Isolated Clostridium saccharobutylicum from the rumen of Holstein cattle and its effect on in vitro ruminal fermentation and microbial population
Michelle A. Miguel1, Lovelia L. Mamuad1, Yeon-Jae Choi2, Chang-Dae Jeong1, Kwang-Keun Cho3, Eun-Tae Kim4, Sang-Bum Kim4, Catherine G. Avedoza1, and Sang-Suk Lee1*
1Department of Animal Science and Technology, College of Bio-industry Science, Sunchon National University, Suncheon, Jeonnam, Republic of Korea
2Department of Animal Biotechnology, College of Agricultural Life Science, Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea
3Department of Animal Resources Technology, Gyeongnam National University of Science and Technology, Jinju, Gyeongnam, Republic of Korea
4Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan, Chungnam, Republic of Korea
*Corresponding author: Sang-Suk Lee, Ph.D., Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, College of Bio-Industry Science, Sunchon National University, 413 Jungangno, Suncheon, Jeonnam 57922, Republic of Korea
Phone/Facsimile: +82-61-750-3237; E-mail: rumen@sunchon.ac.kr
Heading: Butyrate-producing bacteria C. saccharobutylicum
Abstract
Butyrate produced through microbial fermentation is important for energy metabolism and regulating genomic activities influencing rumen nutrition utilization and function. In this study, a butyrate producing bacteria was isolated, identified, and characterized as Clostridium saccharobutylicum RNAL841125 using 16S rRNA gene sequencing and phylogenetic analyses. The strain displayed wide substrate range and high levels of enzyme activity were detected in xylan and starch as substrate. The strain was evaluated on its effects on in vitro rumen fermentation and microbial population. The following concentrations used for the in vitro rumen fermentation were: no addition (control), supplementation with C. saccharobutylicum (106 cfu/mL, 107 cfu/mL, or 108 cfu/mL), and supplementation with 50 mM of butyric acid. Supplementation of C. saccharobutylicum (106 cfu/mL) on in vitro rumen fermentation did not affect total gas production but significantly lowered the pH value and increased the butyrate and total volatile fatty acid concentrations. Quantitative real-time PCR revealed that supplementation of C. saccharobutylicum RNAL841125 increased the number of butyrate-producing bacteria and enhanced the Fibrobacter succinogenes population on in vitro rumen fermentation. However, total bacteria, protozoa and fungi populations was not influenced by the supplementation of C. saccharobutylicum. Additionally, microbial crude protein was increased when supplemented with C. saccharobutylicum (106 cfu/mL). In conclusion, our results indicated that supplementation of C. saccharobutylicum have a potential to improve ruminal fermentation through increased concentrations of butyrate and total volatile fatty acid and enhanced population of butyrate-producing bacteria and F. succinogenes.
Keywords
Butyric acid, Clostridium, Rumen, Microbial population
Introduction
Researchers have evaluated whether ruminant productivity can be increased by manipulating the rumen environment to enhance digestibility and nutrient utilization by animals. One widely used approach is application of probiotics in order to promote digestion and intestinal hygiene (Gournier-Chateau et al. 1994), which enhance animal performance and reduce usage of antibiotics (Wallace et al. 2008). Microbial additives may improve ruminant animal performance in terms of live weight gain and milk production by 7–8% (Wallace 1994). Thus, probiotics have been used for many years to supplement the diets of farm animals and humans (Bernardeau and Vernoux 2013).
Butyric acid is a short-chain fatty acid produced by anaerobic fermentation of dietary substrates in the rumen and large intestine (Aluwong et al. 2013) and a preferred energy source for epithelial cells in ruminants (Bugaut 1987). Most importantly, butyrate stimulates epithelial cell proliferation, resulting in a larger absorptive surface and leading to improved feed utilization. Moreover, butyrate also possesses other important functions in the intestinal epithelium, such as prevention of certain types of colitis (Scheppach 1994). Sodium butyrate supplementation has been reported to improve growth performance in calves (Kato et al. 2011). Furthermore, several studies have shown that butyrate affects several other parameters (e.g., the mucosal barrier, feed passage, microbiota, immune system, and pathogens, among others), and this combination of effects contributes to its general acceptance as beneficial for improving health and performance (Pierce et al. 2014).
Due to the increasing demand of consumers for naturally produced products, the production of butyric acid by microbial fermentation has attracted much attention. Several anaerobic bacteria can produce butyric acid as the major fermentation product from a wide range of substrates. Butyrate-producing bacteria are associated with gastrointestinal health in humans and various animal species (Levine et al. 2013) and play an important role in the degradation of proteins, nucleic acids and structural and storage plant polysaccharides (Mrázek et al. 2006). Thus, we aimed to isolate and identify butyrate-producing bacteria and evaluate its effects on in vitro rumen fermentation and microbial population.
Materials and methods
Isolation, characterization, and molecular identification of butyrate-producing bacteria
Butyrate-producing bacteria were isolated from the rumen contents of a 48-month-old rumen-cannulated Holstein-Friesian cow (600 ± 47 kg). The animal was fed twice daily with concentrate feed (NongHyup Co., Anseong, Korea) and rice straw at a 2:8 ratio. Ruminal fluid was collected before feeding and obtained by straining the rumen contents through four layers of surgical gauze and pooled in an amber bottle with an oxygen free headspace immediately after collection. The collected rumen fluid was sealed, maintained at 39°C, and immediately transported to the laboratory for bacterial isolation. The management of animals was approved by Sunchon National University Committee on Animal Care (2016).
Media preparation and isolation of bacteria from rumen
One milliliter of rumen sample was placed in sterile tube and homogenized in 9 mL of anaerobic medium containing soluble starch, glucose and cellobiose as energy sources (M2GSC) (Miyazaki et al. 1997) pH 6 containing:10.0 g/L of casitone, 2.5 g/L of yeast extract, 4.0 g/L NaHCO3, 2.0 g/L glucose, 2.0 g/L soluble starch, 2.0 g/L cellobiose, 300 ml of clarified rumen fluid, 1.0 g/L of cysteine HCl, 150 mL of Mineral Solution I (3.0 g/L of K2HPO4), 150 mL of Mineral Solution II (3.0 g/L KH2PO4, 6.0 g/L (NH4)2SO4, 6.0 g/L NaCl, 0.6 g/L MgSO4·7H2O, 0.6 g/L CaCl2), and 1.0 g of resazurin (1% w/v). The medium was aseptically added in the bottle and flushed with CO2 according to the anaerobic Hungate method (Hungate 1969). This diluent corresponded to the first 10-fold dilution, which was then mixed by vortexing to form homogenized suspension. From this suspension, it was subsequently diluted by 10-fold serial dilutions through to a 10−9 dilution.
Anaerobic M2GSC medium containing 0.75% agar were prepared in 16×125 mm Hungate tubes sealed with butyl septum stoppers and inoculated with 0.5 mL aliquots of appropriate serial dilutions. Roll tubes were incubated at 37°C for 24 to 48 h prior to the picking of colonies from each sample. Picked colonies were subsequently inoculated in the same medium until purified. Purified cultures were grown in broths of M2GSC at 37°C for 24 to 48 h and used for the determination of fermentation products by high performance liquid chromatography (HPLC) and DNA extraction for molecular identification of the isolates.
Analysis of butyric acid concentration
The butyric acid produced by the bacterial isolates was compared with C. butyricum as positive control and analyzed using HPLC. Short-chain fatty acid concentrations were analyzed using an Agilent 1200 Series HPLC System (Agilent Technologies, USA) with a UV detector set at 210 and 220 nm. Samples were eluted isocratically with 0.0085N H2SO4 at a flow rate of 0.6 mL/min and a column temperature of 35°C.
16S rRNA sequence and phylogenetic analysis
Isolates producing high butyric acid concentrations were identified by sequencing the 16S ribosomal RNA (16S rRNA) gene using the 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primers (Lane 1991).The gene sequences obtained from the isolates were compared with the 16S rRNA sequences available in GenBank using the Basic Local Alignment Search Tool (BLAST) (Madden 2013) and EzBioCloud database (Yoon et al. 2017). Multiple gene sequences were aligned using ClustalW (Thompson et al. 1994) within the Molecular Evolutionary Genetics analysis (MEGA) version 6 (Tamura et al. 2013) to determine the approximate phylogenetic affiliations. The phylogenetic tree was constructed using the Neighbor-Joining method (Saitou and Nei 1987) with pair-wise comparison and with evolutionary distances computed using the Kimura 2-parameter method (Kimura 1980). Reliability of the tree topology was assessed with the bootstrap method using 1000 replications (Felsenstein 1985). Only bootstrap values greater than 50% are shown on the internal nodes.
Cultivation of C. saccharobutylicum RNAL841125
Clostridium saccharobutylicum RNAL841125 was deposited in the Korean Culture Center of Microorganisms (KCCM). The bacterial colonies of the C. saccharobutylicum RNAL841125 were maintained in clostridial growth medium composed of: 2.0 g/L K2HPO4, 0.75 g/L KH2PO4, 1.5 g/L MgSO4∙7H2O, 0.017 g/L MnSO4∙5H2O, 0.01 g/L FeSO4∙7H2O, 2.0 g/L (NH4)2SO4, 5.0 g/L NaCl, 2.0 g/L asparagine, 0.004 g/L p-aminobenzoic acid, 15.0 g/L yeast extract, and 50.0 g/L glucose. The pH of the medium was adjusted to 6.8. The headspace of the bottle was purged with N2 gas. The medium was dispensed anaerobically under an O2-free N2 atmosphere and autoclaved at 121°C for 15 min. Bacterial cultures were grown anaerobically and incubated at 37°C for 24–48 h. Bacterial growth was monitored by optical density (OD) using a spectrophotometer at 600 nm.
Substrate utilization and enzyme activities
Substrate utilization of RNAL841125 isolate was tested using API® 50 CH test kit (Biomérieux, France). Analyses for enzyme activities were carried out for CMCase (EC 3.2.1.4), FPase (EC 3.2.1.91), xylanase (EC 3.2.1.8), pectin methyl esterase (EC 3.2.1.11), polygalactouranase (EC 3.2.1.15) and α-amylase (EC 3.2.1.10) using carboxymethyl cellulose (CMC), filter paper, pectin, xylan and starch as substrate, respectively. The enzyme activities were determined by estimating the amount of reducing sugar liberated from the enzymatic reaction from respective substrate dissolved in appropriate buffer by dinitrosalycilic acid (DNS) (Miller 1959). One unit of enzyme was defined as the amount of enzyme that released 1 μmol of glucose per min.
Rumen fluid collection and in vitro fermentation
All animal care procedures were conducted in accordance with the guidelines approved by the Sunchon National University Committee on Animal Care (2016). Three rumen-cannulated Holstein cows weighing 600 ± 47 kg were fed twice daily with total mixed ration (Table 1) and ryegrass straw (7:3 ratio). Ruminal fluid was collected before feeding and obtained by straining the rumen contents through four layers of surgical gauze and pooled in an amber bottle with an oxygen free headspace immediately after collection. The collected rumen fluid was sealed, maintained at 39°C, and immediately transported in the laboratory.
The buffer medium was prepared according to the method described by Asanuma et al. (1999). The buffer was autoclaved at 121°C for 15 min, maintained in a 39°C water bath, and flushed with CO2 gas, and the pH was adjusted to 6.9 using 10 N NaOH. The experiment was conducted under a constant flow of CO2 gas on the rumen-buffered medium to ensure anaerobic conditions. The particle-free rumen fluid and buffer medium were mixed at a ratio of 1:3 (v/v). After mixing, 100 mL of the mixed buffered rumen fluid was anaerobically transferred into the serum bottles containing 1.0 g dry matter substrate of concentrate and ryegrass straw (7:3 ratio), and the following inocula treatments were conducted under a stream of CO2 gas: no addition (control), supplementation with C. saccharobutylicum (106 cfu/mL, 107 cfu/mL, or 108 cfu/mL), and supplementation with 50 mM of butyric acid (Sigma, St. Louis, MO, USA). The serum bottles were capped with a butyl rubber stopper, sealed with an aluminum cap, and incubated at 39°C with horizontal shaking at 120 rpm. Three replicates were performed for all treatments and incubation time.
Analysis of in vitro rumen fermentation parameters
Rumen fermentation characteristics, including total gas production, pH, and ammonia-nitrogen (NH3-N), microbial crude protein (MCP), and volatile fatty acid (VFA) concentrations were examined at the end of each incubation period. Two one-milliliter of rumen fluid from each serum bottles were collected in a microcentrifuge tubes and maintained at −80°C until further analysis of NH3-N, VFA concentrations and microbial population.
Total gas production was measured from each serum bottle after incubation using a pressure sensor (Laurel Electronics, Inc., Costa Mesa, CA, USA). The pH values of the rumen samples were measured immediately after opening each serum bottle using a digital pH meter (Mettler Toledo, Greifensee, Switzerland). For NH3-N and VFA analyses, ruminal fluid samples were centrifuged at 14,000 rpm for 15 minutes at 4°C and the supernatant was then used for the analysis. Rumen NH3-N levels were determined according to a colorimetric method developed by Chaney and Marbach (1962) using a Libra S22 spectrophotometer (Biochrom Ltd., England) set at 630 nm. MCP was estimated following the method used by Castillo-Lopez et al. (2013). VFA concentrations were analyzed using an Agilent 1200 Series HPLC System (Agilent Technologies, USA) with a UV detector set at 210 and 220 nm. Samples were eluted isocratically with 0.0085N H2SO4 at a flow rate of 0.6 mL/min and a column temperature of 35°C.
DNA extraction
Microcentrifuge tubes containing ruminal fluid samples were centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was then discarded and the isolated pellets were used for the extraction of total microbial genomic DNA using FastDNA SPIN Kit (MP, USA) following the manufacturer’s protocol. The DNA concentration and quality were measured using an Optizen NanoQ spectrophotometer (Optizen, Korea). The DNA samples were stored at −20°C until analysis.
Quantitative real-time PCR analyses
The population sizes of total bacteria, protozoa, general fungi and select bacterial species were quantified using SYBR Green-based quantitative real-time PCR (qPCR) using the Eco™ Real-Time PCR (Illumina, San Diego, CA, USA). The primers used for each microbial group are shown in Table 2. In addition, the butyrate kinase (buk) gene, which is involved in the production of butyrate, was also quantified. The reaction mixture was prepared in a total volume of 20 µL containing 10 µL of 2x QuantiSpeed SYBR No-Rox mix (PhileKorea, Korea), 0.8 µL of each 10 µM primer, and 8.4 µL template DNA at a concentration of 50 ng/µL in sterile distilled water. The qPCR reactions were performed under thermal cycle conditions of one cycle at 50°C for 2 min, and 95°C for 2 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min and 72°C for 30 s. For all PCR runs, standards, negative controls (no DNA), and samples were run in triplicate.
Statistical analysis
Data were subjected to analysis of variance using the general linear model for a randomized complete block design. All treatments were conducted in triplicate and Duncan’s multiple range test was used to identify differences between specific treatments. The linear effects of C. saccharobutylicum supplementation were analyzed using orthogonal polynomial coefficients to describe the functional relationships among the control and treatment groups. Differences with P values less than 0.05 were considered statistically significant. Data analysis were carried out using Statistical Analysis Systems (SAS) version 9.3 (SAS Institute Inc., Cary, NC, USA).
Results
Identification and characterization of butyrate-producing bacteria
Out of 33 samples, only one potential butyrate-producing bacteria was isolated from the rumen. The 16S rRNA gene sequencing and phylogenetic analyses demonstrated that this isolate was 99% similar to C. saccharobutylicum DSM 13864T (Keis et al. 2001). The isolate was deposited in the Korean Culture Center of Microorganisms (KCCM) as C. saccharobutylicum RNAL841125 (Fig. 1) with NCBI GenBank accession number MH032748. Only bootstrap values >50% were shown on the internal nodes, and Butyrivibrio fibrisolvens was used as an outgroup and the bar represents 0.02 substitutions per nucleotide position.
The butyrate production level of C. saccharobutylicum RNAL841125 was compared with the standard butyrate-producing bacteria, C. butyricum. Significantly higher levels (P< 0.05) of propionate and butyrate was produced by C. saccharobutylicum RNAL841125 (20.46 and 42.39 mmol l–1, respectively) than C. butyricum (18.79 and 19.11 mmol l–1, respectively) (data not shown). The substrate utilization test using the API® 50 CH identification system revealed that RNAL841125 isolate could utilize 28 types of sugars as substrates. The C. saccharobutylicum profile revealed that the isolate metabolized D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, glucose, fructose, mannose, inozitol, α-methyl-D-mannoside, α-methyl-D-glucoside, amygdaline, salicin, maltose, lactose, melibiose, sucrose, trehalose, rafinose, starch, glycogen, xylitol, gentobiose, D-turanose, D -fucose, L-fucose, D-arabitol and gluconate. Additionally, analysis of the growth of C. saccharobutylicum RNAL841125 on CMC, filter paper (Whatman filter paper No. 1), xylan, pectin, and starch as substrates revealed that the isolate had weak fermentation on CMC and filter paper and, no fermentation was seen in pectin (data not shown). The enzyme activities of the bacteria using CMC, filter paper (Whatman filter paper No. 1), xylan, pectin and starch are shown in Figure 2. The results indicated that the use of CMC and filter paper as substrates produced low amounts of enzyme in comparison with xylan and starch.
Effects of C. saccharobutylicum supplementation on in vitro rumen parameters
The effects of C. saccharobutylicum on total gas production, pH, NH3-N concentrations, and MCP are shown in Table 3. Total gas production at 24 h was higher (P<0.05) in the treatment supplemented with 106 cfu/mL C. saccharobutylicum and control compared to other treatments. However, increasing the concentration of C. saccharobutylicum supplementation leads to decrease in gas production. The pH value after 24 of incubation was lower (P< 0.05) in treatments supplemented with C. saccharobutylicum compared to the control and treatment with 50mM butyric acid. Moreover, the total gas production and pH was linearly correlated with C. saccharobutylicum supplementation. However, as inclusion rate of C. saccharobutylicum supplementation was increased, the total gas production and pH tended to decrease (P< 0.05). Supplementation with C. saccharobutylicum had no effect on ruminal NH3-N concentrations after incubation for 24 h. However, microbial crude protein and bacterial crude protein increased following treatment with 106 cfu/mL C. saccharobutylicum (15.01 and 10.71 mg/g DM respectively; Fig. 3).
The effect of C. saccharobutylicum on VFA concentrations are shown in Table 4. Higher concentrations of acetate were obtained in treatment with 50 mM butyric acid and 106 cfu/mL C. saccharobutylicum compared to other treatments. In addition, higher (P< 0.05) contents of butyrate and total volatile fatty acid after 24 h of incubation were obtained following treatment with 106 cfu/mL C. saccharobutylicum compared to control and other treatments. After 24 h incubation, significantly higher (P< 0.05) butyric acid concentrations were observed following treatment with 106 cfu/mL C. saccharobutylicum than the control and other treatments. However, total and individual VFA concentrations decreased in concentration when inclusion rates of C. saccharobutylicum was increased.
Effects of C. saccharobutylicum supplementation on the rumen microbial population
Real-time PCR results showed that the treatments did not significantly affect the total quantity of microbes (Fig. 4). Comparable quantities of general bacteria and protozoa were observed among the control and treatment groups. However, there was an increase in the log copies of protozoa and Fibrobacter succinogenes compared with that of the control, although this increase was not significant. Analysis of the buk gene showed that supplementation with C. saccharobutylicum increased butyrate-producing bacteria in the rumen (Fig. 5). Moreover, the butyrate-producing bacteria DNA copies tended to decrease, as increasing inclusion rate of C. saccharobutylicum was added.
Discussion
Butyrate production in the gastrointestinal tract, which has recently been shown to play a special role in modulating bacterial energy metabolism in the gut ecosystem (Li et al. 2008), depends on the diets, microbial species and their relative abundance present in the gut ecosystem, and gut transit time. In the rumen, butyrate-producing bacteria as a functional group have a distinct phylogenetic profile. Consumption of high-fiber diets in cattle tends to increase the population of major butyrate-producing bacteria, Butyrivibrio (Mrázek et al. 2006), resulting in an increase in ruminal butyrate concentrations, whereas high-energy feeds lead to the suppression of these bacteria. Butyrate is a preferred energy source for ruminal epithelial cells (Bugaut 1987). According to a study by Li et al. (2012), introducing butyrate-producing bacteria into the gut ecosystem may be an effective means to treat and prevent colon cancer and enterocolitis, including inflammatory bowel diseases.
In this study, C. saccharobutylicum was found to be capable of utilizing wide range of carbohydrate substrates, including the polymers starch and xylan and saccharides such as glucose, arabinose, xylose, and cellobiose, consistent with the findings of Johnson et al. (1997). Fermentation products of this bacterium included acetic and butyric acids, acetone, butanol, ethanol, CO2, and H2. Weak fermentation was observed in C. saccharobutylicum RNAL841125 on CMC and filter paper; however, no fermentation was seen in pectin, suggesting that these substrates are not suitable for this bacterium. Moreover, the only complex polysaccharide not utilized by C. saccharobutylicum is pectin; consequently, this substrate is useful for discerning this species from C. acetobutylicum, C. saccharoperbutylacetonicum and C. beijerincki (Keis et al. 2001). In a study conducted by Meesukanun and Satirapipathkul (2014), C. saccharobutylicum BAA 117 was able to utilize the hydrolysate of Cassava rhizome, which consists mainly of cellulose, hemicellulose and lignin, as substrate for fermentation in batch culture. We also found that lower concentrations of enzyme were produced using CMC and filter paper as substrates compared with xylan and starch; these findings may be related to the weak fermentation ability of the bacterium on these substrates.
The in vitro gas production technique has been used widely to study feed degradation (Rymer et al. 2005); it can provide valuable information on the kinetics of feed digestion in the rumen and reflect the utilization efficiency of fermentation substrates (Metzler-Zebeli et al. 2012). Our results showed that gas production was higher following treatment with 106 cfu/mL C. saccharobutylicum and control in comparison with other treatments. However, the comparable results between the treatment with 106 cfu/mL C. saccharobutylicum and control suggest that addition of microbes did not significantly affected the increase of gas production.
pH is the main index reflecting internal homeostasis of the rumen environment; maintaining a relatively stable ruminal pH is important to ensure efficient rumen fermentation. Ruminants usually possess highly developed systems to maintain ruminal pH within a physiological range of approximately 5.5–7.0 (Krause and Oetzel 2006). Our results showed that the pH was maintained in the range of 5.53 to 5.69, which according to Stewart et al. (1997) provided suitable conditions for fermentation, microorganism growth, and fiber degradation in the rumen. The direct relationship of rumen pH and VFA is well known. Therefore, lower ruminal pH in treatments supplemented with C. saccharobutylicum might be related to a significant increase in ruminal VFA (Patra et al. 1996).
Ammonia nitrogen is the most important nitrogen source for microbial protein synthesis in the rumen (Bryant 1974). Although NH3-N is an important nitrogen source for microbial growth and protein synthesis, ruminal NH3-N has been shown to have a low efficiency for milk protein synthesis partially due to ammonia N losses in the rumen (Hristov and Ropp 2003). Satter and Slyter (1974) suggested that the lowest concentration of NH3-N in rumen liquor should not be less than 5 mg/dL to maintain higher bacterial growth rate. Deficiency of NH3-N restricts microbial protein synthesis, whereas high concentrations of NH3-N also inhibit microbial utilization of this compound (Hristov et al. 2002). Our results showed that the concentrations of NH3-N following treatment with different concentrations of C. saccharobutylicum ranged from 23.0 to 23.67 mg dL–1, indicating that growth and protein synthesis of microorganisms was not restricted. Moreover, increased NH3-N concentrations reflects greater catabolism of protein and nonprotein nitrogen (Soriano et al. 2014) and result in higher nitrogen concentrations available for microbial utilization and protein synthesis (Mamuad et al. 2014).
Microbial crude protein (MCP) is the most important source of metabolizable protein for ruminant animals because of its quantity and excellent amino acid profile (National Research Council 2001). We obtained the highest amount of MCP (15.01mg/g DM) following treatment with 106 cfu/mL C. saccharobutylicum. According to Block (2006), increased MCP production in the rumen improves the efficiency of feed utilization in cattle and results in the supply of a more ideal protein source. MCP synthesis are influenced by the availability of N, energy availability for rumen fermentation and ruminal passage rate (Purser 1970; Stern and Hoover 1979). In addition, bacterial crude protein (BCP) was also increased following supplementation with C. saccharobutylicum. The numerical increase of BCP might be due to the microbial efficiency during fermentation. According to Castillo-Lopez et al. (2012), readily available N in the form of urea and high starch may have supported more microbial growth leading to more BCP synthesis.
Volatile fatty acids are considered the most important end products of rumen fermentation, providing cows with the majority of energy precursors for metabolic processes (Storm et al. 2012). Their content and composition are important physiological indexes that reflect rumen digestion and metabolism. Many studies have indicated that rumen VFAs can provide 50–80% of the energy needed by ruminants (Sutton 1985). VFAs, particularly butyrate, have important roles in the postnatal development of the ruminal epithelium (Sakata and Tamate 1978). We found that higher concentrations of butyrate concentration was produced following treatment with 106 cfu/mL C. saccharobutylicum, indicating increases in carbon and energy sources for fatty acid synthesis (Mamuad et al. 2017).
The addition of microorganisms in the rumen can alter the microbial community in the rumen, and the microbial community changes in response to changes in feed and feed levels in the rumen (Mao et al. 2008). The microbial population was not significantly affected by the addition of C. saccharobutylicum; however, cellulolytic bacteria, F. succinogenes increased its population with addition of C. saccharobutylicum, which might suggest that addition of C. saccharobutylicum enhances F. succinogenes population. C. saccharobutylicum had exhibited hemicellulolytic activity which enables the microorganism to convert a range of agricultural substrates and monomeric sugars of hemicellulose to solvents and acids. The supplementation with C. saccharobutylicum might have stimulated the growth of F. succinogenes which leads to the increase in population of this microbe. Butyrate-producing bacteria is important to gut homeostasis (Wang et al. 2014), thus, increasing the population might be beneficial in the animal. In this study, the number of copies of butyrate-producing bacteria in the rumen supplemented with C. saccharobutylicum was found to be higher in comparison with that of the control. Our results were consistent with the results of a study by Li et al. (2012), who found that an elevated butyrate levels in the rumen had stimulatory effects on butyrate-producing bacteria populations. Additionally, a study by Flint et al. (2007) showed that an increase in butyrate production may result from a direct stimulation of butyrate producers or indirect effects such as metabolic cross-feeding of fermentation products from other bacterial groups.
Supplementation of C. saccharobutylicum RNAL841125 suggested that supplementation of 106 cfu/mL C. saccharobutylicum improved the in vitro rumen fermentation characteristics through lowering pH, increasing butyric and volatile fatty acids, and enhancing F. succinogenes population. Overall, the butyrate-producing bacteria, C. saccharobutylicum RNAL841125, may be beneficial for animal use and other industrial applications.
Acknowledgement
This research was supported by the Cooperative Research Program for Agriculture Science and Technology Development, (Project No. PJ013448012018), Rural Development Administration, Republic of Korea.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest.
Ethical approval
All applicable international and national guidelines for the care and use of animals were followed.
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Table 1 Composition of total mixed ration (TMR)
Composition Percentage
Soybean meal 9.27
Lupin seed 11.07
Dried distillers’ grains 5.92
Rice bran 5.15
Corn 36.94
Mushroom media 18.40
Protein 11.07
Salt 0.39
Limestone 0.77
Vitamin 1.03
Table 2 Microorganisms, sequences and references of the real-time PCR primers used for the quantification of microbial population and microbial crude protein
Target Sequence (5ʹ→ 3 ʹ) Reference
A. Microbial quantification
General bacteria
F CGG CAA CGA GCG CAA CCC Denman and McSweeney (2006)
R CCA TTG TAG CAC GTG TGT AGC C
Protozoa
F GCT TTC GWT GGT AGT GTA TT Sylvester et al. (2004)
R CTT GCC CTC YAA TCG TWC T
General anaerobic fungi
F GAG GAA GTA AAA GTC GTA ACA AGG TTT C Denman and McSweeney (2006)
R CAA ATT CAC AAA GGG TAG GAT GAT T
F. succinogenes
F GTT CGG AAT TAC TGG GCG TAA A Denman and McSweeney (2006)
R CGC TGC CCCCTG AAC TAT C
B. Microbial Crude Protein
Bacteria
F ACT CCT ACG GGA GGC AGC AG Yu et al. (2005)
Probe FAM/ TGC CAG CAG CCG CGG TAA TAC /TAMRA
R GAC TAC CAG GGT ATC TAA TCC
Protozoa
F GCT TTC GAT GGT AGT GTA TT Sylvester et al. (2005)
Probe FAM/ CGG AAG GCA GCA GGC GC /TAMRA
R ACT TGC CCT CTA ATC GTA CT
C. Butyrate-producing bacteria (butyrate kinase gene)
F TGCTGTWGTTGGWAGAGGYGGA Vital et al. (2013)
R GCAACIGCYTTTTGATTTAATGCATGG
Table 3 Effect of treatments on total gas production, pH, and ammonia-nitrogen during in vitro rumen fermentation at 24 h incubation
Parameters Treatments SEM p value
Control T1 T2 T3 T4 All Linear
Total gas (mL) 84.67a 85.00a 83.00b 81.67b 78.00c 0.480 <0.0001 0.001
pH 5.46a 5.43b 5.42b 5.42b 5.49a 0.007 0.001 0.002
NH3-N (mg dL–1) 23.07 23.63 23.41 23.00 23.31 0.122 0.098 0.540
*Means within a row having the same superscript is not significantly different (P < 0.05)
* SEM, standard error of mean
*Treatments: Control – no addition; T1 – 106 cfu/mL C. saccharobutylicum; T2 – 107 cfu/mL C. saccharobutylicum; T3 – 108 cfu/mL C. saccharobutylicum; T4 – 50mM Butyric acid
Table 4 Effect of treatments on VFA production (mmol l–1) during in vitro rumen fermentation at 24 h
Parameters Treatments SEM p value
Control T1 T2 T3 T4 All Linear
Acetic acid 37.32bc 38.08ab 36.78bc 36.10c 38.89 a 0.346 0.0045 <0.0001
Propionic acid 13.16a 13.04a 12.93a 11.26b 11.43b 0.271 0.0016 0.0008
Butyric acid 16.68b 19.37a 17.86ab 16.67b 16.73b 0.437 0.0128 0.5277
A/P ratio 2.84c 2.92c 2.85c 3.21b 3.41a 0.052 0.0001 0.0421
Total VFA 67.16b 70.49a 67.57b 64.03c 67.06b 0.492 0.0003 0.0004
*Means within a row having the same superscript is not significantly different (P< 0.05)
*SEM, standard error of mean
*Treatments: Control – no addition; T1 – 106 cfu/mL C. saccharobutylicum; T2 – 107 cfu/mL C. saccharobutylicum; T3 – 108 cfu/mL C. saccharobutylicum; T4 – 50mM Butyric acid