Pertanika J. Trop. Agric. Sc. 41 (3): 925 - 940 (2018)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Review Article

Diversity of Nitrogen Fixing bacteria Associated with Various Species

Sarannia Thanganathan and Kamariah Hasan* School of Biological Sciences, Faculty of Sciences and Technology, Quest International University Perak, 30250 Ipoh, Perak, Malaysia

ABSTRACT Nitrogen is one of the vital elements for the growth and survival of many organisms.

Atmospheric N2 can only be used by certain organisms like microbes that supply inorganic form of nitrogen to their host, , or plants via symbiotic or non-symbiotic interaction. are diverse species on the earth, whose guts are inhabited by microbes that help in many physiological activities like N2 fixation. As N2 fixing bacteria ecologically play vital roles, many studies have demonstrated the presence of N2 fixing bacteria in termite gut. Study on termite’s gut omics has also supported a complex systemic understanding of gut digestome that is imperative in understanding the termite holobiome. This review gathers a variety of information from multifarious research which has been done on the isolation and diversity of N2 fixing bacteria in various termite species.

Keywords: Acetylene reduction, nifH, nitrogen, nitrogen fixation, termite gut

INTRODUCTION only be used by certain organisms like Nitrogen is an essential component of microbes that supply inorganic form of N2 to their host, insects, or plants via symbiotic amino acids and proteins. N2 is abundant or non-symbiotic interactions (Khan, in the atmosphere but atmospheric N2 can Mohiuddin, & Rahman, 2008). Thus, N2 fixation process is vital in providing fixed

ARTICLE INFO N2 to organisms. Biological N2 fixation

Article history: (BNF) is the conversion of atmospheric N2 Received: 22 June 2017 Accepted: 26 January 2018 into ammonia (Eskin, Vessey, & Tian, 2014). Published: 29 August 2018 About 90 genera of microbes are able to fix E-mail addresses: [email protected] (Sarannia Thanganathan) N2 by utilizing nitrogenase enzyme (Gaby [email protected] (Kamariah Hasan) * Corresponding author & Buckley, 2012).

ISSN: 1511-3701 e-ISSN: 2231-8542 © Universiti Putra Malaysia Press Sarannia Thanganathan and Kamariah Hasan

N2 fixing bacteria are classified into two According to the recent data, there are categories; symbiotic and non-symbiotic approximately 3106 species of . bacteria. They can be found in soils, gut They are classified into 12 families which of arthropods, or insects and root nodules. are further divided into 282 genera. From Arthropods are diverse species on the earth, the 12 families, the lower temites’ families whose guts are inhabited by microbes are Cratomastotermitidae, Mastotermitidae, that help in many physiological activities Termopsidae, Archotermopsidae, like N2 fixation and cellulose degradation Hodotermitidae, Stolotermitidae,

(Bashir et al., 2013). Since N2 is one of the , Archeorhinotermitidae, limiting nutrients in insects’ diet, most of Stylotermitidae, Rhinotermitidae, and the insects depend on mutualistic bacteria Serritermitidae whereas, the higher termites’ having N2 metabolism in order to obtain family is Termitidae (Krishna, Grimaldi, sufficient amount of N2 (Engel & Moran, Krishna, & Engle, 2013; Kambhampati &

2013). N2 fixing bacteria have symbiotic Eggleton, 2000). interaction in insects’ guts, like in termites, cockroaches, and beetles. As N2 fixing worker soldier bacteria ecologically play vital roles, many studies have demonstrated the presence of N2 fixing bacteria in termite gut that are capable of utilizing nitrogenous wastes excreted by termite and convert them into high-value

N2 (Ohkuma, Noda, & Kudo, 1999). This review gathers a variety of information from various researches, which have been done on the isolation and diversity of N2 fixing bacteria in various termite species.

Termite Classification (a) Termites are classified into a few castes which comprise of queen, soldiers, workers, nymphs, and larvae (Kambhampati & Eggleton, 2000). Soldier class termites have the mandible, which is a jaw like structure on their heads that helps in protecting the colonies as shown in Figure 1 (Watanabe, (b) Gotoh, Miura, & Maekawa, 2014; Watanabe Figure 1. The morphology of termites (worker and & Maekawa, 2012). soldier): (a) The illustration of worker and soldier termite; (b) The mandible of soldier (right) and worker (left). Adapted from Watanabe et al. (2014)

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Termite’s Gut Structure bacteria, and archaea (Ohkuma & Brune, Termite gut is divided into three parts: 2011). Lower termites’ gut is colonized foregut, midgut, and hindgut. The internal by a diverse species of flagellated protists structure of a termite gut is shown in Figure and prokaryotes whereas the gut of higher 2. Termite’s hindgut is colonized by diverse termite is colonized only by prokaryotes microbial symbionts from three different (Brune, 2006; Peterson & Scharf, 2016). domains which are flagellate protists,

Symbiotic Salivary Foregut Midgut gland Hindgut fauna

Figure 2. The internal structure of termite guts showing the foregut, midgut, and hindgut. Adapted and modified from Scharf, (2015)

Termite’s Diet Microbiota of Termite’s Gut Termites act as a decomposer where they The complexity of termite gut leads to the feed on decaying and dead plant material presence of hundreds of phylotypes. Based with relatively high carbon to N2 ratio. They on 16S rRNA studies, most of the species also consume fungi and soil-rich organic isolated from termite guts are novel. Those matter (Engel & Moran, 2013). Such a species are not environmental bacteria and, feeding habit of termites contributes to the are specifically classified as termite gut balanced ecosystem (Freymann, Buitenwerf, specialists. Termites’ gut microbes possess Desouza, & Olff, 2008). mutualistic relationship with their host in According to Inward, Beccaloni and several ways. For instance, gut microbes Eggleton (2007), and Donovan, Eggleton aids in lignocellulose digestion which and Bignell (2001), gut contents and leads to the production of acetate, a main morphology were used to classify termites’ carbon source for the termites. Besides, the feeding habits. Lower termites have simple microbes also supply N2 sources for the host gut and they feed on wood whereas, higher (Breznak, Brill, Mertins, & Coppel, 1973; termites with complex gut feed on wood, Odelson & Breznak, 1983). grass, lichen, litter, epiphytes, and soil.

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About 90% of the termite’s hindgut is termites (Dietrich, Kohler, & Brune, colonized by flagellates from the phylum 2014; Hongoh, 2010). Several cellulolytic Parabasalia that occupy the gut of lower bacteria like Cellulomanas, Citrobacter termites (Ohkuma & Brune, 2011). The and Enterobacter have also been isolated flagellates found in termites’ gut are unique from the gut of termite (Upadhyaya et al., and most of them cannot be found anywhere 2012). Cellulolytic bacteria secrete the else (Brugerolle & Radek, 2006). Some enzymes cellulose and hemicellulose to of the flagellates such as Trichonympha allow them to degrade the cellulose and sphaerica and Trichonympha termosidis hemicellulose materials (Lima et al., 2014). remain uncultured. These two protists were Dysgonomonas termitidis, a lignocellulose isolated from the hindgut of Zootermopsis degrading bacteria, was isolated from the angusticollis (Tai, James, Perlman, & gut of the termite Reticulitermes speratus Keeling, 2013). There are also some (Pramono, Sakamoto, Limo, Hongoh, & protists associated with methanogens in Ohkuma, 2015). A study conducted on termite gut. For instances, Microjoenia the termite Odontotermes formosanus and Dinenympha are two different protists had successfully isolated nine different isolated from the termite Hodotermopsis isolates with cellulolytic property. All sjoestedti. The flagellateSpirotrichonympha the nine isolates were closely related to leidyi which was isolated from the gut of Bacillus cereus, Bacillus thuringiensis, Coptotermes formosanus is associated with Bacillus pumilus, Pseudomonas aeroginosa, endosymbiotic methanogens (Hongoh & Citrobacter freundii, Serratia marcescens, Ohkuma, 2011). Salmonella enterica, Staphylococcus The gut of higher termites, especially gallinaum, and Enterococcus casseliflavus soil-feeding termites of the family (Kavitha, Vijayarani, & Kumanan, 2014). Termitidae, is colonized by a wide range There are also some acetogenic bacteria in of archaea. However, their presence is the termite gut. Most of them are from the minute compared to bacteria (Brauman et phylum Firmicutes and genera Clostridium al., 2001). At present, methanogens from and Acetobacterium (Drake, Gobner, & the genus Methanobrevibacter and non- Daniel, 2008). methanogens like Thermoplasmoles and Crenarchaeota have been isolated from N2 Fixing Bacteria in Termite Gut termite gut (Friedrich, Schmitt-Wagner, Termites have a symbiotic relationship Lueders, & Brune, 2001; Leadbetter, Crosby, with microbes and play a vital role in N2 & Breznak, 1998). fixation. Microbial fixation in termite gut The most abundant bacteria found is the main source of N2 for the termite in the gut of wood-feeding termites are colony (Vecherskii, Kostina, Gorlenko, Spirochaetes whereas, Bacteroideres are Dobrovol’skaya, & Umarov, 2008). Several abundant in the gut of fungus-cultivating studies have proved the presence of N2 fixing

928 Pertanika J. Trop. Agric. Sc. 41 (3): 925 - 940 (2018) Nitrogen Fixing Bacteria Diversity in Termites bacteria in the gut of various termite species. hindgut. Using acetylene reduction assay

Several bacterial endosymbionts that have (ARA), N2 fixation activity was discovered the ability to fix 2N have also been found in all the isolates obtained from all three 15 in the gut of lower termites (Peterson & species. Based on N2 incorporation test, Scharf, 2006). Since termites grow well in isolate from Mastotermes drawiniensis 15 nitrogen-poor diet, there are many types of showed highest N2 incorporation. All symbiotic N2 fixing bacteria, living in the the isolates obtained from this study were gut of termites (Masepohl et al., 2002). characterized as Citrobacter freundii. In the study conducted by Doolittle, Another study was conducted by Eutick,

Raina, Lax and Boopathy (2008), Klebsiella O’Brien and Slaytor (1978), to isolate N2 pneumoniae was isolated from Coptotermes fixing bacteria from few species of Australian formosanus which was the Formosan termites including Coptotermes lacteus, subterranean termites (FST). N2 fixation Coptotermes acinaciformis, Cryptotennes assay which was done using basic salt media primus Hill, Mastotennes darwiniensis, supplemented with NaMo and Fe2(SO4)3 Nasutitermes exitiosus, Nasutitermes walker, revealed that K. pneumonia was able to Nasutitermes graveolus, Heterotermes fix N2 anaerobically which contributes ferox, and Schedorhinotermes intermedius to the N2 source of FST. Apart from this, intermedius. Based on their morphological another study conducted by Potrikus and and biochemical characteristics, all the Breznak (1977), demonstrated the presence isolates were identified asEnterobacter spp. of Enterobacter agglomerans in the gut Two facultative anaerobes, namely, of C. formosanus and proved its ability to Clostridium sp. and Klebsiella sp. with the fix N2 through acetylene reduction assay. ability to fix N2 have been isolated from Inhibition of acetylene reduction by oxygen Mactotermes sp., the fungus cultivating proved that E. agglomerans can only fix N2 termite. In this study, N2 fixing bacteria anaerobically. were isolated from the gut of queen, soldier Few studies have demonstrated the and worker termites. These bacteria were presence of N2 fixing bacteria in various isolated using Hill’s medium and were Australian termite species. In order to characterized using Hino and Wilson confirm the presence of N2 fixation in medium (Gomathi, Ramalakshmi, & Australian termite, French, Turner and Ramasamy, 2005). This research showed Bradbury (1976) used three different that, compared to others, the amount of termite species including Mastotermes bacteria enumerated was highest in worker darwiniensis, Nasititermes exitiosus, and termites. Previously, Breznak et al. (1973), Coptotermes zactues which were obtained also discovered similar findings whereby, in from mounds at Townsville, Seymour, worker termites fed with wood, the N2 fixing and Canberra, respectively, to isolate and activity was higher, compared to the soldier characterize N2 fixing bacteria from the termites. This was demonstrated using ARA.

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N2 Fixation Activity in Termite Guts Several other researches were also The distribution of sulfate reducing bacteria conducted on the isolation of N2 fixing in termite gut was studied using various bacteria from termites. The isolates with termite species that includes Mastotermes N2 fixing ability which were isolated darwiniensis, jouteli, Neotermes from various termite species have been castaneus, Nasutitermes nigriceps, tabulated in Table 1. These researches Zootermopsis angusticollis, Zootermopsis showed the variety of N2 fixing bacteria nevadensis, Kalotermes flavicollis, that occupy termites gut and form symbiotic Heterotermes indocola, Reticulitermes relationship, which benefits both hosts and santonensis, and Odontotermes obesus. A microorganisms. total of seven pure culture isolates were Culture independent molecular method obtained in this study. Nitrogenase activity was used to study the expression of N2 of those isolates was tested using ARA. fixation genes in the gut microbiota of Neotermes koshunensis. As a primary All the isolates had the ability to fix N2 screening, the worker larvae were tested which eventually provide N2 source for the termites. Based on biochemical and for N2 fixation activity using ARA. The physiological characteristics, the seven relationship between N2 fixation activity and isolates were identified asDesulfovibrio sp. quantity of mRNA was tested by feeding (Kuhnigk et al., 1996). the termite with two different diets; filter paper with added N2 source and filter paper

Table 1

N2 fixing bacteria isolated from various termite species

N2 Fixing Bacteria Host Reference Klebsiella pneumoniae Coptotermes formosanus Doolittle et al., 2008 Enterobacter agglomerans Coptotermes formosanus Potrikus & Breznak, 1977 Mastotermes darwiniensis Citrobacter freundii Nasititermes exitiosus French et al., 1976 Coptotermes zactues Clostridium sp. Mactotermes sp Gomathi et al., 2005 Klebsiella sp. Coptotermes lacteus Coptotermes acinaciformis Cryptotennes primus Hill Enterobacter sp. Mastotennes darwiniensis Eutick et al., 1978 Nasutitermes graveolus Heterotermes ferox Schedorhinotermes intermedius intermedius Desulfovibrio sp. Mastotermes darwiniensis Desulfovibrio desulfuricans Reticulitermes santonensis Kuhnigk et al., 1996 Desulfovibrio termitidis Heterotermes indicola Desulfovibrio sp. Odontotermes obesus

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without N2 source. The acetylene reduction Ohkuma (2007). The nifH homologs from activity was higher in termites fed with filter hindgut of I. marginipennis and N. castaneus paper without any N2 source. Nitrogenase were clustered in group I and were closely activity was reduced in termites fed with related to Azoarcus strain BH72 and actively filter paper containing N2 source (Noda, expressed. NifH homologs in group II were Ohkuma, Usami, Horikoshi, & Kudo, 1999). obtained from all three termites species This finding was similar to the experiment except for I. marginipennis and had a close performed by another researcher using identity with anfH gene of Clostridium Coptotermes formosanus (Breznak et al., pasteurianum. Some homologs were related 1973). to Spirochaetes but not expressed. The

The N2 fixing activity was also studied nifH homologs in group III had a close by Desai and Brune (2012), to identify the relationship with A. pseudotrichonymphae presence of diazotrophs in four different and few homologs clustered together with termites, Cryptotermes longicollis, Treponema azotonutricium where the nifH Incisitermes marginipennis, Neotermes gene in both clusters was expressed. castaneus and Kalotermes flavicollis which In another study, lower termites of belong to genera Kalotermitidae. Termites Reticulitermes speratus were used to were fed under two different diet conditions investigate the diversity of nifH genes in (wood and filter paper) followed by ARA their intestinal microbiota (Ohkuma et to test nitrogenase activity. Although the al., 1996). The DNA of mixed microbiota termites were able to reduce acetylene in from termites’ hindgut was extracted and both conditions, the acetylene reduction nifH gene was amplified using four primer activity was higher when termites were combinations (KAD-GEM, KAD-YAA, fed with filter paper compared to wood. IGK-GEM and IGK-YAA). Four clones, PCR and RT-PCR were performed using TDG, TDY, TKG and TKY were isolated universal primers IGK and YAA for hindgut respectively. A total of 27 nifH amino acid homogenate of four termite species followed sequences were obtained from R. speratus by creation of a clone library. Based on the and all the sequences were not similar to clone library, it was revealed that the nifH any of the published sequences in database. homologous are diverse in hindgut of those Phylogenetic analysis was performed using termite species. Based on the phylogenetic neighbor joining tree constructed using 25 analysis of nifH homologs, it was found nifH sequences (Figure 3). that the gut of those termites is colonized by Based on the phylogenetic tree, although Treponema sp., Bacteriodales, and Azoarcus most of nifH amino acid sequences from sp. All the nifH homologs obtained in this the termite group are closely related to the study were clustered into respective groups sequences of Clostridium pasterenium and as referred to in the group nomenclature also Desulfovibrio gigas and Chromatium of Yamada, Inoue, Noda, Hongoh and buderi, there are no similarities between

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Figure 3. Neighbor-joining relationship constructed using nifH amino acid sequences of 24 sequences from database and 25 sequences from termites. Two chlorophyll sequences were used as outgroups. The nifH fragments used correspond to 45‑153 amino acid residues of K. pneumoniae sequence. Scale bar 0.2 denotes substitutions of 20 nucleotides over 100 nucleotides. Numbers shown in the internal branches are the bootstrap values derived from 1000 replications when above 50% is shown at each node. Adapted from Ohkuma et al., 1996

932 Pertanika J. Trop. Agric. Sc. 41 (3): 925 - 940 (2018) Nitrogen Fixing Bacteria Diversity in Termites the nucleotide sequences of clones and C. had 95% similarity with Candidatus pasterenium. The nifH sequence of clone Azobacteroides pseudotrichonymphae TDG1 and TKY17 are grouped together in genomovar, CFP2 which is endosymbionts gamma subclass of proteobacteria which of Coptotermes formasanus. One more includes Azotobacter sp., Vibrio sp. and sequence is related to D. vulgaris. Another Klebsiella sp. The sequence of TDY3 is 14 nifH sequences are clustered together clustered with C. buderi and D. gigas. The into alternative nitrogenases groups where sequences of other clones are distantly six of them are related to proteobacteria, related to the sequences obtained from Rhodospirillum rubrum. Five sequences database and they formed their own clusters having higher similarity with spirochetes (Ohkuma et al., 1996). There are also many were obtained from Z. angusticollis. copies of nifH sequence in one single Besides, another three sequences are related organism due to the presence of many nifH to Methanosarcina barkeri. Although genes like alternative nitrogenase genes. nitrogenase activity was not performed for Although a few sequences were obtained all the isolates obtained from this study, from the same organism, there is a unique there are diverse groups of N2 fixer in the diversity among them. intestinal microbiota of R. chinensis (Du et Previous study proved that al., 2012). Reticulitermes speratus was contributed by The microaerophilic nature of Termite

N2 source through N2 fixation of microbes in Associated Verrucomicrobium 2 (TAV2) its gut (Ohkuma et al., 1996). Likewise, the was studied by Isanapong et al. (2013). diversity of N2 fixing microbes in intestinal Through proteomic and transcriptome microflora of Reticulitermes chinensis method, the genes and proteins expressed

Synder were studied using the culture in different oxygen, O2, concentration were independent method. Primary screening identified. This study revealed the presence was done using nifH gene amplification of peptides corresponding to nifH genes with few primers combinations (IGK– in the cells which were grown at 2% O2 YAA, IGK–GEM, KAD–YAA, and KAD– concentration. This indicates that TAV2 GEM). From 63 clones, six chimeric is involved in the BNF and contributes to sequences, and several similar sequences the metabolism of microbial community were eliminated. About 34 sequences were in the gut of termites. In another study, the used for phylogenetic analysis. About 20 N2 fixation activity of TAV2 was confirmed nifH sequences clustered together which through genomic analysis. The TIGRFAM were true functional nifH and 17 of them and Pfam protein family databases were had a close relationship with Clostridium sp. used to perform protein model comparisons. with the similarity of 75‑88%. One sequence In addition, Kyoto Encyclopedia (KEGG) had a higher relationship with a spirochetes, and Clusters of Orthologous Groups (COG) Treponema primitia ZAS-2, one sequence databases were also used to justify the

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presence of N2 fixing activity. Based on the of Zootermopsis angusticollis has two results obtained, the presence of nifHDK homologous nifH genes with nitrogenase and anfHDGK operons in TAV2 was activity (Hongoh et al., 2003). Apart from confirmed (Wertz, Kim, Breznak, Schmidt, this, the nifH gene was also found in the & Rodrigues, 2012). bacteria isolated from Hodotermopsis Other than that, the ability of symbiotic sjostedti and Zootermopsis nevadensis.

N2 fixation in fungus growing termites was In another study, Endobacterium tested using ARA. Two termite species, proavitum strain Rsa215 was isolated from Mactotermes natalensis and Odontotermes Reticultitermes santonensis (Rsa) and badius were used in this study. The study Zootermopsis nevadensis and was tested for revealed that there was a positive acetylene the ability to fix N2. This strain contains a reduction activity in live termites of those single gene cluster encode for nitrogenase. two termite species and not in the fungus The phylogenetic analysis of nifD, nifK, and comb. This indicated the presence of N2 fixer nifH demonstrated that Rsa215 has group in termite guts. The fixation was higher in IV nitrogenase. The group IV nitrogenase worker than in soldier in both the species gene is known as nif-like gene with some

(Sapountzis et al., 2016). of these genes involved in N2 fixation. Endobacterium proavitum has a single set of nifHDK genes which are functional in N N2 Fixing Bacteria and NifH Gene 2 fixation (Zheng, Dietrich, Radek, & Brune, NifH gene is responsible for N2 fixation 2016). and used as identification marker to detect According to Noda et al. (2002), a the presence of N2 fixing microbes (Zehr, strong N fixation activity had been shown Jenkins, Short, & Steward, 2003). The 2 by C. formosanus in Japan. The culture genome of spirochetes strains ZAS-1, independent studies of nifH gene show ZAS-2, and ZAS-9 were examined in the that there are diverse species of microbes previous study to check for the presence of such as spirochetes, clostridia, archaea, nifH gene and the ability to fix 2N (Noda, and proteobacteria which are able to fix N Ohkuma, & Kudo, 2002). The study showed 2 available in the gut of C. formosanus. the presence of two nifH homologs in each Besides, the termite Neotermes strain. koshunensis has abundant nifH genes in its Study conducted on Spirochaeta gut and it possesses high N fixation activity aurantia, Spirochaeta zuelzerae, and 2 (Noda et al., 1999). There is also potential treponeme ZAS-9 showed their ability to N2 fixer which has symbiotic interaction in fix N2 due to the presence of nifH gene the gut of Reticulitermes chinensis. This (Hongoh, Ohkuma, & Kudo, 2003; Lilburn was demonstrated from the phylogenetic et al., 2001). The ZAS-strain which belongs analysis of the clones of nifH gene isolated to Treponema sp. isolated from the gut from R. chinensis (Du et al., 2012).

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Role of Metagenomic Technologies in and life processes of an organism. Omics

Studying the N2 Fixing Bacteria research has also provided advances in In termites, gut consortium play direct roles understanding symbiotic roles of individual microbial species. Currently, 82 termite in N2 fixation, amino acid biosynthesis and lignocellulose digestion. However, a species have been studied using several majority of gut microbes are unculturable omics methods, with bigger representation thus molecular methods such as single- by lower (72%) compared to higher termites species-targeting metagenomics analysis (28%). and other omics approaches are crucial in Recently, studies on metatranscriptome capturing and revealing the diversity of using ribodepletive strategy on lower termite the termite’s gut microbe. Metagenomics Reticulitermes flavipes substantiate earlier studies employing bacterial 16S rRNA discoveries of physiological contribution sequences have been used to catalog bacteria of bacteria with regard to biosynthesis, and archaea in termites (Do et al., 2014; catabolism, and transport of major organic He et al., 2013; Peter & Scharf, 2016; molecules and ions (Peterson & Scharf, Rajarapu, Shreve, Bhide, Thimmapuram, 2016). The existence of N2 metabolism & Scharf, 2015; Tartar et al., 2009). Large genes like nitrogenase, nitroreductases, metagenomic dataset will allow in-depth and ureases for N2 recycling and fixation analysis of microbial functions and could in lower termites gut bacteria were also offer resources for advancing integrative identified through genome sequencing sociogeomic, digestomic, and termitosphere (Hongoh et al., 2008; Inoue et al., 2015). in order to better understand the intricate Metagenomic approach is an important symbiotic relationships between termites tool to uncover the diversity of uncultured and their gut microbes. Overall, six main N2 fixing microbes associated with termites’ bacterial phyla are represented across higher gut. Currently, metagenomic studies focusing and lower termites, namely Bacteroides, on N2 fixing ability of guts microbiome Firmicutes, Sphirochetes Proteobacteria, utilizing gene encoding nitrogenase enzyme, Fibrobacteres, and Elusimicrobia (Brune, nifH are still lacking as many studies 2014). Metagenomic study on wood and focus more on lignocellulase and xylanase soil-feeding higher termites revealed screening from genome sequence compared that community structure and functional to N2 metabolism (Bastien et al., 2013; Liu potential of microbes in gut compartments et al., 2013). Although the nature of N2 are determined by digestive approach of fixation in termites is still in the dark, they the host (Rossmaler et al., 2015). Omics deserve attention because of their potential science collectively catalog, enumerate, influence on N2 metabolism in tropical soil. and illustrate biological molecules that By uncovering the diversity of N2 fixing transform into organization, function, bacteria in termite gut through metagenomic

Pertanika J. Trop. Agric. Sc. 41 (3): 925 - 940 (2018) 935 Sarannia Thanganathan and Kamariah Hasan and other omics study, we might uncover a Bastien, G., Arnal, G., Bozonnet, S., Laguerre, S., Ferreira, F., & Fauré, R. (2013). Mining for unique and exceptional N2 fixer that could be incorporated into soil and plant to promote hemicelluloses in the fungus-growing termite Pseudacanthotermes militaris using functional plant growth. metagenomics. Biotechnology for Biofuels, 6(1), 78. CONCLUSION Brauman, A., Dore, J., Eggleton, P., Bignell, The main source of N2 for termites is D., Breznak, J. A., & Kane, M. D. (2001). from the microbial fixation of N2. There Molecular phylogenetic profiling of prokaryotic is an abundance of microbes in the gut communities in guts of termites with different of various termite species which help in feeding habits. FEMS Microbiology Ecology, 35(1), 27‑36. N2 fixation. They are detected by looking at the presence of N2 fixing genes, most Breznak, J. A., Brill, W. J., Mertins, J. W., & Coppel, commonly nitrogenase encoded by nifH. H. C. (1973). Nitrogen fixation in termites. Nature, 244(5418), 577380. The importance of N2 fixation in termite should be analyzed further using other Brugerolle, G., & Radek, R. (2006). Symbiotic housekeeping genes, for instance, other protozoa of termites. In H. Konig & A. Varma (Eds.), Intestinal microorganisms of termites and nif genes and fix genes that are involved other invertebrates (pp. 244–269). Heidelberg, in N2 fixation. The importance of 2N fixing Germany: Springer. genes should be emphasized because Brune, A. (2006). Symbiotic associations between those genes can be manipulated to provide termites and prokaryotes. Prokaryotes, 1, significant impact on agriculture since 439‑474. N2 fixing bacteria have the potential to Brune, A. (2014). Symbiotic digestion of increase plant productivity. The bacteria lignocellulose in termite guts. Nature Reviews can be released into plants as free living Microbiology, 12(3), 168. bacteria. If there is significant improvement Desai, M. S., & Brune, A. (2012). Bacteroidales in the plants’ nutrients, the nifH gene from ectosymbionts of gut flagellates shape the these bacteria can be manipulated and nitrogen-fixing community in dry-wood termites. incorporated into non-leguminous crops International Society for Microbial Ecology, for better conversion of N2 gas. At the same 6(7), 1302‑1313. time it may reduce contamination caused by Dietrich, C., Kohler, T., & Brune, A. (2014). The chemical fertilizers. cockroach origin of the termite gut microbiota: Patterns in bacterial community structure REFERENCES reflect major evolutionary events. Applied and Environmental Microbiology, 80, 2261–2269. Bashir, Z., Kondapalli V. K., Adlakha N., Sharma A., Bhatnagar R. K., Chandel G., & Yazdani S. Do, T. H, Nguyen, T. T, Nguyen, T. N, Le, Q. G, (2013). Diversity and functional significance of Nguyen, C., Kimura, K., & Troung, N. H. cellulolytic microbes living in termite, pill-bug (2014). Mining biomass-degrading genes and stem-borer guts. Scientific Reports, 3, 2558. through Illumina-based de novo sequencing and

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