Diversity Analysis Of The Culturable Anaerobic And Facultative Anaerobic Bacteria From Different Intestinal Compartments Of Nasutitermes Hainanensis And Characterization Of Two Lactococcus Strains

Wood-feeding higher termites are the natural decomposers that can degrade lignocellulose effectively. The ability to degrade lignocelluloses is associated with symbiotic bacteria inhabited the intestinal tract of termites. The different physiological conditions in different compartments of termite gut, such as pH, oxygen partial pressure and hydrogen partial pressure also affect the population structure and spatial distribution of symbiotic microorganisms. A lot of symbiotic microorganisms inhabit the enlarged paunch with nearly anaerobic environment, where lignocellulose is digested. Therefore, the isolation and characterization of anaerobic and facultative anaerobic bacteria will help us to understand the metabolic mechanisms of wood-feeding termites. In this paper, we studied the diversity of culturable anaerobic and facultative anaerobic bacteria in different intestinal compartments of Nasutitermes hainanensis and characterized two new Lactococcus isolates with polyphasic approach, which makes great significance to further elucidation of the symbiotic relationship between the intestinal microbiota and termites. The main results of this study are as follows:1. The diversity of culturable anaerobic and facultative anaerobic bacteria from the gut of N. hainanensisA total of 342 anaerobic and facultative anaerobic bacteria were isolated from six intestinal compartments:the crop, midgut and hindgut P1, P3, P4, P5 using EG and AM media with different pH designed according to the difference of physiochemical conditions. Based on morphological characteristics and 16S rRNA genes sequences analysis, these 342 bacterial strains belong to 16 distinct bacterial genera (Lactococcus, Clostridium, Leuconostoc, Bacillus, Cellulomonas, Enterobacter, Citrobacter, Serratia etc). Among these isolates, Lactococcus (43.6%), Clostridium (19%), Citrobacter (7.0%) and Bacillus (10.8%) were the most dominant ones.Comparative studies showed that the amount, species and dominant culturable bacterial groups isolated from different gut compartments were obviously distinct. The dominant bacteria were Lactococcus in the crop and midgut with the proportions of 83.0% and 68.9%, respectively. The proportion of isolated Lactococcus significantly reduced in the hindgut, whereas the proportion of isolated Clostridium and Leuconostoc increased and became the dominant groups. The analysis of the diversity of cultivable bacteria showed that:there were 3 and 6 bacterial genera isolated from the crop and the midgut, whereas bacteria belonging to 10,7,8 and 9 genera were isolated from the hindgut P1, P3, P4 and P5 compartment, respectively. The results indicated that the diversity of anaerobic and facultative anaerobic bacteria in the hindgut was significantly higher than that in the foregut and midgut.2. The characterization of two Lactococcus strains isolated from the gut of N. hainanensis with a polyphasic approachCells of Strain M15 are Gram-staining-positive, short rods, catalase- and oxidase-negative, non-motile, facultative anaerobic and grow well on MRS agar at 30?. Colonies on MRS agar plates are milky white, opaque and with a diameter about 0.2-0.3 mm. Growth occurs at 10-40? (optimum,30?), at pH 4.0-9.0 (optimum, pH 7.0). Strain M15 can not grow at NaCl concentrations of 3% (w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M15 was affiliated with members of the genus Lactococcus, with sequence similarities ranging from 84.5 to 88.8%. The closest relatives was Lactococcus fujiensis JCM 16395T with sequence similarity of 88.8%. Acid is produced from ribose, galactose, D-glucose and so on. The strain is positive for acid phosphatase and esterase in API ZYM tests. The major fatty acids were Ci4:0 (12.6%), Ci6:o (34.8%), cyclo-C19:0?8c (17.6%), C18:1?9c (15.5%). The DNA G+C content is 40.6 mol%. Most of the characteristics were accordant with the other members of the genus Lactococcus. However, strain Ml5 were different in the utilization of carbon sources such as xylose, lactose, starch and in the composition and proportion of cellular fatty acids. Based on the above data presented, strain Ml5 represents a novel species of the genus Lactococcus.Cells of Strain Ml9 are Gram-staining-positive, coccoid or ovoid-shaped, catalase-and oxidase- negative, non-motile, facultative anaerobic and grow well on MRS agar at 30 ?. Colonies on MRS agar plates are light yellow, opaque and with a diameter about 0.2 mm. Growth occurs at 10-45? and at pH 5.0-7.5. Cells can not grow at NaCl concentrations of 3% (w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M19T was related to members of the genus Lactococcus, with sequence similarities ranging from 84.8% to 95.5%. The closest relatives were L. fujiensis JCM 16395T with sequence similarity of 95.5%. Acid is produced from ribose, galactose, D-glucose and so on. The strain is positive for acid phosphatase and esterase in API ZYM tests. The major fatty acids were C16:0 (43.6%), cyclo-C19:0?8c (13.1%), C18:1?9c (9.1%), summed feature 7 (10.1%) and summed feature 8 (9.5%). The DNA G+C content is 39.6 mol%. Most of the characteristics were accordant with the other members of the genus Lactococcus. However, strain Ml 9 were different in the utilization of carbon sources such as xylose, lactose, potassium gluconate and in the composition and proportion of cellular fatty acids. Based on the above data presented, strain M19 represents a novel species of the genus Lactococcus, for which the name Lactococcus nasutitermitis sp. nov. is proposed.