تعیین دما و pH بهینه رشد باکتری‌های تجزیه کننده مواد لیگنوسلولزی جدا شده از دستگاه گوارش موریانه‌ و اثر آن‌ها بر قابلیت هضم و فراسنجه‌های تخمیر آزمایشگاهی برخی پسماندهای کشاورزی

Introduction: There is a shortage of animal feed and water resources in several developing countries. Thus, proper use of agro industrial by products is a useful means to overcome this problem. Agricultural by products can use as animal feeds, but because of high lignin and low protein content, their digestibility and palatability is low. Several processing method has been used to improving nutritive value of such materials (Rahal et al. 1997 and Fazaeli et al. 2004). Recently, biological delignification of lignocellulosic material has been considered as an alternative approach (Fazaeli et al. 2004). Termites gut contains different kinds of lignocellulose degrading microbes, which the possibility of their use in improving the nutritive value of agricultural by-products is a major step in compensation of animal feed shortage.

Material and methods: The present study was conducted to determine the optimum temperature and pH for growth of symbiont bacteria degrading lignocellulosic material isolated from termite gut, and to investigate their effects on the digestibility and in vitro fermentation parameters of wheat straw (WS) and date leaf (DL). For this purpose, three bacteria including Bacillus licheniformis, Ochrobactrum intermedium and Microbacterium paludicola were used,which isolated by culture of termite guts contents in mediums containing different types of lignin and lignocellulosic materials. Sterile basal media was used as a medium for isolation process and contained the following ingredients per litre: 7.0 g K₂HPO₄, 3.0 g KH₂PO₄, 1.0 g (NH₄)₂SO₄, 0.1 g MgSO₄·7H₂O (Borji 2003). Growth curves of isolated bacteria for optimum temperature and pH were obtained using nutrient broth medium. Three temperatures of 30, 37 and 40 ^oC and four pH of 5, 6, 7 and 8 were evaluated. After that, WS and DL were processed with these isolates in media 9 (M9) medium with following composition (per litre): 6.2 g Na₂HPO₄, 3.0 g KH₂PO₄, 0.5 g NaCl, 1.0 g NH₄Cl (Kato et al. 1998). After processing, residues were collected, dried and used for in vitro gas production experiment according to Makkar (2010). For this purpose, rumen liquor was collected from two ruminally fistulated cows. Animals were maintained on a 60% hay and 40% concentrate diet according to their requirements for two weeks. Rumen contents were collected from the ventral sac of rumen before morning feeding. A completely randomized design was used to determine the optimum temperature and pH for bacterial growth as well as effects of different bacteria on in vitro gas production and fermentation parameters of treated substrates. Data were analyzed by General Linear Model (GLM) procedures of SAS (SAS Institute, 2001),

Results and discussion:All of isolates were capable of grow at all of examined temperatures (i.e., 30, 37 and 40 ^oC). The optimum growth of B. licheniformis occurred at 40 ^oC, but the optimum growth of O. intermedium and M. paludicola was observed at 37 ^oC. All of isolates grew at different pH ranges (i.e., 5, 6, 7 and 8) and their optimum growth was observed at neutral pH of 7.0. At this neutral pH, highest growth for B. licheniformis, O. intermedium and M. paludicola was obtained after 30, 34 and 36 h of incubation, respectively. After processing, in both WS and DL, highest and lowest gas production and organic matter (OM) digestibility were observed by B. licheniformis and control treatment, respectively (P<0.05). In vitro dry matter (DM) digestibility, truly digested substrate, metabolizable energy, partitioning factor, pH and ammonia nitrogen concentration were not affected by the experimental treatments. Factors such as temperature, pH, atmosphere gas composition, bacterial type, composition of media culture, number of bacteria used and end products bacteria and their interaction has an important effect on growth rate of isolates (Mohan and Manuselis 1995). Similar to our results, by isolation of Bacillus sphaericus, Ochrobactrum anthropi and Enterobacter cloacae from termite gut, Borji (2003) reported that their optimum temperature and pH were observed at 25-37 ^oC and 7-8, respectively. Other researchers also reported similar results (Giroux et al. 1988 and Yang et al. 1995). Totally, obtaining optimizing pH and temperature of isolated bacteria for processing agricultural by-products can maximize their nutritive value. Improving OM digestibility and fermentation parameters in both substrates was maybe due to effect of isolates on changing lignin structure and loosening its bounds with carbohydrate compounds (VanSoet 2004). Consistent to results of present study, borji (2003) indicated that inoculation of wheat straw and barley straw with different bacteria isolated from termite gut increased their DM digestibility compared to control treatment. Increasing DM and OM digestibility and volume of gas production in WS than DL was probably because of lower lignin and higher carbohydrate content in the former (Azizi-Shotorkhoft et al. 2016).     

Conclusion:Results of present study showed that optimum growth for Ochrobactrum intermedium and Microbacterium paludicola isolated from termite gut was observed at 37 ^oC while it was 40 ^oC for Bacillus licheniformis. Optimum pH for growth of all of isolates was 7. Processing wheat straw and date leaf with bacteria, especially B. licheniformis improved rumen fermentation parameters in vitro. Research for upgrading the nutritive value of by products with ligninolytic bacteria isolated from termite gut is in infancy. Thus, further works in this field of study is needed.