RESUMO
The objective of this study was to evaluate feed intake and digestibility and ruminal characteristics of development-stage calves fed mulatto II (Brachiaria sp.) grass hay (MGH) and a protein supplement (PS) consisting of increasing levels of the parota (Enterolobium cyclocarpum) pod (PP). We used eight Swiss-zebu calves in growth stage with an average age of 11 months and initial average weight of 157.6 ± 8.5 kg. They were distributed in a repeated 4 × 4 Latin square design with 4 treatments (period 30 days): 0% (PP0), 25% (PP25), 50% (PP50), and 75% (PP75) of the PP. Calves in the PP0 and PP25 treatments had higher intake of PS and MGH as dry matter (DM) than those in the PP50 and PP75 treatments (p < 0.05). Organic matter intake (OMI) of the PP75 calves was lower than that of PP0 and PP25 calves. Crude protein intakes (CPI) of PP0 and PP25 calves were higher than those of PP50 and PP75 calves (p < 0.05). Apparent digestibility of crude protein was higher in the PP0, PP25, and PP50 treatments compared with that in treatment PP75 (p < 0.05). The treatments did not affect total bacterial count, cellulolytic bacterial count, cellulase enzymatic activity, volatile fatty acids, or the acetate/propionate ratio (p > 0.05). Rumen pH in the PP0 calves was higher than that of the PP25 calves, whereas the protozoa count and ammonia content were higher in PP0 calves than in PP75 (p < 0.05). In conclusion, the inclusion of 25% PP in the PS for forage-fed calves is a feeding alternative.
Assuntos
Ração Animal , Rúmen , Ração Animal/análise , Animais , Bovinos , Dieta/veterinária , Fibras na Dieta/metabolismo , Digestão , Ingestão de Alimentos , Fermentação , Rúmen/metabolismo , DesmameRESUMO
This study assessed the potential of termite gut inhabiting bacteria towards bioconversion of cellulosic waste into biofuel. Total seven bacterial isolates from the gut of Heterotermes indicola were isolated. Among all the isolates, HI-1 produced the largest zone upon primary screening. Untreated paper had more cellulose content (73.03%) than acid (0.5%) treated paper that was used as a lignocellulosic substrate for saccharification. Among all the isolates tested, glucose yield (1.08mg/mL) was high for HI-1 isolate. Several factors were considered for optimizing augmented glucose yield (8.57mg/mL) and growth (8.07×108cfu/mL), such as temperature 37°C, pH 4.5, 5% (w/v) substrate concentration, 6 % bacterial inoculum size, agitation 150 rpm with PEG 0.25 % and Ca2+ ions 0.002 g/L. Overall 8-fold increase in glucose yield was achieved. Enzyme activity of HI-1 showed higher endoglucanase 0.29 ± 0.01 (U/mL/min) and exoglucanase 0.15±0.01 (U/mL/min) activity under optimum conditions, mentioned above. temperature 37°C, pH 4.5, substrate concentration 5%, inoculum size 6%, surfactants PEG 0.01%, ions Ca2+(0.002g/L) and agitation (120 rpm). Simultaneous saccharification and fermentation (SSF) of hydrolyzed office paper yielded 5.43mg/mL bioethanol. According to 16S rRNA sequence homology, the bacterial isolate H1 was identified as Alcaligenes faecalis. Bioethanol production from office paper untreated waste proved an effective strategy. Bacteria having natural tendency towards cellulosic waste consumption are promising for bioconversion of cellulosic waste to valuable products.
Assuntos
Isópteros/microbiologia , Alcaligenes faecalis , BioetanolRESUMO
BACKGROUND: Cellulose as a potential feed resource hinders its utilization because of its complex structure, and cellulase is the key to its biological effective utilization. Animal endogenous probiotics are more susceptible to colonization in the intestinal tract, and their digestive enzymes are more conducive to the digestion and absorption of feed in young animals. Min pigs are potential sources of cellulase probiotics because of the high proportion of dietary fiber in their feed. In this study, the cellulolytic bacteria in the feces of Min pigs were isolated and screened. The characteristics of enzymes and cellulase production were studied, which provided a theoretical basis for the rational utilization of cellulase and high-fiber food in animal production. RESULTS: In our study, 10 strains of cellulase producing strains were isolated from Min pig manure, among which the M2 strain had the best enzyme producing ability and was identified as Bacillus velezensis. The optimum production conditions of cellulase from strain M2 were: 2% inoculum, the temperature of 35°C, the pH of 5.0, and the liquid loading volume of 50 mL. The optimum temperature, pH and time for the reaction of cellulase produced by strain M2 were 55°C, 4.5 and 5 min, respectively. CONCLUSIONS: Min pigs can be used as a source of cellulase producing strains. The M2 strain isolated from feces was identified as Bacillus velezensis. The cellulase from M2 strain had a good activity and the potential to be used as feed additive for piglets.
Assuntos
Animais , Porco Miniatura , Bactérias/enzimologia , Celulase/biossíntese , Bacillus , Fibras na Dieta , Probióticos , Digestão , Fezes , Ração AnimalRESUMO
Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.
Cohnella es un género de bacterias celulolíticas que puede ser encontrado en una variedad de hábitats. El propósito de este estudio fue registrar su presencia en el tracto digestivo de termitas (Nasutitermes aquilinus, N. corniger y Cortaritermes fulviceps) colectadas en el noreste argentino (NEA). Se incubaron homogenados de intestinos en sustrato celulósico para multiplicar las bacterias. Utilizando nuevos cebadores para Cohnella spp., se amplificó una porción del ADN ribosomal 16S bacteriano, el cual fue posteriormente clonado. Las secuencias obtenidas mostraron su mayor porcentaje de similitud (97,2-99,9%) con Cohnella spp., previamente reportadas en diversos ambientes. El análisis filogenético tendió a agrupar a los clones de acuerdo a la especie hospedante y al sitio de muestreo. Estos resultados indican que especies de termitas frecuentes en el NEA albergan simbiontes intestinales relacionados con el género Cohnella. Las potenciales aplicaciones industriales de estos microorganismos animan a profundizar los estudios.
Assuntos
Isópteros/microbiologia , Paenibacillus/isolamento & purificação , Paenibacillus/crescimento & desenvolvimento , Crescimento Bacteriano/análise , Análise de Sequência de DNA/métodos , Trato Gastrointestinal/microbiologiaRESUMO
Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.
Assuntos
Bacillales/genética , Bacillales/isolamento & purificação , Isópteros/microbiologia , Animais , Argentina , Bacillales/fisiologia , DNA Bacteriano/análise , DNA Ribossômico/análise , Trato Gastrointestinal/microbiologia , Isópteros/fisiologia , SimbioseRESUMO
Leaf-cutter ants (Atta and Acromyrmex) use fresh leaves to cultivate a mutualistic fungus (Leucoagaricus gongylophorus) for food in underground gardens. A new ant queen propagates the cultivar by taking a small fragment of fungus from her parent colony on her nuptial flight and uses it to begin her own colony. Recent research has shown that the ants' fungus gardens are colonized by symbiotic bacteria that perform important functions related to nitrogen fixation and have been implicated in contributing to plant biomass degradation. Here, we combine bacterial culturing in several media for counts and identification using the 16S rRNA gene with electron microscopy to investigate the process of cellulose degradation in the fungus garden and refuse dumps, and to assess the potential role of symbiotic bacteria. We show through electron microscopy that plant cell walls are visibly degraded in the bottom section of fungus gardens and refuse dumps, and that bacteria are more abundant in these sections. We also consistently isolated cellulolytic bacteria from all sections of fungus gardens. Finally, we show by culture-dependent and electron microscopy analysis that the fungus garden pellets carried by recently mated queens are colonized by fungus garden-associated bacteria. Taken together, our results indicate that cellulose is degraded in fungus gardens, and that fungus garden bacteria that may contribute to this deconstruction are vertically transmitted by new queens.
Assuntos
Formigas/microbiologia , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Celulose/metabolismo , Fungos/metabolismo , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/ultraestrutura , Biodiversidade , Biomassa , Metabolismo dos Carboidratos , Fungos/crescimento & desenvolvimento , Fungos/ultraestrutura , Microscopia Eletrônica , Filogenia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , RNA Ribossômico 16S/genética , Eliminação de Resíduos , Análise de Sequência de DNA , SimbioseRESUMO
ABSTRACT The wide variety of bacteria in the environment permits screening for more efficient cellulases to help overcome current challenges in biofuels production. This study focuses on the isolation of efficient cellulase producing bacteria found in pulp and paper mill effluent contaminated soil which can be considered for use in large scale biorefining. Four different bacterial strains were isolated and screened for cellulase production by using CMC agar medium. All isolates showed cellulase activity these strains were further characterized by morphological, physiological, biochemical and 16S rRNA gene analyses. These isolates were identified as two Bacillus subtilis sub sps, Bacillus mojavensis and Bacillus cereus.
RESUMO
ABSTRACTIn this study, a potential novel cellulolytic bacteriumArthrobacter sp. HPG166 was isolated from the hindgut of root-feeding larvaeHolotrichia parallela. Optimization of fermentation factors for endoglucanase production byArthrobacter sp. HPG166 was carried out via response surface methodology. Sodium carboxymethylcellulose 1.19% (w/v) and beef extract 0.35% (w/v) were the ideal combination of carbon and nitrogen sources for enzyme production; the optimum temperature and pH for cellulase production were 34°C and pH 8.0 respectively. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.411 U mL-1 was obtained. The crude endoglucanase was thermotolerant as it retained 50.31% of its activity after incubation at 70°C for an hour. Metal profile of the enzyme indicated that Mg2+ and Na+ were strong stimulators while Mn2+ and Co+ drastically inhibited its activity. Due to its particular characteristics, this enzyme could have potential for industrial applications.
RESUMO
The enzymatic hydrolysis of cellulose by cellulases is one of the major limiting steps in the conversion of lignocellulosic biomass to yield bioethanol. To overcome this hindrance, significant efforts are underway to identify novel cellulases. The snail Achatina fulica is a gastropod with high cellulolytic activity, mainly due to the abundance of glycoside hydrolases produced by both the animal and its resident microbiota. In this study, we partially assessed the cellulolytic aerobic bacterial diversity inside the gastrointestinal tract of A. fulica by culture-dependent methods and evaluated the hydrolytic repertoire of the isolates. Forty bacterial isolates were recovered from distinct segments of the snail gut and identified to the genus level by 16S rRNA gene sequence analysis. Additional phenotypic characterization was performed using biochemical tests provided by the Vitek2 identification system. The overall enzymatic repertoire of the isolated strains was investigated by enzymatic plate assays, containing the following substrates: powdered sugarcane bagasse, carboxymethylcellulose (CMC), p-nitrophenyl-ß-D-glucopyranoside (pNPG), p-nitrophenyl-ß-D-cellobioside (pNPC), 4-methylumbelliferyl-ß-D-glucopyranoside (MUG), 4-methylumbelliferyl-ß-D-cellobioside (MUC), and 4-methylumbelliferyl-ß-D-xylopyranoside (MUX). Our results indicate that the snail A. fulica is an attractive source of cultivable bacteria that showed to be valuable resources for the production of different types of biomass-degrading enzymes.
RESUMO
The raw materials used to produce bioethanol mostly are food crops, which has led to conflicts on food security. It is, therefore, recommended the gradual replacement for second generation substrates such as lignocellulosic materials. Herein, cellulolytic bacteria were isolated from the gut content of native larvae from Lepidoptera, Coleoptera, and adults of Isoptera. Few environmental samples from the pulp and paper feedstock were also assessed. A total of 233 isolates were obtained using enrichment cultures and classic criteria. Interestingly, several halo-forming colonies were found to be bacterial consortia that presented difficulties to take apart the microbial members. Those pure isolates which hydrolyzed cellulose in larger extend (45 strains) were selected and identified by means of 16S rRNA sequence analysis. Firmicutes was the prevalent phylum (62.2%) being Bacillus spp. the most frequent genus, while Paenibacillus, Brevibacillus, Cohnella, and Staphylococcus species were less frequent. The phylum Actinobacteria (6.7%) was represented by isolates related to Agromyces spp. and Microbacterium spp. Regarding Gram-negative bacteria (31.1%), the more depicted genus was Pseudomonas spp., and members of Achromobacter spp., Enterobacter spp., and Bacteroidetes phylum were also selected. These native bacterial strains are expected to enlarge the cellulolytic toolbox for efficient biomass deconstruction.
Assuntos
Celulose/metabolismo , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/isolamento & purificação , Bactérias Gram-Positivas/metabolismo , Insetos/microbiologia , Animais , Argentina , Biomassa , Celulase/metabolismo , DNA Ribossômico/genética , Microbioma Gastrointestinal , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Isópteros/microbiologia , Larva/microbiologia , Filogenia , RNA Ribossômico 16S/genéticaRESUMO
Fiber digestibility is an important factor regulating DMI in ruminants. Additionally, the ensiling process can also affect digestibility and chemical composition of the forage. The objective of this study was to investigate effects of sugarcane NDF digestibility (NDFD) and conservation method on intake, rumen kinetics, and the ruminal ecosystem of steers. Eight ruminally cannulated Nellore steers (275±22 kg BW) were used in a replicated 4×4 Latin square design with a 2×2 factorial arrangement of treatments. Two sugarcane genotypes divergent for stalk NDFD were used: IAC86-2480 with high NDFD and SP91-1049 with low NDFD. Experimental diets were formulated with 40% sugarcane, either freshly cut or as silage, and 60% concentrate on a DM basis. Each experimental period lasted for 14 d, with the last 4 d used for determination of intake, ruminal evacuation, and ruminal fluid collection. The effect of fiber digestibility on DM and NDF intake was dependent on the forage conservation method (P=0.01). High NDFD increased (P<0.01) DMI only when sugarcane was offered as silage, having no effect (P=0.41) on DMI when offered as freshly cut. Conservation method had no effect on total ruminal mass, with only a tendency (P<0.10) for greater NDF and indigestible NDF ruminal mass in steers fed the low-NDFD genotype. The NDF turnover and passage rates were greater (P<0.05) for the genotype with high NDFD but only when offered as silage. Liquid turnover rate in the rumen was greater (P=0.02) for diets containing silage, with no effect of genotype (P=0.87). There was no effect of NDFD genotype on ruminal pH (P=0.77); however, diets containing sugarcane as silage increased (P<0.01) ruminal pH. Total concentration of short chain fatty acids (P=0.05) and proportions of propionate (P=0.01) were greater for diets containing fresh sugarcane. Diets with fresh sugarcane increased the ruminal population of Streptococcus bovis (P<0.01) and Ruminococcus albus (P=0.03). The relative population of R. albus was also greater (P=0.04) for diets containing the sugarcane genotype with high NDFD. Feeding diets containing the sugarcane genotype with high NDFD increased Fibrobacter succinogenes population but only when sugarcane was fed as freshly cut (P=0.02). Using sugarcane genotypes with high NDFD can increase intake and benefit fiber-degrading bacteria in the rumen.
Assuntos
Regulação do Apetite/efeitos dos fármacos , Bovinos/crescimento & desenvolvimento , Dieta/veterinária , Fibras na Dieta/farmacologia , Digestão/efeitos dos fármacos , Rúmen/metabolismo , Amônia/metabolismo , Ração Animal/análise , Animais , Ácidos Graxos Voláteis/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Distribuição Aleatória , Rúmen/microbiologia , Ruminococcus/isolamento & purificação , Saccharum/metabolismo , Silagem/análise , Streptococcus bovis/isolamento & purificaçãoRESUMO
Microbial biomass and activity in soils are frequently studied in tropical dry forests, but scarce information is available about the relationships between functional bacterial groups and soil fertility, where relief interacts with rainfall seasonality. The culturable-bacterial groups and nutrients were studied during two consecutive years in soils from two topographic areas of different relief (hilltop vs hillslope) in a tropical dry forest from Chamela Jalisco, Mexico. We expected that seasonal and spatial variation in soil resources availability affects the abundance of functional culturable-bacterial groups. To evaluate this, fifteen soil cores (1kg), 0-5cm depth, were taken in the dry, early rainy and rainy seasons, from each of the ten replicate plots in hilltop and hillslope areas located in three microbasins. We found that hilltop soils were more organic and had higher concentration of labile C and total nutrient forms than hillslope soils, for which these soils had higher counts of colony-forming units (CFU) of total heterotrophic and P solubilizing bacteria. In both hilltop and hillslope soils, C and nutrient concentrations, as well as the counts of CFU of heterotrophic and P solubilizing bacteria generally decreased from the dry to the rainy season during the two study years. In contrast, the counts of CFU nitrifying and cellulolytic bacteria were higher at the hillslope than at the hilltop soils. The seasonal pattern of both groups was opposite to that of heterotrophic bacteria, presumably associated with a decrease in soil labile C and organic matter quality. In conclusion, our study suggests that available C appears to be the main factor that controls the structure of soil bacterial groups and soil fertility, where relief, rainfall seasonality and intraand inter-annual variations are critical factors that interactively modify bacterial dynamics related to soil C availability in the tropical dry forest.
Los bosques tropicales secos tienen una amplia heterogeneidad ambiental y por lo tanto se podría esperar una variación temporal y espacial en la abundancia y tipo de grupos bacterianos activos. Se evaluó, durante dos años consecutivos, el efecto de la estacionalidad de la lluvia sobre algunos grupos bacterianos cultivables y nutrimentos del suelo en dos posiciones topográficas (crestas vs laderas) en el bosque tropical seco, Chamela Jalisco, México. La hipótesis fue que la variación temporal y espacial de la disponibilidad de recursos afectaría la abundancia de los grupos bacterianos funcionales (heterótrofas totales, celulolíticas, solubilizadores de fosfatos y nitrificantes) del suelo. En las crestas, el suelo fue más orgánico y con mayor concentración de nutrimentos totales y C orgánico lábil que en la ladera, registrando más bacterias heterótrofas y solubilizadoras de fosfato. En ambas posiciones topográficas la concentración de C y nutrimentos, así como el número de bacterias heterótrofas y solubilizadoras de P, decrecieron de la estación seca a la lluviosa en ambos años. En contraste, las bacterias nitrificantes fueron mayores en el suelo de la ladera, y al igual que las celulolíticas, tuvieron un patrón estacional opuesto a las bacterias heterótrofas, asociado a la disminución del C lábil y de la calidad de la materia orgánica. En conclusión, se sugiere que el relieve, la estacionalidad y la variación inter-anual de la lluvia regulan interactivamente la relación entre el C disponible y la estructura de las comunidades bacterianas del suelo en el bosque tropical seco de Chamela.
Assuntos
Cianobactérias/classificação , /genética , Microbiologia do Solo , Árvores/microbiologia , Cianobactérias/genética , México , Estações do Ano , Clima TropicalRESUMO
Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.