gut region, which may create micro niches favorable for H2 dependent acetogenesis. Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites. Wagner and Brune (1999) reported that methanogenesis and reductive acetogenesis in the hindgut of the wood feeding termite, Reticulitermes flavipes, is based on the microbial population and relatively high hydrogen partial pressure in the gut lumen. Cellulolytic bacteria Cleveland (1924) was unsuccessful in isolating cellulolytic bacteria from the gut of termites, but Eutick Velu et al. 59 Table 1. Different groups of microorganisms present in the gut of termites. Microorganisms Insect Reference Aerobes Arthrobacter sp. Reticulitermes hesperus Paul et al. (1990) Arthrobacter sp. M.darwiniensis Thayer (1976) Bacillus cereus Reticulitermes virginicus Thayer (1976) Bacillus sp. R. hesperus Eutick et al. (1978) Citrobacter freundii M. darwiniensis Eutick et al. (1978) Micrococcus sp. Odontotermes sp. Paul et al. (1986) Serratia marcescens Coptotermes formosans Thayer (1976) Streptomyces sp. Cubitermes severus Bignell et al. (1979) Streptomyces sp. Soil feeding termites Pasti and Belli (1985) Facultative anaerobes Bacteroides sp. R. flavipes Schultz and Breznak (1978) Cellulomonas sp. Odontotermes sp. Paul et al. (1990) Cellovibrio sp. Odontotermes sp. Paul et al. (1990) Clostridium termitidis N. lujae Saxena et al. (1993) Enterobacter sp. M. darwiniensis Eutick et al. (1978) Staphylococcus sp. M. darwiniensis Eutick et al. (1978) N2 fixing bacteria Citrobacter freundii R. flavipes French et al. (1976) E. agglomerans Kalotermes minor Bennemann (1973) CO2 acetogenic bacteria Acetonema longum Pterotermes accidensis Kane and Breznak (1991) C. mayombei Cubitermes species Kane and Breznak (1991) Sporomusa termitida N. nigriceps Breznak et al. (1988) Methanogenic bacteria Methanobrevibacter sp. R. flavipes Lee et al. (1987) M. curvatus R .flavipes Leadbetter and Breznak (1996) M. cuticularis R. flavipes Leadbetter and Breznak (1996) M. arboriphilicus N. nigriceps Yang et al. (1985) M. bryantii N. nigriceps Yang et al. (1985) Protozoa Trichomitopsis termosidis N. nigriceps Yamin (1978) Trichonymphs sphareica N. nigriceps Yamin (1978) (1978) and Schultz and Breznak (1978) were successful and reported the presence of cellulose degrading bacteria from termites. They further reported that cellulose digestion in the gut of termites was a slow process. Most of the energy available to termites from cellulose digestion appears to come from oxidation of the acetate derived cellulose. Many cellulolytic microbes like Bacillus cereus, Serratia marcescens and Arthrobacter sp., from Reticulitermes sp., Staphylococcus and S. saprophyticus from Odontotermes obesus, Cellulomonas and Micrococcus sp., which are also from the hindgut of Odontotermes sp were isolated (Hino 1958; Thayer, 1976; Paul et al., 1986; Saxena et al., 1991). Cellulolytic actinomycetes were isolated from the hindgut of four different termites Macrotermes, Armitermes, Odontotermes and Microcerotermes spp. The isolated actinomycetes (Streptomyces sp. and Micromonospora sp.) were grown on cellulosic substrates and their extracellular cellulase (Cl , Cx and cellobiase) activity were evaluated (Hydo et al., 2003, Korb and Aaanen, 2003); using filter paper as a substrate for Cl ; carboxymethylcellulose (CMC) for Cx and d-cellobiose for cellobiase. All 60 Afr. J. Environ. Sci. Technol. Polymers Monomers Primary fermentati on products Acetate H2 + CO2 CH4 CO2 Homoacetogens O2 Termi te Methanogen s Protozoa Protozoa (Fermenting) ((())))bacteria) Figure 2. Fermentation of polymers in the termite guts (Tholen et al., 1997). strains were shown to degrade soluble and insoluble cellulose, whereas optimum pH for growth was 6.2 -- 6.7 at 28° C. However, the three strains could grow at 48° C on cellulosic substrates (Pasti and Belli, 1985). CO2 reducing acetogenic bacteria Acetogenic bacteria capable of fermenting glucose and or cellobiose to acetate are present in the guts of higher and lower termites. These bacteria are capable of forming acetate by the reduction of CO2. Acetate is not only an oxidizable energy source for termites, but an important precursor for synthesis of amino acids, cuticular hydrocarbons and terpenes. H2 oxidizing CO2, reducing Sporomusa termitida sp. nov. acetogenic bacteria, was isolated from the gut of Nasutitermes nigriceps (Breznak, 1984). Methanogenic bacteria Termites inhabit many different ecological regions, but they are concentrated primarily in tropical grasslands and forests. Symbiotic micro-organisms in the digestive tracts of termites (flagellate protozoa in lower termites and bacteria in higher termites) produce methane (CH4). Methane has been considered to be an important greenhouse gas (GHG) contributing significantly to global warming (Thakur et al., 2003). Termites may emit large quantities of methane, carbon dioxide and molecular hydrogen into the atmosphere (Zimmerman et al., 1982), though considerable uncertainty exists regarding the true estimates of methane emission. Significant studies are available on diversity, social structure, physiology and ecology, still termites as source of methane contributing to the sources of atmospheric greenhouse gas is yet to be explored in India. An attempt has been made to focus on the importance of termites and their global contribution in GHG. There is need for a detailed study in India for abatement of termites to reduce the GHG emission from termite source, as termites also to some extent contribute to land degradation and have serious implications for desertification. Methane production by termites was first reported by Cook (1932) who observed the evolution of a gas from a species of termite. Studies during the following years indicated that methane is produced in a termite's digestive track during the breakdown of cellulose by symbiotic micro-organisms (Figure 3). Later, studies showed large variations in the amount of CH4 produced by different species. More recent research by Zimmerman et al. (1982) found average CH4 production rates of 0.425 µg CH4/termite/day for the lower termite species and 0.397 µg CH4/termite/day for the higher termite families. Environmental conditions such as light levels, humidity, temperature, and CO2 and O2 concentrations play a part in methane production. Termites prefer the absence of solar radiation, an immobile Velu et al. 61 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Larvae Soldier Worker Queen Winged Fungal comb Lower termites Higher termites CO2 emission (µ moles/g/h) Figure 4. Carbondioxide emission in situ by different castes of termites. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Larvae Soldier Worker Queen 1 Winged Lower termites Higher termites Methane emission ( µ moles) Figure 3. Emission of methane in situ by different castes of termites. atmosphere, saturated or nearly saturated relative humidities, high and stable temperatures and even elevated levels of CO2. Although termite populations are active in the middle latitude environments, the vast concentrations of mounds and nests are found in the lower latitude tropical forests, grasslands and savannahs of Africa, Asia, Australia and South America. It is estimated that these regions contribute approximately 80% of global termite emissions. Gomathi and Ramasamy (2001) reported maximum CO2 emission from worker, larvae and queen of higher termite compared to lower termites (Figure 4). The results are in agreement 62 Afr. J. Environ. Sci. Technol. with those reported in the literature (Zimmerman et al., 1982; Collin and Wood, 1984). Fraser et al. (1986) performed an experiment using 6 different species of termites from the United States and Australia. Termite mounds under glass enclosures were studied in a laboratory setting, with diet and temperature allowed to vary, while all other variables were controlled. It was found that the capacity of termites to produce CH4 varied from species to species, within groups from different mounds or nests of a particular species, and also with temperature. The 6 different species studied produced methane at rates that ranged over more than two orders of magnitude. Raising the temperature by 5° C within each species' preferred temperature range caused a 30 - 110% increase in the measured CH4 emissions. Prior laboratory and field research seems to show that termites prefer temperatures in excess of 10° C above the ambient air temperatures determined by their geographical locations. A positive correlation between amounts of biomass consumed and CH4 emitted was observed, with the average being 3.2 mg CH4 per gm of wood. Seiler et al. (1983) performed a field research project near Pretoria, South Africa, to study termite methane production. His team placed aluminum framed boxes covered by plastic over termite mounds with the goal of separating the mounds from the ambient conditions while keeping the termite colonies in their natural environments. CH4, CO2 and temperature levels were monitored inside the mounds and flux rates of the carbon compounds were measured within the boxes by extracting air samples by means of syringes. Also monitored was the exchange of CH4 and CO2 at the soil surface within the vicinity of the nests. The calculated flux rates from termite mounds into the atmosphere showed significant variations which were related to the size of the mounds, the population density of the termites, termite activity and termite species. It was found that the flux rates exhibited diurnal variations, with maximum values during the late afternoon and minimum values during the early morning. The CH4 flux rates from individual mounds were directly proportional to the corresponding CO2 rates, with methane increasing linearly with increasing carbon dioxide. It was also shown that the ratios of CH4 and CO2 flux rates measured at different days, mounds and weather conditions were relatively constant for each species, but differed considerably from species to species. Most interestingly were measurements performed on the soil surface at distances of 1 to 20 m from the center of the termite nests, which generally showed a decrease in CH4, indicating that CH4 is decomposing in the soil. This observed destruction of atmospheric methane in the termite-free soil areas has led some researchers to suggest that such adjacent areas are a sink for CH4, but it is agreed that much further tests and measurements are needed to fully more understand the effects of termites on atmospheric levels of methane. Methanogenic bacteria have been visualized in association with protozoa in termites. Though methanogens are generally strict anaerobes, their metabolic responses to the presence of oxygen and their sensitivity to it vary with the species. Methanobacterium sp. was isolated from the termite hindgut (Lee et al., 1987). Leadbetter and Breznak (1996) isolated Methanobrevibacter cuticulam and M .curvatus from the hindgut of the termite Reticulitermes flaviceps. The presence of M. arboriphilicus and Methanobacterium bryantii in the guts of wood eating higher termites has been reported also (Veivers et al., 1991). Termites are one of the contributors of methane to the atmosphere. They also produce more methane, carbon dioxide and molecular hydrogen (Darlington, 1994). Termite mounds and galleries in the natural environment contain high levels of CO2 and humidity depending on the types of termites involved and their habitats. Termite guts are the world's smallest bioreactors. It was generally believed that the enlarged hindgut serves as an anaerobic digester where a symbiotic gut microflora ferments cellulose and hemicelluloses to short chain fatty acids, which are then absorbed and oxidized by the host (Breznak and Pankratz, 1977). The presence of carbohydrate--fermenting bacteria and protozoa, high levels of volatile fatty acids in the gut fluid and the occurrence of typical anaerobic activities such as homoacetogenesis and methanogenesis resemble the situation encountered in the rumen of sheep and cattle. Methane (CH4) is a metabolic end product in the hindgut of most termites. It has been estimated that these insects contribute appro-ximately 2 to 4% to the global emissions of this important greenhouse gas. Methanogenic archaea, which are easily identified by their coenzyme F 420 auto fluorescence, have been located in several microhabitats within the hindgut. Depending on the termite species, these organisms can be associated either with the hindgut wall or with filamentous prokaryotes attached to the latter, or they can occur as ectosymbionts or endosymbionts of certain intestinal flagellates. Methane emission from termites has often been debated to be a significant source of global atmospheric CH4. Methanogens produce about one billion tons of methane every year. They thrive in oxygen-free environments like the guts of cows and sheep, humans and even termites (Zeikw et al., 1977). Methanogenic bacteria share physiological and biochemical characters such as the ability to anaerobically oxidize hydrogen (H2) and reduce carbon dioxide (CO2) to CH4. Methane is formed in the rumen by methanogens (part of the domain Archaea), mainly from H2 and CO2. The methanogens from a wide range of habitats are being genome-sequenced to gain a better understanding of their biology and, in particular, to identify targets for inhibition technologies for gutassociated methanogens. Within the rumen microbial food web, methanogens perform the beneficial task of removing H2, which allows reduced cofactors to be reoxidized and recycled, thereby enhancing the breakdown Velu et al. 63 Table 2. Enumeration of total anaerobes from the different gut regions of termite. Organisms Foregut (CFU ml -1 ) Midgut (CFU ml -1 ) Hindgut (CFU ml -1 ) Total anaerobes 10 5 44.7 27.7 22.3 Celluloysers 10 3 14.0 16.3 21.0 Acid formers 10 4 40.3 30.3 20.4 Methanogens 10 4 10.3 15.6 28.0 Klebsiella 10 4 20.0 17.0 11.6 Clostridium 10 4 39.7 22.3 26.7 SD 0.92 0.81 0.81 CD (0.05) 1.62 1.70 1.77 and fermentation of plant material. Methanogens occur on and within the cells of symbiotic protists. Okuma (2001) identified the endobiotic methanogens as novel phylotypes of the genus Methanobrevibacter, of which it utilizes H2 plus CO2, but use other substrates poorly. Brauman et al. (1992) studied the probes for methanogenic archaea which detected members of two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these accounted for 60% of all archaeal probe signals in methane emission. In four species of termites, Methanosarcinaceae were found to be dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogens family profiles and termite diet or taxonomy. Gomathi and Ramasamy (2001) reported that methanogens were predominantly present in the hind gut of fungus growing termites (Table 2). Conclusion Termites are a complex assemblage of species showing considerable variation in terms of social behavior and nutritional ecology. The digestive tract of termites normally paves a suitable niche for a variety of microorganisms to inhabit and multiply. The nutritive metabolism of termite is based on the exploitation of cellulosic materials by facultative anaerobes and obligate anaerobes, which are symbiotically associated with the termites and exist in the hindgut of the insect. Cellulolytic microorganisms are responsible for the digestion of cellulose and the nitrogen requirements are met out by the presence of nitrogen fixing aerobic and anaerobic microorganisms present in their gut. Therefore, rumen methane mitigation strategies need to consider alternative routes of H2 utilizations in the absence (or decreased levels) of methanogenesis to maintain rumen function. Two main alternatives are possible: enhancing rumen microorganisms that carry out reductive acetogenesis (combining CO2 and H2 to form acetate) or promotion of organisms that consume reducing equivalents during the conversion of metabolic intermediates (malate, fumarate and crotonate) into propionate and butyrate. A better understanding of the role and scale of methane oxidation in the rumen may help to develop mitigation strategies. Reducing ruminant methane emissions is an important objective for ensuring the sustainability of ruminant-based agriculture. The authors' studies suggest that although overall methane fluxes from soil invertebrates under study cannot substantially influence a methane budget in most ecosystems, methane production is significant at least in some millipedes and therefore can impact meso and microenvironments inhabited by these invertebrates. This work also confirms that methane production is not only supported by tropic soil invertebrates, but also by temperate species.