Expression of a Recombinant Lentinula edodes Xylanase by Pichia pastoris and Its Effects on Ruminal Fermentation and Microbial Community in in vitro Incubation of Agricultural Straws.
ABSTRACT: Agricultural straws, such as rice straw, wheat straw, and corn straw, are produced abundantly every year but not utilized efficiently in China. An experiment was conducted to determine the effects of recombinant xylanase on ruminal fermentation and microbial community structure in in vitro incubation of these straws. The recombinant xylanase from Lentinula edodes (rLeXyn11A) was produced in Pichia pastoris. The optimal temperature and pH for rLeXyn11A were 40°C and 4.0, respectively. The rLeXyn11A featured resistance to high temperature and showed broad temperature adaptability (>50% of the maximum activity at 20-80°C). Supplemental rLeXyn11A enhanced the hydrolysis of three agricultural straws. After in vitro ruminal incubation, regardless of agricultural straws, the fiber digestibility, acetate concentration, total volatile fatty acids (VFAs) production, and fermentation liquid microbial protein were increased by rLeXyn11A. Supplemental rLeXyn11A increased the ammonia-N concentration for corn straw and rice straw. High throughput sequencing and real-time PCR data showed that the effects of rLeXyn11A on ruminal microbial community depended on the fermentation substrates. With rice straw, rLeXyn11A increased the relative abundance of fibrolytic bacteria including Firmicutes, Desulfovibrio, Ruminococcaceae and its some genus, and Fibrobacter succinogenes. With wheat straw, rLeXyn11A increased the relative abundance of Ruminococcus_1 and its three representative species F. succinogenes, Ruminococcus flavefaciens, Ruminococcus albus. With corn straw, the fibrolytic bacteria Firmicutes, Christensenellaceae_R_7_group, Saccharofermentans, and Desulfovibrio were increased by rLeXyn11A. This study demonstrates that rLeXyn11A could enhance in vitro ruminal digestion and fermentation of agricultural straws, showing the potential of rLeXyn11A for improving the utilization of agricultural straws in ruminants.
Project description:Background:Urea pretreatment is an efficient strategy to improve fiber digestibility of low quality roughages for ruminants. Nitrate and oil are usually used to inhibit enteric methane (CH4) emissions from ruminants. The objective of this study was to examine the combined effects of urea plus nitrate pretreated rice straw and corn oil supplementation to the diet on nutrient digestibility, nitrogen (N) balance, CH4 emissions, ruminal fermentation characteristics and microbiota in goats. Nine female goats were used in a triple 3?×?3 Latin Square design (27 d periods). The treatments were: control (untreated rice straw, no added corn oil), rice straw pretreated with urea and nitrate (34 and 4.7?g/kg of rice straw on a dry matter [DM] basis, respectively, UN), and UN diet supplemented with corn oil (15?g/kg soybean and 15?g/kg corn were replaced by 30?g/kg corn oil, DM basis, UNCO). Results:Compared with control, UN increased neutral detergent fiber (NDF) digestibility (P?<?0.001) and copies of protozoa (P?<?0.001) and R. albus (P?<?0.05) in the rumen, but decreased N retention (-21.2%, P?<?0.001), dissolved hydrogen concentration (-22.8%, P?<?0.001), molar proportion of butyrate (-18.2%, P?<?0.05), (acetate + butyrate) to propionate ratio (P?<?0.05) and enteric CH4 emissions (-10.2%, P?<?0.05). In comparison with UN, UNCO increased N retention (+34.9%, P?<?0.001) and decreased copies of protozoa (P?<?0.001) and methanogens (P?<?0.001). Compared with control, UNCO increased NDF digestibility (+8.3%, P?<?0.001), reduced ruminal dissolved CH4 concentration (-24.4%, P?<?0.001) and enteric CH4 emissions (-12.6%, P?<?0.05). Conclusions:A combination of rice straw pretreated with urea plus nitrate and corn oil supplementation of the diet improved fiber digestibility and lowered enteric CH4 emissions without negative effects on N retention. These strategies improved the utilization of rice straw by goats.
Project description:Straws are agricultural residues that can be used to produce biomethane by anaerobic digestion. The methane yield of rice straw is lower than other straws. Steam explosion was investigated as a pretreatment to increase methane production. Pretreatment conditions with varying reaction times (12-30 min) and maximum temperatures (162-240 °C) were applied. The pretreated material was characterized for its composition and thermal and morphological properties. When the steam explosion was performed with a moderate severity parameter of S0 = 4.1 min, the methane yield was increased by 32% compared to untreated rice straw. This study shows that a harsher pretreatment at S0 > 4.3 min causes a drastic reduction of methane yield because inert condensation products are formed from hemicelluloses.
Project description:The present study evaluated the effects of 4 typical subtropical forages on ruminal microbial community composition to formulate a better diet for buffalo. Corn straw silage, elephant grass, cassava residues and sugarcane tail silage were used as substrates for in vitro fermentation. Eight replicates were set up for every substrate, and fermentation was carried out in a 100-mL glass syringe, using buffalo rumen inoculum. Every replicate was anaerobically dispensed with 10 mL of rumen inoculum, 20 mL of McDougall's buffer and 200 mg of dried substrate, and placed in a water bath at 39 °C. Gas production was recorded at 0, 2, 6, 12, 24, 36, 48 and 72 h of incubation. After 24 h, fermentation was ceased for 4 replicates and samples were collected. Volatile fatty acids (VFA) concentrations were measured using gas chromatography. Microbial populations were quantified using quantitative real-time PCR (qRT-PCR), and microbial community was analyzed using high throughput sequencing technology. The results showed, cassava residues as substrate had the highest gas production, acetate, propionate and total VFA concentrations (P < 0.05), and corn straw silage had the lowest acetate:propionate ratio (P < 0.05). The lowest numbers of fungi, Ruminococcus albus and Fibrobacter succinogenes, and the highest number of protozoa were observed with cassava residues (P < 0.05). The least abundances of bacterial phyla Firmicutes, Bacteroidetes and genus Prevotella, and substantially higher abundance of phylum proteobacteria (56%) and genus Succinivibrio (52%) were observed with cassava residues. The most abundances of Methanobrevibacter gottschalkii and Entodinium were observed with cassava residues. Spearman's correlations analysis showed, Succinivibrio had strong positive correlations with propionate, butyrate, Metadinium and M. gottschalkii, indicating fermentation products were related to microbial community. In conclusion, incubation with cassava residues resulted in lower number of fiber degrading microbes but higher protozoal population because of its low fiber contents. The microbial community was highly altered by in vitro incubation with cassava residues, whereas remained similar for the other 3 high fiber containing substrates.
Project description:Comparative transcriptional profiling of N. crassa grown on five major crop straws of China (barley, corn, rice, soybean and wheat straws) revealed a highly overlapping group of 430 genes, the Biomass commonly Induced Core Set (BICS). A large proportion of induced carbohydrate-active-enzyme (CAZy) genes (82 out of 113) were also conserved across the five plant straws. Excluding 178 genes within the BICS that were also up-regulated under no-carbon conditions, the remaining 252 genes were defined as the Biomass Regulon (BR). Interestingly, 88 genes were only induced by plant biomass and not by three individual polysaccharides (Avicel, xylan, and pectin); these were denoted as the Biomass Unique Set (BUS). Deletion of one BUS gene, the transcriptional regulator rca-1, significantly improved lignocellulase production using plant biomass as the sole carbon source, possibly functioning via de-repression of the regulator clr-2. Thus, this result suggests that rca-1 is a potential engineering target for biorefineries, especially for plant biomass direct microbial conversion processes. Conidia of Neurospora crass wild type were inoculated at 10^6 conidia/mL into 100 mL 1×Vogel’s salts with 2% (w/w) ground crop straws, barley straw, corn straw, rice straw, soybean straw and wheat straw respectively for 30 h or 2% sucrose for 16 h. Then, mycelia were harvested through filtration and immediately frozen in liquid nitrogen.Total RNA from frozen sample was isolated with TRIzol reagent (Invitrogen) and further treated with DNase I (RNeasy Mini Kit, QIAGEN). The qualified RNA was prepared with standard protocol from Shenzhen BGI (China) and sequenced on the Illumina HiSeqTM 2000 platform.
Project description:Feed fiber composition is usually considered as one of the factors that have an impact on digestive tract microbiota composition. The investigations on the level of fermentation and in-vitro digestibility of different fibers are not well understood. The aim of the current study is to determine the effect of different fiber sources on intestinal nutrient digestibility, hindgut fermentation, and microbial community composition under in vitro conditions using pigs' hindgut as a model. The experimental treatment diets contained alfalfa hay, cornstalk, and rice straw. Cornstalk treatment displayed higher digestibility compared to alfalfa hay and rice straw; similar results were observed with in-vitro digestibility using intestinal digesta. Firmicutes were the most abundant phyla (Firmicutes = 89.2%), and Lactobacillus were the prominent genera (75.2%) in response to alfalfa compared to rice straw and cornstalk treatments. In simulated in-vitro digestion, corn stalk fiber improved dry matter digestibility; rice straw fiber improved volatile fatty acid content and fermentation efficiency. Alfalfa fiber improved the thickness of deposited Firmicutes and Lactobacillus.
Project description:Improvement of the food value of rice straw is urgently required in rice crop growing areas to mitigate pollution caused by rice straw burning and enhance the supply of high-quality forages for ruminants. The aims of the present study were to compare the effects of fresh corn Stover and rice straw co-fermented with probiotics and enzymes on rumen fermentation and establish the feasibility of increasing the rice straw content in ruminant diets and, by extension, reducing air pollution caused by burning rice straw. Twenty Simmental hybrid beef cattle were randomly allotted to two groups with ten cattle per group. They were fed diets based either on rice straw co-fermented with probiotics and enzymes or fresh corn Stover for 90 days. Rumen fluid was sampled with an esophageal tube vacuum pump device from each animal on the mornings of days 30, 60, and 90. Bacterial diversity was evaluated by sequencing the V4-V5 region of the 16S rRNA gene. Metabolomes were analyzed by gas chromatography/time-of-flight mass spectrometry (GC-TOF/MS). Compared to cattle fed fresh corn Stover, those fed rice straw co-fermented with probiotics and enzymes had higher (P?<?0.05) levels of acetic acid and propionate in rumen liquid at d 60 and d 90 respectively, higher (P?<?0.05) abundances of the phyla Bacteroidetes and Fibrobacteres and the genera Ruminococcus, Saccharofermentans, Pseudobutyrivibrio, Treponema, Lachnoclostridium, and Ruminobacter, and higher (P?<?0.05) concentrations of metabolites involved in metabolisms of amino acid, carbohydrate, and cofactors and vitamins. Relative to fresh corn Stover, rice straw co-fermented with probiotics and enzymes resulted in higher VFA concentrations, numbers of complex carbohydrate-decomposing and H2-utilizing bacteria, and feed energy conversion efficiency in the rumen.
Project description:The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.
Project description:The development of agricultural residue ethanol has a profound effect on the environment protection and energy supply. To increase the production efficiency of straw ethanol and reduce operation progress, the single-enzyme-system-three-cellulase gene (sestc) engineered Aspergillus niger and sestc engineered Saccharomyces cerevisiae were combined to produce ethanol using the pretreated rice straw as the substrate. The present results showed that both the step-by-step and in situ saccharification and fermentation can effectively produce ethanol using rice straw as the carbon substrate. The conversion rates of ethanol were 12.76 and 14.56 g per 1 kg of treated rice straw, respectively, via step-by-step and in situ processes. In situ process has higher ethanol conversion efficiency of rice straw and fewer operation processes as compared with step-by-step process. Therefore, in situ saccharification and fermentation is a more economical and effective pathway to convert rice straw into ethanol. This study provides a reference to the conversion of lignocellulosic residues into ethanol with a combination of two kinds of sestc engineered strains.
Project description:We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H(2)-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H(2)-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H(2) utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.
Project description:Supplementation of appropriate probiotics can improve the health and productivity of ruminants while mitigating environmental methane production. Hence, this study was conducted to determine the effects of Enterococcus faecium SROD on in vitro rumen fermentation, methane concentration, and microbial population structure. Ruminal samples were collected from ruminally cannulated Holstein-Friesian cattle, and 40:60 rice straw to concentrate ratio was used as substrate. Fresh culture of E. faecium SROD at different inclusion rates (0, 0.1%, 0.5%, and 1.0%) were investigated using in vitro rumen fermentation system. Addition of E. faecium SROD had a significant effect on total gas production with the greatest effect observed with 0.1% supplementation; however, there was no significant influence on pH. Supplementation of 0.1% E. faecium SROD resulted in the highest propionate (P?=?0.005) but the lowest methane concentration (P?=?0.001). In addition, acetate, butyrate, and total VFA concentrations in treatments were comparatively higher than control. Bioinformatics analysis revealed the predominance of the bacterial phyla Bacteroidetes and Firmicutes and the archaeal phylum Euryarchaeota. At the genus level, Prevotella (15-17%) and Methanobrevibacter (96%) dominated the bacterial and archaeal communities of the in vitro rumen fermenta, respectively. Supplementation of 0.1% E. faecium SROD resulted in the highest quantities of total bacteria and Ruminococcus flavefaciens, whereas 1.0% E. faecium SROD resulted in the highest contents of total fungi and Fibrobacter succinogenes. Overall, supplementation of 0.1% E. faecium SROD significantly increased the propionate and total volatile fatty acids concentrations but decreased the methane concentration while changing the microbial community abundance and composition.