Introduction

Termites are the main force of lignocellulose degradation in the continental ecosystem, and they play a crucial role in the formation of terrestrial ecosystems. Termites depend upon the microbes in their gut or digestive tract to obtain end products such as sugars, hydrogen, and acetate, which are further metabolized by symbiotic microbes as energy and nutrition sources for termites [1]. Termites are considered to be the most destructive insect pests in the world [2]. The damage to residential and commercial buildings that are caused by termite in the U.S. costs more than $1 billion annually. Compared with physical and chemical termite control measures that tend to be inconvenient and hazardous to animals, plants, and humans, biological control of termites has great potentially due to its minimal contamination of the environment.

Studies have revealed that the termite gut is a “gold mine” of microbial flora, consisting of a large number of symbiotic microorganisms, including bacteria, fungus, actinomycetes, and other microorganisms. According to anatomical and social organizational characteristics, the termite can be divided into two groups: lower termites (symbiotic protozoa) and higher termites (non symbiotic protozoa). The lower termites contain six families, which mainly depend on their wood-feeding, and mainly rely on their own and other symbiotic protozoa to degrade cellulose. The higher termites which consist of 75% of all the species of termites, have no protozoan symbiosis in their gut, but only composed of termitidae [3]. The gut of termite is divided into three parts, namely, foregut, midgut, and hindgut. There are many processes in the intestinal tract which can prolong the digestion time of digestive enzymes in termites [4]. There are many microorganisms isolated and identified from the gut of termite, such as Clostridium mayombei and Acetonema longum from the gut of Macrotermes gilvus (Hagen) [5]. Additionally, our group has isolated many microorganisms from the gut of Odontotermes formosanus, and four of them have lignin and cellulose degrading activities [6]. The results showed that symbiotic microorganisms play a crucial role in the life of termites. In this paper, we summarized the diversity of symbiotic microorganisms in the gut of termites, and their role in the digestion of lignocellulose, and biotechnological applications of these symbiotic microorganisms.

Diversity of Symbiotic Microorganisms in the Gut of Termite

There are mainly three kinds of microorganisms in the gut of termites, namely bacteria, archaea, and eukaryotes. There are diverse microbes in the termite as shown in Table 1, many of which are unidentified due to the challenge of growing them outside of the termite gut. Additionally, many of the microbes are exclusively found in termite guts. Researchers have isolated several flagellates (Trichomonadida, Hypermastigote, and Oxymonad) from the gut of lower termites (Mastotermitidae, Hodotermitidae, and Kalotermitdae), which were not found in nature [7, 8]. Symbiotic microorganisms play an indispensable role in the gut of termites and, therefore, are called a mutualistic relationship [9]. Sometimes neither partner can live without the other; hence, the relationship is called an obligate symbiosis [10].

Table 1 The isolated and identified microbes from termite gut
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Termite Gut Protozoan

There are many protozoans in the hindgut of lower termite; most of them are mastigophora, sporozoa, and ciliata. The difference is rarely found among the protozoans of higher termite hindgut. The symbiotic protozoa in lower termite have an important role in the digestion of lignocellulose [21]. Lignocellulose is degraded by the protist in the gut of termite, and then utilized as a source of nutrients for the termites. Studies have identified a variety of protozoans containing the cellulase gene [7]. Flagellates are the most important of the large number of protozoans that are symbiotic in the gut of termites [22]. In 1877, Leidy first reported that they found flagellates in the gut of Reticulitermes flavipes. In 1981, Trichomitopsis termopsidis was isolated from the gut of Zootermopsis angusticollis [11]. The flagellates occur in high numbers in the paunch (103–107 cfu/ml) and they can occupy >90% of the paunch volume. Majority of flagellates belong to the Trichomonadida, Hypermastigida, and Oxymonadida family [12, 23].

Termite Gut Spirochete

The spirochetes are the dominant bacteria in the termite gut. For example in Reticulitermes speratus (Isoptera; Rhinotermitidae), the spirochetes accounted for 42–63% of the total intestinal bacteria [24]. In 1996, Leadbetter first discovered spirochetes in the gut of Zootermopsis angusticollis, and successfully isolated the spirochetes. Later on, spirochetes similar to the genus Treponema were reported [14]. After that, Treponema isoptericolens and Spirochaeta coccoides were isolated from the gut of Incisitermes tabogae [14]. Droge isolated a spirochete from the hindgut contents of the lower dry-wood termite Neotermes castaneus. These spirochetes were isolated by culture in a medium with different carbon sources. They also found out that this spirochete produces formate, acetate, and ethanol, which are involved in the degradation of lignocelluloses [13]. The spirochete is closely related to cellulose decomposing bacteria in lower termites, and can promote the formation of acetic acid and also has the function of hydrogen consumption [25,26,27]. The symbiotic spirochetes have been isolated from five dry-wood feeding termites (Cryptotermes cavifrons, Heterotermes tenuis, Kalotermes flavicollis, Neotermes mona, and Reticulitermes grassei). To determine the closest relatives, Berlanga obtained the DNA sequence of spirochete, and the results indicated that spirochetes were specific symbionts that have coevolved with their respective species of termites [28].

Termite Gut Actinomycete

There are a large number of symbiotic actinomycetes in the gut of both higher and lower termites, nest and surrounding soil [29]. The actinomycetes in the gut can help the lignocellulose digestion and maintain the micro-environment of termite gut. In 1946, Hungate first isolated Micromonospora propionici from the termite gut. Consequently, other actinomycetes have been found in the gut of all kinds of termites, including Streptomyces naraensis from the Coptotermes formosanus and Streptomyces filamentosus from the O. formosanus [15, 16]. It has been found that most of the actinomycetes found in the intestinal tract of termites are Streptomyces. For example, it’s reported there are five close relatives of actinobacteria in the gut of Nasutitermes corniger (Propionibacteriaceae, Cellulomonadaceae, Corynebacteriaceae, Sterptomycetaceae, and Coriobacteriaceae). PCR-DGGE and 16S rDNA sequence analysis have revealed Streptomyces to account for the largest proportion of the gut of the termite N. corniger [30]. The actinomycetes have adapted to the intestinal environment of termites and can help the host to digest food. In particular, Streptomyces can secrete cellulase at the stage of lignocellulose hydrolysis.

Termite Gut Bacteria and Archaea

In addition to the symbiotic flagellate, bacteria and archaea also play a crucial role in the digestion of lignocellulose by termites [18, 31]. Researchers have found that symbiotic bacteria have a high density (109–1011 cfu/ml) in the gut of termite [32, 33]. Intestinal bacteria are involved in the degradation of cellulose, hemicellulose, oligosaccharides, aromatic compounds, and nitrogen fixation [34].

The intestinal microorganisms can produce a large number of acetic acid [35]. 1/3 of acetic acid needed by the respiration of termites is produced by acetic acid producing bacteria such as Sporomusa termitida, Acetonema longum, and Clostridium mayombei [17]. The identity of the lactate-producing microorganisms is not clear. A limited amount of acetic acid may be formed by the intestinal protozoa. Because Lactococci and Enterococci have the ability to ferment cellobiose and xylose, they may also be candidates for acetic acid production [36].

Termites, cockroaches, and scarab beetles are the insects known to emit methane by using its symbiotic microorganism [37]. The methanogens colonizing the gut of termite fall into three major phylogenetic groups, namely, Methanobacteriales, Methanosarcinales, and Methanomicrobiales. Leadbetter isolated two strains of Methanogenic archaesa from the hindgut of Reticulitermes speratus, namely Methnobrevibacter cuticularis and Methnobrevibacter curvans [18, 38]. The two strains can produce methane just utilizing H2 and CO2, without the need for methanol, acetic acid, and ethanol as the carbon source.

Our group has isolated more than 230 strains of endophytic bacteria which have the activities of cellulase, ligninase, and nitrogen fixation in the gut of O. formosanus from the Logia Hill of Wuhan in China. Strains with degradation activities for lignocellulose are identified as Penicillum, Fusarium oxysporum, Bacillus cereus, and Paenibacillus polymyxa [6]. Strains with nitrogen fixing activities are identified as Klebsiella variicola and Klebsiella pneumoniae [20, 39].

Termite Gut Fungi

Visser et al. isolated Xylaria from the gut in wood-feeding higher termites (Nasutitermes spp.), and proved that Xylaria was rich in termites. The researchers investigated that the synergistic activities of the symbiotic fungus Termitomyces can significantly improve the cellulose degradation ability of Odontotermes formosanus [40]. The researchers have also identified a total of 33 enzymes in Termitomyces albuminosus, of which five were cellulases. The fungi play an important role in the termite gut, most probably in cellulose and hemicelluloses degradation. Therefore, in the evolutional process of termites, termites and fungi have established interdependent relationships [41]. Our group has also isolated a laccase-producing endophytic fungus from the gut of O. formosanus, and the strain is identified as Trichoderma virid [42].

Roles of Symbiotic Microorganisms in the Gut of Termite

Termite depend upon the microbes in their gut or digestive tract to produce digestive enzymes to decompose lignocelluloses and convert them to end products such as sugars, hydrogen, and acetate, which are further metabolized by symbiotic microbes as energy and nutrition sources for termites. Cellulose is a major sugar in wood and it is broken down in the hindgut of the termite by microbes into molecules called short-chain fatty acids [43]. The nitrogen content of the organs of termites is similar to that of other animals. Nitrogen-fixing process happens in the gut of termite, and up to 60% of the nitrogen in the organs of termite comes from the nitrogen fixation of symbiotic microorganisms living in the hindgut [20].

Cellulase Activity

All the species of termites are able to produce their own cellulase [44]. The endoglucanases are the enzymes involved in the first step of the process of cellulose hydrolysis. The produced tissues of endoglucanase in lower termites and higher termites are different. In lower termites, endoglucanase is secreted by the salivary glands, and in higher termites, it is secreted in the midgut epithelial cells. The lower termites, which harbor symbiotic flagellates, need cellulose decomposition symbiosis to increase the efficiency of cellulose decomposition. Research shows glycosyl hydrolase genes expressed by the flagellates in the hindgut of Reticulitermes speratus comprises cellulases, xylanases, arabinosidase, mannosidase, and arabinofuranosidase [45]. Termites degrade lignocellulose through efficient cellulase and glycoside hydrolase activity, which is a combination of their own enzymes and that of their symbiotic microorganism. In the study of how the intestinal tract of termites digests cellulose, researchers have been using carboxymethyl cellulose as the substrate for the detection of cellulase activity. They also proved that when the flagellates in the termite gut were treated with metronidazole, the termites died after 2 weeks. Arakawa has successfully characterized the functional xylanase originating from the flagellate C. formosanus [46]. The study confirmed that the protists, especially the flagellates, could help the lower termites digest lignocellulose in the food.

The higher termites have almost non-existent flagellates in the termite gut, so the cellulase originates mainly from the termites itself and other intestinal symbiotic microorganisms. The actinomycetes in the hindgut of termite are associated with termite lignocellulose digestion [22]. During the hydrolysis stage of lignocellulose, actinomycetes can secrete 1,4-β-xylanase and 1,3-β-glucan to decompose cellulose and hemicellulose [47]. At the oxidation stage, Streptomyces sp. and Nocardia sp. can secrete β-d-glucosidase, β-galactosidase, α-l-arabinofuranosidase, and β-d-xylosidase to further degrade the hydrolysis products and aromatic compounds [48].

In the gut of termite, the spirochetes are freely moving or moving with the protozoa. Treatment with antibiotics for the termite to reduce the spirochete abundance, and they found that the degradation ability of cellulose was significantly reduced. Quite a few spirochetes isolated from the termite gut are not capable of reductive acetogenesis, but they can produce a variety of monosaccharides, disaccharides, and oligosaccharides including formate, acetate, and ethanol, through fermentation.

Nitrogen Fixation

Cleveland is the first to prove that the nitrogen nutrition of termites is not obtained from food [49]. After that, Hungate also proved that termites did not have the ability of nitrogen fixation [50]. Breznak and Benemann studied the termite nitrogen fixation by the acetylene reduction method, and they obtained the experimental evidence of nitrogen fixation of symbiotic bacteria in the termite’s gut [51,52,53]. Research shows that the C. formosanus nitrogenase activity is associated with intestinal microorganisms. The symbiotic bacteria which have nitrogenase activities were Citrobacter freundii and Enterobacter agglomerans. The ability of nitrogen fixation in the gut of termite is associated with termite feeding; intestinal commensal bacteria of soil-feeding termites generally do not have the ability of nitrogen fixation. When termites are mainly wood-feeding, the biological activities of nitrogen fixing bacteria in the gut of termite are more pronounced [19, 54].

Applications of Symbiotic Microorganisms in the Gut of Termite

Degradation on Lignocellulosic Materials

Microbes in the gut of termites not only play a crucial role in the life of termites, but also can be used by people. As one of the few insects that can degrade cellulose, termites can efficiently degrade lignocellulose and turn it into nutrition and energy for their survival [34]. This ability is mainly based on the microbial symbiont placed in a specialized region of the intestine. Metabolic collaboration between the termite and its symbionts make the hindgut an efficient bioreactor that produces a variety of cellulase, hemicellulase, and auxiliary enzymes, capable of efficiently liberating sugar from cellulose and hemicellulose. Termites have a highly efficient lignocellulose degradation system in their gut.

It is still unclear how termites deal with lignin, whether it is by the termites themselves, or possibly by unidentified microbes in the gut of termites. Although there is lack of understanding concerning lignin degradation, this has not stopped people from researching termite gut symbiosis. More knowledge gathered on lignocellulose digestion in termites could inspire a more efficient production of biofuels in the future [55].

The reaction condition of cellulase is high specific and mild, which will not promote environmental pollution as characteristic of chemical methods. The waste produced by human activities also contains a lot of cellulose, such as agricultural waste, food processing waste, wood waste, and municipal waste. As a biological enzyme preparation, cellulase has vast potential for future development in the food industry, feed industry, detergent industry, new energy development, and so on. Cellulase has great potential in paper making, geological drilling, extraction of herbs, and so on [56]. Proper treatment of pulp with cellulase can increase the amount of micro fiber and increase water holding capacity and may promote the tensile resistance of some paper. In addition, cellulase is also used in the pharmaceutical industry, making digestive agents, and so on. The economic benefits created are immeasurable.

Biofuel Production

With the shortage of fossil fuels, energy is a common issue facing mankind. The search for new energy sources is relevant to the sustainable development of the economy and even to the survival of mankind. Cellulose is the most abundant and cheapest renewable resource in the world [57]. Every year, photosynthesis produces large amount of cellulose and hemicellulose. Provided that we can effectively use cellulase to biotransform cellulose into simple sugars, and consequently fermented to produce ethanol and other source of energy, cellulose will become a valuable energy source. Cellulose can be hydrolysed into glucose and other substance. For mankind, this is an effective way to produce new forms of bioenergy [58].

For example, in the production of environmentally friendly fuel ethanol, cellulose can be degraded into reducing sugar by cellulase, and the reducing sugar can be fermented to produce more sustainable second generation biofuels [59]. This efficient transformation system provides a new possibility for human beings. We can use cellulose, which is abundant in nature, as a new type of biological resource. The microbial cellulase can be effectively used to decompose and transform cellulose in the natural world. The conversion of cellulosic materials into energy sources has great practical significance in providing renewable energy, protecting the environment, and sustaining the development of human society.

Conclusions

The symbiotic microorganisms in the gut of termite play an important role in the digestion of cellulose and nitrogen metabolism. Prevention or elimination of these symbiotic microorganisms will serve as an effect way of termite control. The study on the symbiotic microorganisms will also provide a new method for the biological control of termites by the endophytic bacteria in the gut of termites. Our group has isolated some endophytes from the natural anti-termite tree Eastern Red Cedar (ERC, Juniperus virginiana) [42] and Port Orford Cedar (POC, Chamaecyparis lawsoniana) [6], and through antibacterial assay, 24 endophytic bacteria with antibacterial activity have been obtained out (Table 2) [60], five highly effective compounds for killing termites have been obtained, including α-terpineol, nootkatone, τ-muurolol, torreyol, and α-cadinol [6, 42]. Further studies on the function and mechanism of cellulose digestion by symbiotic microorganisms will be helpful to clarify the relationship between intestinal symbiotic microorganisms and intestinal commensal microorganisms. This is a novel way to produce an effective, environmental friendly, low toxicity, and low-cost termiticide for termite control, which has practical significance for the development and application of beneficial microorganisms.

Table 2 The antibacterial activities of endophytic bacteria
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