Int. J. Biosci. 2013 International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print) 2222-5234 (Online) http://www.innspub.net Vol. 3, No. 4, p. 27-35, 2013 RESEARCH PAPER OPEN ACCESS Screening of fermentative symbiotic microorganisms from digestive tract of Macrotermes subhyalinus and Macrotermes bellicosus D.T. Yoro1, NK Kouassi2, S. Dabonné3*, LP Kouamé3, R. Koffi-Nevry1 1Laboratoire de Biotechnologie et de Microbiologie des Aliments de l’Université d’Abobo-Adjamé (Abidjan, Côte d’Ivoire), 02 BP 801 Abidjan 02, Côte d’Ivoire 2Centre National de Recherche AgronomiquesKM17 Route de Dabou / 01 BP 1740 Abidjan 01 (Côte d'Ivoire 3Laboratoire de Biocatalyse et des Bioprocédés de l’Université d’Abobo-Adjamé (Abidjan, Côte d’Ivoire), 02 BP 801 Abidjan 02, Côte d’Ivoire Key words: Fermentative microflora, lactic bacteria, Macrotermes bellicosus, Macrotermes subhyalinus, Symbiotic microorganisms. doi: http://dx.doi.org/10.12692/ijb/3.4.27-35 Article published on April 22, 2013 Abstract Diversity of fermentative microorganisms in the digestive tract of Macrotermes bellicosus and Macrotermes subhyalinus, two species of tropical termites was investigated. Morphological, phenotypic and biochemical parameters were used for this preliminary study. Positive Gram bacteria were revealed on Man Rogosa Sharpe agar MRS and Chalmers agar while sabouraud chloramphenicol agar showed yeasts. Cultural techniques added to the use of API 50 CHL, had allowed the identification of lactic acid bacteria strains of the genera Lactobacillus, Lactococcus and Pediococcus. The API 20 CAUX revealed presence Candida and Cryptococcus only for M. bellicosus worker. These first results highlight the presence of a variety of fermentative microorganisms in the midgut of the two termites. * Corresponding Author: S. Dabonné [email protected] 27 Yoro et al. Int. J. Biosci. 2013 Introduction relationship between termites and microorganisms With earthworms and ants, termites are one of the involving (protozoa, bacteria, fungi) (Abo-Khatwa, most important representatives of the group of 1978 ; Brauman et al., 2001).Various organisms which activity significantly modify microorganisms are present in the midgut of all environment (Lavelle et al., 1997; Lavelle and Spain, termites (Brauman et al., 2001) with a density of 106- 2001). The termite’s nets are regarded as the volume 107 cells per µl of intestinal content (Brauman et al., of soil and organic resources affected by termites, 2001). either directly or through their mandatory or voluntary associations with microorganisms. The We here focused on the microbial diversity of M. flora of the digestive tract, their composition, subhyalinus and M. bellicosus. It is now known that variability, and their reciprocal interactions with they are involved in the complex process of these their host, make a digestive system that is still not insects feeding. Due to the diet of termites they could well known. Termites are social insects that live in an therefore be interesting sources of microorganisms organization structured into families with castes of for food process and biotechnological use. This study small soldiers, grand soldiers and workers. They aims to highlight the diversity of the fermentative belong to the order of Isoptera. They are arthropods microbial population. of the insect class known as "white ants" (Klambhampati and Eggleton, 2000). Termites live Materials and methods in nests built in warm regions (Rouland, 1994). Sampling Termites M. subhyalinus and M. bellicosus identified Because of their social life and their distribution over by an entomologist were from the savannah of Lamto the globe, termites represent the largest biomass of (Toumodi, Côte d’Ivoire). Twenty (20) insects of soils in tropical Africa (Woods and Sands, 1978). each caste (worker, small and grand soldier) were They are also one of the mayor components of the collected without damaging the nets. tropical and subtropical savannas and forests ecosystem from southeastern Asia and Africa. Their Isolation and enumeration of microorganisms diet and their impact on physico-chemical properties Twenty (20) insects from each caste (worker, small of the soil (Woods and Sands, 1978 ; Bignell and and big soldier) were separately sterilized in 70% Eggleton, 2000 ; Brauman et al., 2000) play a very ethanol (w/v) and washed with sterile water. They important role in the recycling of organic matter and were then dissected under aseptic conditions using a formation of humus (Boyer, 1973 ; Bachelier, 1978). stainless steel blade and a sterile forceps to extract So they have an important ecological and economic the contents of the digestive tract. Homogeneous influence. Previous studies have shown that termites mixing of the contents of the digestive tract of twenty are able to use substrates hemicelluloses without the insects was investigated for symbiotic help of their symbiotic microflora (Rouland and microorganisms. Lactic acid bacteria, yeast and mold Lenoir-Labe, 1998). Termite’s osidases therefore are were isolated on nutritive and selective broths using varied. These enzymes are produced either by the classic bacteriology methods (Bahiru et al., 2006). 9 termite itself or by its symbiotic microorganism mL of a sterile physiological water solution (AES (Rouland 1986 ; Matoub, 1993 ; Mackenzie et al., Laboratory, COMBOURG France) was added to 1 ml 2007). of every sample. Serial dilutions (10-1-10-8) are performed and 0.1 mL aliquot of the appropriate The enzymatic equipment from several species of dilution is directly inoculated in triplicate on the African termites with different diets showed that following media. most of these insects are capable of digesting various compounds of plant material. It is due to a symbiotic 28 Yoro et al. Int. J. Biosci. 2013 Man Rogosa Sharpe agar (Laboratory CONDA, 0.5, 5 and 10% (w/v) NaCl. Only the isolates positive spain) (De Man et al., 1960): (AFNOR, NF ISO Gram, catalase and negative oxydase have been 15214), incubated anaerobically at 37°C for 48 h for considered as being potential lactic bacteria and have enumeration of des Lactobacillus and Pédiococcus; been kept at -80°C in the MRS broth (Laboratory Bile esculine Azide agar (BEA, ISO 7899/1) CONDA, Spain) in 20% of glycerol for the ulterior incubated at 37°C for 48 h for enumeration of studies. Enterococcus; Chalmers agar incubated at 37°C for 48 h enumeration of Lactococcus; Sabouraud- Identification of the Lactobacillus Chloramphenicol agar (Laboratory CONDA, spain) Strains isolated on MRS were identified using the (Norme NF ISO 6611) incubated at 30°C for 2-5 days API 50CH galleries and API 50 CHL medium for enumeration of yeasts; Potato Dextrose Agar (bioMerieux, l’étoile, France). Tests were performed (PDA), (Laboratory CONDA, Spain) incubated 25°C according to the manufacturer’s instructions. at 2-6 days for enumeration of molds. Characterization of Yeasts The colonies have been counted on the basis of some Yeasts structures were directly observed on agar- morphological and biochemical criteria Results were agar under the objective (x 40) of the microscope expressed by means of ± SD. (color of the colonies, after incubation at 27°C for 24 h to 48 hours. Yeasts Gram coloration, shape, size of the cells, no selected on Sabouraud-Chloramphenicol were sporulation, cytochrom oxydase and catalase for the identified through the system API bioMérieux using lactic bacteria. Production of catalase and budding the galleries API 20 C AUX (bioMerieux, l’étoile, for the Yeasts). France). Tests were performed according to the manufacturer’s instructions. Phenotypic characterization of lactic acid bacteria Presumptive lactic acid bacteria strains isolated from Results different media were assigned to a genus on the basis Enumeration of the fermentative flora of keys characteristics and tests (Kostinek et al., Enumeration of the fermentative flora is resumed in 2005 ; Ricciardi et al., 2005 et Bahiru et al 2006). table 1 for M. Subhyalinus and Table 2 for M. Morphological and arrangement of cells were Bellicosus. Lactobacillus and Pediococcus were the examined by microscopy. Growth at 10, 45 and 51°C most frequent in both species of termite. Pediococcus in MRS broth was monitored by spectrophotometer was lower in little soldier of M. subhyalinus. Yeasts at 600 nm determinate after 1-3 days of incubation. were isolated only in the workers of M. subhyalinus. Gas production from glucose was assessed in MRS No Enterococcus or mould was observed. broth containing Durham tubes. The ability to grow at different pH (3.0, 6.5 and 9.0) was tested. The salt tolerance was evaluated using MRS broth containing Table 1. Fermentative microflora of the intestinal tract of M. subhyalinus (cfu/mL). Genus Lactobacillus Pediococcus Enterococcus Lactococcus Yeasts Mould Castes worker 2.8 107± 0.7 107 4.2 104±1.2 104 0 3.7 106±0.84 106 2.1 105± 0.21 105 0 Small 3.1 104±0.92 104 1.3 102±0.84 102 0 3.104±0.35 104 0 0 soldier Big 2.5 104±0.77 104 3.2 103±0.49 103 0 2,8.102±0.49 102 0 0 Soldier Each value is an average e of three replicate, Values are mean ± standard deviation. 29 Yoro et al. Int. J. Biosci. 2013 Phenotypic characterization and identification observed but not Pediococcus and Lactococcus. At Identification of lactic bacteria 51°C with 10% NaCl, there was no growth at all. The Fermentation and physiological profiles (Table 3) majority of heterofermentative strains are observed, showed that only the presumptive Lactobacillus as Lactobacilli that ferment hexoses lactate, acetate produced gas, so they are heterofermentative. (or ethanol) and CO2, suggesting that these Lactococcus and pediococcus not producing gas are microorganisms are capable of converting glucose to homofermentative. No Lactococcus was observed at lactate, acetate or ethanol and CO2. Indeed termites 45°C while Lactobacillus and Pediococcus growth at can effectively hydrolyze cellulose using their 10°C. Between pH 3.0 and pH 9.0 Lactobacillus was intestinal microflora. Table 2.