Bioresource Technology 145 (2013) 337–344

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Bioresource Technology

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Synergistic collaboration of gut symbionts in Odontotermes formosanusfor lignocellulosic degradation and bio-hydrogen production

0960-8524/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2012.12.055

⇑ Corresponding author at: Department of Life Sciences, National Chung HsingUniversity, Taichung 40227, Taiwan. Tel.: +886 4 22840416x404; fax: +886 422874740.

E-mail address: cchuang@dragon.nchu.edu.tw (C.-C. Huang).

Gincy Marina Mathew a, Dony Chacko Mathew a, Shou-Chen Lo a, Georgy Mathew Alexios b, Jia-Cih Yang a,Jagathala Mahalingam Sashikumar c, Tanveer Mahamadali Shaikh d, Chieh-Chen Huang a,e,⇑a Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwanb Mount Zion College of Engineering for Women, Kozhuvalloor 689521, Kerala, Indiac College of Natural and Computational Sciences, Haramaya University, Ethiopiad Department of Chemistry, National Chung Hsing University, Taichung 40227, Taiwane Agricultural Biotechnology Centre, National Chung Hsing University, Taichung 40227, Taiwan

h i g h l i g h t s

" The paper reports hydrogen production using gut symbionts to bio-mimic the termite gut of Odontotermes formosanus." NMR studies of the different layers of the fungus comb to study their aging." Lignocellulosic degradation observed in mango tree substrates in the presence of Clostridium, Bacillus and Termitomyces." Bacillus acts as a potential mutualist.

a r t i c l e i n f o

Article history:Received 5 October 2012Received in revised form 8 December 2012Accepted 10 December 2012Available online 20 December 2012

Keywords:Odontotermes formosanusClostridiumBacillusTermitomycesSolid-state NMR

a b s t r a c t

In this work, gut microbes from the macrotermitine termite Odontotermes formosanus the cellulolyticBacillus and fermentative Clostridium were studied in batch experiments using different carbon substratesto bio-mimic the termite gut for hydrogen production. Their fungus comb aging and the in vitro lignocel-lulosic degradation of the mango tree substrates by the synergistic interaction of Bacillus, Clostridium andTermitomyces were detected by Solid-state NMR. From the results, Bacillus species acted as a mutualist, byinitiating an anaerobic environment for the growth of Clostridium, for bio-hydrogen production and thepresence of Termitomyces enhanced the lignocellulosic degradation of substrates in vitro and in vivo.Thus, the synergistic collaboration of these three microbes can be used for termite-derived bio-fuel pro-cessing technology.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The biodegradation and bio-modification of lignocellulosicmaterials are necessary to elucidate the carbon recycling on theearth. Termites are considered to be the smallest and the most effi-cient natural bioreactor, among the other living organisms in theworld that are known to help in carbon circulation (Scarf et al.,2011; Velu et al., 2011). Their ability to convert recalcitrant plantbiomass to bio-fuels has drawn attention among researchers dueto the increase in the oil prices and global warming issues. Recentstudies have revealed the production of hydrogen and ethanol via

microbial fermentation as an eco-friendly and economical method-ology (Chang et al., 2008).

The termite gut contains various microorganisms that symbiot-ically co-exist with each other (Scarf et al., 2011). In lower ter-mites, it is observed that cellulosic degradation is effectivelycarried out with the help of protozoan symbionts, whereas in high-er termites, cellulosic and hemicellulosic degradation is mainly as-sisted with the cooperation of gut microbiota and the host cellulasegenes (Yang et al., 2004; Huang et al., 2008). The functions of thesegut symbionts, in both lower and higher termites have been effec-tively studied recently by metagenomic and metatranscriptomicanalyses (Breznak and Brune, 1994; Warnecke et al., 2007). Theseanalyses reveal that hindgut microbiota in higher termites consistof various endoglucanases, endoxylanases, glucosidases, nitrogen-ases and enzymes that are involved in carbon dioxide reductionand acetate production (Warnecke et al., 2007). Hydrogen acts as

338 G.M. Mathew et al. / Bioresource Technology 145 (2013) 337–344

an intermediate that links the carbohydrate fermentation withmethanogenesis and reductive acetogenesis (Wagner and Brune,1999). Reports have indicated that the daily hydrogen turnoverrates are 9–33 m3 H2 per m3 of the hindgut volume with respectto their respiratory activity (Velu et al., 2011). The paunch of thetermites is about 108 times smaller than the rumen resulting in500 times larger oxygen influx per unit volume. Owing to theirsmaller size, the transit time of the food particles through theintestinal tract is just 24 h (König, 2006). Therefore, it is easier tobio-mimic the microbial co-culturing under in vitro conditionsdue to the lesser time required for incubation.

In the macrotermitine termite Odontotermes formosanus, theplant cellulolytic degradation is facilitated by the double digestivesymbiosis between the gut symbionts, termite and the ectosymbi-otic fungi Termitomyces (Rouland-Lefèvre et al., 2006; Mathewet al., 2012). The termite excretes metabolites along with degradedplant matter form a ventilated sponge like structure known as thefungus comb. Based on their aging, they are classified as old, ma-ture and fresh combs (Rouland-Lefèvre et al., 2006). These funguscombs are inoculated with Termitomyces basidiospores that arebrought into the nest by the worker termites and develop intofruiting mushroom, which are dependent on the termites for theirsurvival (Ohkuma et al., 2001; Aanen et al., 2007). Evidently, thesebasidiomycete fungi have the unique property of degrading ligninby oxidation and act as an external rumen for plant cell wall diges-tion and fungus comb aging (Ohkuma et al., 2001).

The gut symbionts of O. formosanus are detected by cultureindependent and culture dependent methods. By RFLP analysis,gene-targeted PCR and clonal selection, Firmicutes are found tobe the most dominant phyla in the termite gut of O. formosanus,where the Clostridiales subphyla contributed to form the majorityof the species in that phyla (Shinzato et al., 2007; Mathew et al.,2012). Bacillus species isolated from the termite gut and the funguscomb of this termite are identified by culture dependent methodsand are qualitatively analyzed for lignocellulosic degradation(Mathew et al., 2012). It has been assumed that the coexistenceof anaerobic Clostridia and aerobic Bacilli, facilitated in effectivecellulosic degradation as observed in rice paddy soils (Liesacket al., 2000), water retting pond (Tamburini et al., 2003) and frombrewery yeast waste (Chang et al., 2008). It has been proven thatsyntrophic culturing of cellulolytic Bacillus and hydrogen produc-ing Clostridium using brewery yeast waste could enhance the bio-hydrogen production (Chang et al., 2008). Therefore, the co-cultur-ing of cellulolytic Bacillus and fermentative Clostridium can be con-sidered as an ideal combination of microbes for bio-hydrogenproduction.

On the other hand, Bacillus species are considered to be indus-trially important microbes because of their ability to secrete extra-cellular enzymes involved in saccharification and proteolysis andfor antibiotics production. They also act as an antagonist againstvarious plant pathogens (Chaurasia et al., 2005)and are knownfor their enhanced growth rate and ability to survive in differentenvironments. The mutualistic role of Bacillus has been studied inprotecting the termite’s fungus comb against invasive microfungilike Trichoderma harzianum (Mathew et al., 2012). The function ofBacillus in O. formosanus needs to be further investigated with re-spect to their cooperation with other microbes to promote the pro-duction of hydrogen. In this study, synergetic interaction of aerobicBacillus and anaerobic Clostridium has been monitored to under-stand the termite-fungus comb lignocellulose conversion andbio-fuel production system in vitro. For the present work, in vitrostudies involving the production of hydrogen by the co-culturesof Bacillus and Clostridium and the degradation of the mango treeresidues by co-cultures of Bacillus and Clostridium with and with-out Termitomyces were studied using in situ Solid-state nuclearmagnetic resonance (NMR) with 13C cross-polarization magic angle

spinning (13C CP/MAS). The maturation of the different layers ofthe fungus comb is also studied by Solid-state NMR to predictthe degradation of lignin and hemicelluloses by Termitomyces, un-der in vivo conditions. This is the first study to bio-mimic the ter-mite gut-fungus comb scenario in macrotermitine termites.

2. Methods

2.1. Termites and fungus comb

O. formosanus worker termites and fungus combs were collectedfrom the subterranean nests, located near the mango orchards ofNational Museum of Natural Science (24.156112� N latitude and120.666275� E longitude), Taichung, Taiwan, in June, 2009. Workertermites were surface sterilized with 70% ethanol and deguttedusing sterile forceps according to Long et al. (2010). Their gutswere homogenized, and dilution series (up to a dilution of 10�12)were spread-plated on LB medium at 37 �C under aerobic andanaerobic conditions.

2.2. Aging of the fungus combs of O. formosanus

The fungus combs that were aseptically collected and separatedas fresh, mature and old, were analyzed by CP/MAS 13C NMR(Hyodo et al., 1999) with respect to their lignocellulosic degrada-tion, in order to study the in vivo conditions of the different layersof the termite feces (fungus comb) by aging. The 13C NMR spectrawere obtained using Varian Inova 300 spectrometer operating at75.3 MHz with the following experimental parameters: pulse rep-etition time, 3.2 s; cross-polarization contact time, 1.5 ms; sweepwidth, 50 kHz and acquisition time, 40 ms. The chemical shiftswere referenced to methyl-carbon signal (17.3 ppm) ofhexamethylbenzene.

2.3. Detection of hydrogenase gene in the isolated Clostridial species

In order to identify whether the Clostridial culture containedhydrogen producing genes, the total genomic DNA was isolatedfrom the pure culture of the isolate by using the Easy Tissue andcell genomic DNA purification kit, (Genemark, USA) according tothe manufacturer’s instruction. After identifying the isolate asClostridial species, the DNA was amplified using FeFe hydrogenasecluster 2-specific primers HydA2F: 50 TGY YTA YTA TGY GGW CARTG 30 and HydA2R: 50 GCT TCC ATW ACK CCR CCD GT 30 (Calusinskaet al., 2011). Based on the result, a phylogenetic tree was con-structed using the spatial amino acid sequences MEGA 5 software(Tamura et al., 2011).

2.4. Bacterial strains and growth conditions

The Clostridium (Clos 3) and Bacillus (MGB 5) strains isolatedfrom the termite gut by culture dependent methods were used inthis study, in order to bio-mimic the termite gut environment.The bacterial DNA was identified using the Eub 968F and Univ1392R primers (Nielsen et al., 1999) and ligation and transforma-tion was done according to the protocol of Mathew et al., 2012.

In order to replicate the gut environment, the basal mediumused, comprised (per liter composition) of 2.04 g K2HPO4,0.0192 g MgSO4�7H2O, 0.008 g CaCl2�2H2O, 0.04 g KH2PO4, 0.4 gNaHCO3, 0.08 g NaCl, with a pH of 7.0. Bacillus and Clostridiumstrains were inoculated in the ratio of 1:2 (1.7 � 1014 CFU/mL:3.4 � 1014 CFU/mL) into basal medium consisting of 5 g/L peptone,5 g/L tryptone and 10 g/L yeast extract and incubated at 30 �C inserum bottles with an active volume of 150 mL, under aerobic con-ditions, to replicate the environment in the termite’s foregut and

G.M. Mathew et al. / Bioresource Technology 145 (2013) 337–344 339

midgut. Resazurin dye was added to monitor the transition fromaerobic to anaerobic conditions. After a period of 4–6 h, the med-ium changed from aerobic to anaerobic as the Bacillus species uti-lized the oxygen, thus providing an anaerobic environment for thegrowth of Clostridium. The anaerobic environment indicated themovement of regurgitated food materials through the hindgut.This experiment was monitored to know if the presence or absenceof Bacillus with the Clostridium, helped in bio-hydrogen production.

2.5. Experimental conditions

Prior to the experiments, Bacillus (MGB5) and Clostridium (Clos3) were cultivated in LB medium and PYG medium, overnight at30 �C. Their growth was monitored at an absorbance of 600 nmand the Bacillus and Clostridium were inoculated into the basalmedium containing the substrates in the ratio of 1:2.

2.6. Addition of carbon sources

Preliminary studies using various carbon sources like cellulose,humic acid, lignin, pectin, xylan, glucose and mango tree substrateswere added to the basal medium with a concentration of 1 g/100 mL, to check if hydrogen was produced under aerobic condi-tions by co-culturing Bacillus with Clostridium and also by individ-ually culturing with pure cultures of Bacillus and Clostridium.

For the preparation of the mango tree substrates, dried leavesand fallen branches of the mango tree (Magnifera indica) were col-lected and dried at 60 �C, to kill small insects and other inverte-brates. The leaves and stems were separately powdered using apulveriser (Taiwan), passed through 420 lm sieve and were mixedin the ratio of 1:1 in the following experiments. Unautoclavedmango tree residues was used as the substrate, as termites werecollected from the nests situated near the mango trees, as auto-claved mango tree substrates released furfural and hydroxymethylfurfural, which inhibited the growth of the inoculated microorgan-isms. Depending on the intake of mango substrates by the termites,different concentrations of mango powder (1 g and 3 g/100 mL)were added to the basal medium with co-cultures of Clostridiumand Bacillus and pure cultures of Bacillus and Clostridium, to studytheir effect on the hydrogen production under in vitro conditions.

2.7. Hydrogen gas determination

The bio-hydrogen production was measured by a plunger dis-placement method according to the protocol of Owen et al.,1979. The collected gas was tested for hydrogen by using GC–TCD (GC-8A gas chromatograph, Shimadzu do Brasil ComércioLtda.) equipped with a thermal conductivity detector and a2 mm � 2 mm (internal diameter) stainless steel column packedwith Porapak N (80–100 mesh). The working temperature of thecolumn, injector and detector were set at 60 �C, 100 �C and100 �C, respectively.

Table 1Microorganisms from the termite gut to the fungus comb in O. formosanus isolated by culisolates used in this study are highlighted in bold.

I stage Hydrolytic MO II stage Fermentative MO III stage AceMethanogenreductive M

Bacillus sp. yeast species likeCandida sp. Pichia sp. andDebaromyces sp.

Bacillus sp. Enterobacter, Lactococcussp. Bacteriodetes, Termitomycesconidia, Proteobacterium

ClostridiumBacteriodetes

2.8. NMR studies of the mango tree substrates with gut symbionts inthe presence and absence of Termitomyces

For the in vitro studies, the chemical constituents of the mangotree substrates were analyzed by NMR (Hyodo et al., 1999), byincubating them in the presence and absence of Termitomyces, afterco-culturing Clostridium and Bacillus for 2 days to study thechanges that occurred in the degradation of wood material, whiletraversing through the termite’s gut. After co-culturing with Clos-tridium and Bacillus to mimic the termite gut for 48 h, the environ-ment became anaerobic. The serum bottles were opened to createan aerobic environment and Termitomyces spores (2 � 107 CFU/mL)from Termitomyces isolate T-983, were inoculated to study the lig-nin and hemicellulosic degradation for 2 weeks, since the funguscomb composting was strictly an aerobic process. In order to ana-lyze the Solid-state NMR spectra, the mango tree substrates werefiltered and dried at 70 �C, for a time period of one week. Uninoc-ulated mango tree substrates were used as the control.

3. Results and discussion

3.1. Identification of the isolates by culture dependent method

Different bacterial cultures were isolated from the termite gutof O. formosanus by culture dependent methods and their potentialfunction in various stages of hydrolysis and fermentation of ligno-cellulosic substrates were compiled in Table 1. It was reported andobserved that phylum Firmicutes (Bacillales and Clostridiales) wasthe most dominant group of bacteria detected in the termite gutof the macrotermitine termite O. formosanus (Mathew et al.,2012; Shinzato et al., 2007). In the termite gut, Bacillus sp aidedin oxidative fermentation of cellulosic and hemicellulosic sub-strates (I stage and II stage) and aromatic compounds, whereasClostridial species assisted in carrying out hydrolysis of the cellu-losic substrates (I stage), carbon dioxide reduction, acetogenesisand hydrogen production in termites (III stage) (Kane et al.,1991; König, 2006). Both Clostridia and Bacillus were involved inthe hydrolysis of cellulolytic substrates (I stage), and so they wereinoculated simultaneously under aerobic conditions to mimic thegut environment, in vitro. The Bacillus MGB5 isolated and used inthis study, under aerobic conditions, was known to exhibit pectin-ase, xylanase, CMCase and lignin peroxidase activity and showedsimilarity to other soil Bacillus (Mathew et al., 2012). Hence, thisisolate was ideally used along with Clostridial strain to study thehydrogen production with respect to termite-derived bio-fuel pro-cessing technology.

Although the microecology of the termite gut of O. formosanuswas yet to be studied, it was necessary to set up a model systemto bio-mimic the termite-gut fungus comb system of this termite.Normally in wood-feeding lower termites like Coptotermes formos-anus, cellulose was effectively digested more than hemicelluloses.In O. formosanus endogenous cellulases helped in the hydrolysisof cellulose which was located in the midgut of the termite

ture dependent and independent methods are grouped according to König, 2006. The

togenic,ic Sulfur

O

IV stage Hydrolytic Acetogenic, MethanogenicSulfur reductive MO

sp. Fungi like Termitomyces, Minor fungi like, Trichoderma sp.Pestalotiopsis Yeast species like Candida and Pichia Bacterial specieslike Bacillus cereus, Bacillus sp. E. coli Clostridium species,Bacteriodetes and Planctomycetes

340 G.M. Mathew et al. / Bioresource Technology 145 (2013) 337–344

(Katsumata et al., 2007). In the wood-feeding termites, polysaccha-ride hydrolysis was greater in the midgut of the termite than in therumen. In order to study the biodegradability of the wood sub-strates, powdered mango tree substrates were used to bio-mimicthe mechanical grinding of the wood by the mandibles and the giz-zard (foregut); thereby, increasing the surface area of these ligno-cellulosic substrates for enzyme saccharification by the termite(Ke et al., 2012). The foregut and the midgut regions of the termitewere aerobic in nature whereas most parts of the hindgut acted asanoxic fermentors. From this study and previous studies, the Clos-tridia were capable of producing hydrogen, which was detected inthe central part of the hindgut, which was anaerobic in nature.Therefore in current research, there was a shift from an aerobicsystem to an anaerobic system to mimic the termite gut whichwas attained by the co-culturing of Bacillus and Clostridium. In or-der to depict the fungus comb degradation, the serum bottles weretreated with oxygen and Termitomyces was inoculated along withthe co-cultures of Bacillus and Clostridium. Earlier, it was observedby Ke et al., 2010, that the process of lignin degradation and mod-ification in the whole gut passage of the lower termite, C. formos-anus, required the presence of oxygen. Exposure of the lignincompounds to oxygen in the presence of Termitomyces mycosym-biont initiated lignin degradation. To develop the environmentalconditions in the termite gut, the temperature in the study wasmaintained as 30 �C, as the temperature of the macrotermitinemound and the optimum temperature required for the growth ofTermitomyces (Kobe, 2011; Rouland-Lefèvre et al., 2006). SinceTermitomyces was regarded as the major nutrient for the macroter-mitine termite, this temperature was used throughout experiment.The pH of the medium used in this study was 7.0, as the pH rangein the fungus-growing termite gut varied from 6.5 to 7.5 and theiralimentary canal was structurally similar to that of the lower ter-mites (Bignell and Eggleton, 1995).

3.2. Chemical composition of the fungus comb on the basis of theiraging (in vivo conditions)

Since the fungus combs of the macrotermitine termites weredifferentiated as three layers, based on their maturation; as fresh,mature and old, it was necessary to study which layer was effi-ciently involved in the degradation of lignocellulosic compoundsby Termitomyces, which enhanced their aging. The presence ofthese Termitomyces species was detected on the fungus comb ofO. formosanus using Termitomyces specific primers (Aanen et al.,2007). CP/MAS 13C NMR was done to characterize the conversionof lignocellulosic compounds present in the different layers ofthe fungus comb. The samples were collected individually fromthe fresh, mature and old comb and their NMR results showed thatthe chemical composition of the different layers of the funguscomb were similar to the fungus comb in Macrotermes gilvus(Hyodo et al., 1999).

Biochemical transformation of lignocellulosic compounds wasobserved as the fungus comb ripened. See Supplementary informa-tion, Fig. S1, showed that as the fungus comb aged, there was asharp decrease in the signal at 72.2 ppm and 75 ppm due to thebreakdown of cellulosic and hemicellulosic moieties. The intensityof the peak signals at 80–86 ppm, corresponding to C4 non crystal-line cellulose showed a prominent decrease. The signal at 56 ppmcorresponding to aryl methoxyl carbons of lignin (coding forsyringyl and guaiacyl units) reduced with aging. Also, there wasa reduction in the nitrile signal (105 ppm) and more number of sig-nals associated with lignin compounds in the fresh comb (peak sig-nals ranging from 149 to 173 ppm) than in the old comb. Evidently,there was a decrease in the carbonyl groups of lignin in the oldercomb than in the fresh comb. As a result of degradation, more sig-nals associated with alkyl C (signals ranging from 16 ppm to

33 ppm) were observed in the old comb than in the fresh and ma-ture comb of O. formosanus.

The fungus combs of macrotermitine termites were rich inhumus, which contained humin, humic acid and fulvic acid. InM. gilvus, the CP/MAS 13C NMR of the fungus comb, based on theirdifferent ages and degrees of maturation proved that the lignindegradation took place progressively in the fungus comb (Hyodoet al., 1999). Also, the various organic fractions obtained from thefungus comb of Macrotermes michaelseni were characterized by13C NMR and found to contain paraffins, proteinaceous com-pounds, carbohydrates, aromatics and carboxylic groups (Arshadet al., 1987). Similar research was conducted in the wood termiteMicrocerotermes parvus and soil feeding termite Thoracotermesmacrothorax using Solid-state 13C NMR to investigate the transfor-mations and biodegradation of their food in the gut and feces(Hopkins et al., 1998). Previous research showed that, the workertermites being humivorous in nature, utilized the oldest part ofthe fungus comb (Ohkuma et al., 2001). Therefore, there was lignindegradation and a drastic reduction of polysaccharide residues;also possibly because other microbes and Termitomyces in the olderfungus comb helped in the enhanced degradation of lignocellulosiccompounds.

3.3. Detection of hydrogenase gene in the Clostridial species (Clos 3)

In order to identify the bacteria, the pure culture of the isolateClos 3 was amplified using universal primers Eub 968F and Univ1392R (Nielsen et al., 1999) and showed similarity to Clostridiumbifermentans (HQ013322). The isolate Clos 3 belonged to Group AClostridial [FeFe] hydrogenases, which was identified using Clos-tridial hydrogenases specific primers (Calusinska et al., 2011). Itwas observed that the partial amino acid sequence of Clostridiumsp Clos 3 showed similarity to Clostridium perfrigens(ZP_02631057.1) using BLASTX. A phylogenetic tree was con-structed using partial amino acid sequences with MEGA5 program(Fig. 1) and the results confirmed that the Clostridial strain Clos 3belonged to Cluster A2 hydrogenases (Tamura et al., 2011).

According to previous research, Clostridial species were foundin the intestine of rumen, humans, animals and insects and werepredominant in the hindgut of the termites, both lower and highertermites. A novel H2/CO2 acetogenic bacterium Clostridium mayom-bei sp. nov. was isolated from the gut of African soil feeding ter-mite, Cubitermes speciosus (Kane et al., 1991). Also, sequenceanalysis revealed that the Clostridial species dominated the P1 seg-ments of higher termites like Termes comis, Pericapritermes latigna-thus, Microcerotermes and Speculitermes sp. with their gutenvironment having the capacity to withstand higher concentra-tions of hydrogen than other biological systems. In situ studieswere also performed to study the hydrogen turnover and intestinaldistribution of hydrogen in lower and higher termites (Pester andBrune, 2007).

3.4. Hydrogen production using various substrates

According to the co-culture studies by Chang et al., 2008, it wasobserved in that higher population of Bacillus species consumedthe carbon sources required for the growth of Clostridium, thereforea lower proportion of Bacillus was preferred for the co-culturestudies. Also, it was noted that among the Firmicutes present inthe termite gut of O. formosanus, Clostridial sp constituted a higherratio than Bacillus sp (Shinzato et al., 2007). Therefore, in the cur-rent research, the concentration of Bacillus to Clostridium was usedas 1:2.

For the preliminary studies, Clostridium was inoculated in ser-um bottles in the presence and absence of Bacillus with differentcarbon substrates under aerobic initiation conditions. Pure

C. kluyveri (YP 001395640.1)

C. tyrobutyricum (ACI42788.1)

C. acetobutylicum (NP 346675.1)

C. cellulovorans (YP 003845523.1)

C. saccharobutylicum(AAA85785.1)

C. butyricum (ZP 02951233.1)

C. botulinum (ZP 02621445.1)

C. novyi (YP 878332.1)

Clostridium (Clos 3)Clostridium (Clos 3)

C. perfringens(ZP 02631057.1)100

98

95

94

80

94

51

0.1

Fig. 1. Neighbor joining phylogenetic tree, specific for Clostridial Fe Fe Cluster A2 hydrogenases based on partial amino acid alignments. The partial [FeFe] hydrogenase geneamplified in this study is shown in bold type and designated as Clos 3.

G.M. Mathew et al. / Bioresource Technology 145 (2013) 337–344 341

cultures of Clostridium and Bacillus were not able to produce hydro-gen under aerobic conditions, whereas their mixed cultures pro-duced hydrogen, under aerobic conditions using the basalmedium (data not shown). A concentration of 1 g/100 mL of thecarbon substrates was added to the basal medium, to test theirhydrogen production. From the co-culture studies, after 12 h, thehydrogen accumulated was 2.79 mmol/mL using glucose as thesubstrate and increased to a maximum yield of 4.08 mmol/mL after18 h (Fig. 2). The maximum hydrogen production using the unau-toclaved mango tree substrates, cellulose and humic acid was1.605 mmol/mL, 1.42 mmol/mL and 0.706 mmol/mL, respectively,and was observed after 12 h incubation. Hemicellulose like xylanproduced the maximum hydrogen of 0.395 mmol/mL after 18 h,where as pectin could produce 1.779 mmol/mL of hydrogen after36 h of incubation, suggesting that pectin had to be degraded to

Time in hours

Cum

ulat

ive

Hyd

roge

n pr

oduc

tion

in m

mol

/mL

Fig. 2. Hydrogen production of co-cultures of Bacillus and Clostridium in

simple sugars in order to be directly converted to hydrogen bythe Clostridium. The reason for the reduction in the H2 peak after12 h was due to substrate utilization by the microorganisms, whichresulted in the substrate depletion.

Apparently, it was observed that the Bacillus species could func-tion as an oxygen consumer by changing the medium’s environ-ment from aerobic to anaerobic in 6 h, and which was visualizedby using the anaerobic indicator, resazurin. Since they were co-cul-tured with Clostridium species, they were able to convert the sim-ple as well as the complex sugars to hydrogen only after ananaerobic environment was attained, although they appeared tobe dormant during the aerobic phase. It was noticed that maxi-mum production of the hydrogen usually occurred after 12 h,showing the probable time required for the food particles to reachthe termite’s hindgut before expulsion. The current experiments

Glucose

Pectin

Xylan

Lignin

Cellulose

Humic acid

Mango tree substrates

the presence of various carbon substrates, under aerobic conditions.

342 G.M. Mathew et al. / Bioresource Technology 145 (2013) 337–344

were monitored only for 48 h, as the feces were to be expelled outof the termite’s gut within 24 h (König, 2006). It was noted thatClostridial and Bacillus species exited via the termite gut along withthe termite feces to deposit on the fungus comb; as there weresimilarities in the termite gut and fungus comb Clostridia (Mathewet al., 2012). Apparently from the previous studies, it was seen thatthe Bacillus species found on the fungus comb formed an entirelydifferent clade compared to the Bacillus isolated from the termitegut of other lower and higher termites (unpublished data), showedthat the fungus comb Bacillus were acquired from their surround-ings. As Bacillus species assisted in creating an anoxic environmentby utilizing the oxygen, it could implicate to function as a mutual-ist by supporting the growth of Clostridial species for hydrogenproduction in the termite’s gut. From the studies, it was observedthat co-cultures of Clostridium and Bacillus using glucose as sub-strate could produce the maximum yield of hydrogen (Fig. 2), thusproposing that the glucose moieties produced by the degradationof cellulosic and hemicellulosic substances by the Bacillus wouldbe directly used up by the Clostridium to produce hydrogen. Hence,it can be proposed that the synergistic association of these mi-crobes could help in production of bio-fuels at an enhanced rate.

Lignin and humic acid were formed by plant microbial degrada-tion and breakdown of the fungus comb of termites (Arshad et al.,1987); therefore, they were used as carbon sources to study thehydrogen production. The worker termites fed on the parts of thefungus comb and nodules, thus ingesting humic acid in their ter-mite gut (Ohkuma et al., 2001) and lignin was ingested in thegut as the termites fed on woody plant based substrates. Pectinwas relatively less prominent in the woody plant biomass thancompared to cellulose and hemicelluloses. Therefore, it was foundto be quantitatively less in the termite’s gut, considering their foodhabits. Different concentrations of unautoclaved mango tree resi-dues were used as the substrate, to study the effect on hydrogenproduction under in vitro conditions; to mimic the consumptionof mango tree substrates by the termites (Fig. 3). In this experi-ment, co-cultures of Clostridia and Bacilli were inoculated alongwith the unautoclaved mango wood substrates. From the overallstudy, co-cultures of Clostridium and Bacillus produced maximumhydrogen than inoculating the mango tree substrates with purecultures of Clostridium or Bacillus species alone. Maximum produc-tion of hydrogen (1.707 mmol/mL) was produced using 3% of man-go tree substrates, co-cultured with Bacillus and Clostridium.

Time in hours

Cum

ulat

ive

hydr

ogen

pro

duct

ion

in m

mol

/ mL Ma

MaMMaMaM

Fig. 3. Hydrogen production using different concentrations of unautoclaved mango trecultures of Clostridium and Bacillus.

Hydrogen production was detected even when pure culture ofBacillus (MGB5), was inoculated into the unautoclaved mango treesubstrates. This was probably due to the presence of inherent Clos-tridial and Bacillus species in the unautoclaved mango tree sub-strates that could be detected using Clostridium specific primers(Calusinska et al., 2011) and Bacillus specific primers (Wu et al.,2006).

3.5. Chemical composition of mango tree substrates under in vitroconditions

Degradation of the mango tree substrates by co-cultures of Clos-tridium and Bacillus, in the presence and absence of Termitomyceswere monitored by Solid-state NMR (See Supplementary informa-tion, Fig. S2 and Table S1), to check the in vitro scenario in the ter-mite’s gut on consumption of mango wood. Compared to thecontrol mango tree substrates, there was a decrease in the peaksignals (1–11) of the mango substrates co-cultured with Clostrid-ium and Bacillus, inoculated with and without Termitomyces. Itwas noted that in the control samples, the peak signals from 18to 38 ppm were lower before the inoculation of the microbes andslightly increased with the inoculation of Clostridium and Bacillus.After 48 h, hemicellulosic and cellulosic degradation were initiallyobserved by co-culturing with Clostridium and Bacillus without Ter-mitomyces. Before the addition of Termitomyces, there was a de-crease in OMe of hemicellulose and lignin (56.3 ppm) and C4 noncrystalline cellulose (80–86 ppm). It was observed that after theaddition of Termitomyces culture, there was a slight decrease inthe peak signals 10 (152 ppm) and 9 (143 ppm), which encodedfor C5 lignin with OMe (guaiacyl and syringyl units of lignin) andC5 lignin without OMe and also drastic reduction was observedin peak 4 (72.2 ppm) and peak 5 (75 ppm), which encoded for cel-lulosic and hemicellulosic degradation. Thus, it clearly showed thatTermitomyces contributed mainly in the degradation of lignin andalso hemicelluloses, which could correlate with the aging of thefungus comb. The lignocellulosic degradation of the Termitomycesstrain (T-983) used in this study was qualitatively assayed, to con-firm their function (See Supplementary information, Fig. S3).

By parallel comparison, the degradation of the mango tree sub-strates and fungus comb aging showed a similar pattern in thereduction of lignin and hemicellulosic peak signals (56 ppm andbetween 143 and 152 ppm) and weakened peak signals 72.2 ppm

ngo tree substrates containing Clostridiumngo tree substrates containing Bacillus

ango tree substrates containing CBngo tree substrates containing Clostridiumngo tree substrates containing Bacillus ango tree substrates containing CB

e substrates with co-cultures of Clostridium and Bacillus (marked as CB) and pure

Lignocellulose

Glucose

H2 + CO2 (Bio-fuels production)

Gut symbionts , host cellulase genes and chewing of wood particles by termites

Hydrolytic Bacillus ,Clostridial species and Termitomyces

Lignin

Simple forms of aromatic compounds

Termitomyces

Cellulose Hemicellulose

Carbon recycling andIndustrial applications

Oxidative/fermentative Clostridial species

Fig. 4. Flowchart for the functional role and industrial applications of the macrotermitine termite gut and fungus comb symbionts.

G.M. Mathew et al. / Bioresource Technology 145 (2013) 337–344 343

and 75 ppm encoding cellulosic and hemicellulosic moieties.Hyodo et al., 1999, studied the effect of lignin degradation on thepassage of wood particles through the termite gut of C. formosanusby Solid-state CP/MAS 13C NMR. A recent study showed that mod-ification of the lignin moieties and partial degradation of the carbo-hydrates in the softwood occurred with the physical chewing ofthe wood material by C. formosanus (Ke et al., 2012). From theirstudy, a rapid release of C6 sugars occurred within the first 12 hof hydrolysis. Therefore, it was important to study the effect ofthe bacterial strains on various hemicellulosic, cellulosic and ligninsubstrates that pass through the termite’s gut. It was found outthat the termite’s mechanical chewing increased the hydrolysablecapacity of lignin, by cleavage of specific bonds from the lignin net-work, thus increasing the release of cellulose.

In this research, mango tree substrates degradation by NMRgave a new insight into the mode of action in termite’s complexlignocellulolytic system (See Supplementary information, Fig. S2),to detect the chemical changes occurring in the lignocellulosic bio-mass and gave a pattern of the various components present in thewood, before and after microbial degradation. Therefore, mangotree substrates were used as an ideal energy crop because of itsperennial nature and distribution throughout Taiwan and also be-cause the termites and fungus comb samples used in this studywere collected from the subterranean nest, situated near the man-go orchards. Being a hardwood, mango tree consisted of lignin withsyringyl and guaiacyl units in contrast to softwood lignin contain-ing mostly p-hydroxyphenyl and guaiacyl units. Evidence for in-creased level of guaiacyl derived moieties by CP/MAS 13C NMRspectroscopic analysis revealed cellulose degradation (Ke et al.,2011), followed by the release of lignin molecules from the plantsubstrates. Depolymerization and demethylation of the ring meth-oxyl group and hydroxylation of aromatic rings of lignin were de-tected in the food particles present in the gut of the lowerdampwood termite Zootermopsis augusticollis (Geib et al., 2008)just as detected in the degraded mango tree substrates inoculatedin the presence and absence of Termitomyces. The feces ofC. formosanus termites were studied by CP/MAS 13C NMR spectro-scopic analysis to study the lignocellulosic mechanism for break-down of carbohydrates under in situ conditions (Hyodo et al.,1999; Ke et al., 2011). The in vitro and in vivo degradation capabil-ity of lignin and other related aromatic compounds were studied inlower termites like Mastotermes darwiniensis and Reticulitermessantonensis and higher termites like Nasutitermes nigriceps(Kuhnigk and König, 1997; Katsumata et al., 2007).

The overall cellulosic and hemicellulosic degradation was ob-served by decrease in the chemical resonance signals between

75 ppm and 73 ppm, which corresponded to C2, C3 and C5 sugarcarbon resonances and also slight decrease in C1, C4 and C6 whichshowed signals at 102.4, 80–86 and 62.3–64 ppm, respectively.Reduction of the peaks corresponding to C5 lignin with OMe(152 ppm) and C5 lignin without OMe (143 ppm) were also ob-served in the mango tree substrates inoculated with Clostridium,Bacillus and Termitomyces. Termites digested the hemicellulosicand cellulosic moieties of the wood materials effectively, usingtheir gut symbionts, whereas Termitomyces was specifically re-quired for lignin degradation, although it degraded hemicelluloses.Thus, the results suggested that Termitomyces assisted in the lignindegradation, hemicellulosic digestion of the mango tree substratesand helped in the aging of the fungus combs of O. formosanus. Thiswas the first study to bio-mimic the termite gut-fungus comb sce-nario in macrotermitine termites. The combined action of thesethree microbes in lignocellulosic degradation and their role inindustrial applications was highlighted in Fig. 4. The synergisticscenario of Bacillus, Clostridium and Termitomyces in the termitegut-fungus comb of O. formosanus, could be further applied forthe development of bio-industrial processes.

4. Conclusion

To conclude, this study showed that the termite gut Bacillus ofO. formosanus acted as a mutualist by creating an anaerobic condi-tion in the termite’s gut for the survival of Clostridium species andtheir lignocellulosic degradation pattern was observed underin vitro and in vivo conditions in the presence and absence of Ter-mitomyces. Thus, the termite gut-fungus comb scenario of the mac-rotermitine termite O. formosanus was bio-mimicked by threemicrobes and this process may be envisioned to produce lignocel-lulosic bio-energy in the future.

.

Acknowledgements

The authors wish to thank Dr. Yu-Ming Ju for providing the Ter-mitomyces isolate (T-983), Dr. Chi-Yung Lai for his valuable sugges-tions on cultivation of Odontotermes formosanus, by the laboratoryconditions and Dr. Cheng-Yu Ho and Sheng-Ping Yang for theirtechnical assistance in this research. The authors would also liketo thank the anonymous reviewers for their valuable suggestionsfor the betterment of this paper. This work is financially supportedby the IPMB Summit Team Project, Academia Sinica, Taiwan andthe Ministry of Education, Taiwan R.O.C, under the ATU plan.

344 G.M. Mathew et al. / Bioresource Technology 145 (2013) 337–344

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2012.12.055.

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