Maintenance of the Rumen Microbial Population in Continuous Culture

The design and use of an apparatus for continuous in vitro cultivation of the mixed bovine rumen microbial population are described. Data relative to the concentrations and proportions of volatile fatty acids produced, methanogenesis, carbon dioxide production, and survival of bacteria and protozoa indicated that an essentially normal in vivo fermentation pattern was maintained in vitro for experimental periods of from 3 to 10 days. The continuous cultures were responsive to major changes in type of feed intake. A change from grain to hay resulted in increased acetate and decreased propionate production, whereas decreased acetate and increased propionate resulted when feed intake was changed from hay to grain. Methanogenesis, volatile fatty acid production, and the numbers of microorganisms in the cultures were proportional to the amount of feed materials added up to levels calculated to approximate the in vivo maintenance requirement of the adult bovine.     

Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.The rumen is a complex microbial ecosystem that is composed of an immense variety of bacteria, protozoa, fungi, and viruses (5). Among these microorganisms, bacteria are the most investigated population and have a significant effect on the animal''s performance. However, our understanding of how rumen bacteria change and adapt to different ruminal environments is in its infancy.In the feedlot cattle industry, when animals on a forage diet are directly put on a high-grain diet, a decrease in ruminal pH due to lactate production has been observed (23, 31, 42), which leads to the possibility of digestive disorders, which can cause a decrease in the animal''s performance (23, 45). Therefore, feeding programs have been implemented to adapt feedlot cattle from a high-forage diet to a high-concentrate diet by gradually increasing the concentration of grain in the diet and decreasing the fiber content (2, 35). During this adaptation to high-grain diets, significant changes in the ruminal environment and rumen bacterial population structure have been reported (17, 46, 48). However, the microbial changes that occur during this transition phase are poorly understood (17, 21, 26, 46). Studies performed to date have utilized culture-based techniques or have looked at the fluctuation of a few indicator bacteria (48, 47) to evaluate bacterial population changes. Due to limitations in culturing rumen bacteria, the use of culture-based techniques to evaluate bacterial populations substantially underestimates the diversity of microorganisms within the rumen. In this study, we have utilized culture-independent approaches to evaluate bacterial population structure and diversity using terminal restriction fragment length polymorphisms (T-RFLPs) and sequence analysis of 16S rRNA gene libraries to compare the rumen bacterial population structure in animals on prairie hay against that in animals adapting to a high-concentrate (high-grain) diet. We have also quantified the fluctuations in the populations of previously reported indicator bacterial species using quantitative real-time PCR (qRT-PCR) to assess the role of these organisms during adaptation to a high-concentrate diet.     

Copper Sulfate-induced Fermentation Changes in Continuous Cultures of the Rumen Microbial Ecosystem

The effect of CuSO(4) on fermentation was studied in a continuously cultured rumen ecosystem. CuSO(4), introduced at a level of 50 mg/500 ml of culture volume twice daily, caused a marked inhibition of fermentation of concentrates. Fermentation of alfalfa hay was not inhibited by the same CuSO(4) concentration when the inoculum for the culture was obtained from a cow maintained on a normal concentrate ration. When the inoculum was from a cow on a high concentrate ration, hay fermentation was partially inhibited by CuSO(4). Concentrations of CuSO(4) that did not inhibit the fermentation of alfalfa hay or hay-concentrate mixtures caused preferential production of propionic acid and decreased production of methane.     

Microbial Phospholipid Synthesis as a Marker for Microbial Protein Synthesis in the Rumen

Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with (33)P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of (33)P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with (33)P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs.     

A Continuous Culture Device for Protozoan Cells

SYNOPSIS. A continuous culture device suitable for controlled growth of Euglna gracilis is described. It could probably be adapted to other similar cell types with generation times no greater than 150–200 hrs.     

Electronically Controlled Continuous Culture Device

A photocell-controlled continuous culture device, a Nephelostat, is described that maintains a wide variety of cultures of microorganisms in balanced growth. This Nephelostat controls concentrations of bacteria within +/-3% over a cell concentration range of 10(6) to 10(9) cells per ml. Growth rates are recorded so that changes in the growth rate are observed over small increments of time. Spontaneous and caffeine-induced mutation rates of two strains of Escherichia coli were compared under Nephelostat and chemostat conditions.     

A Continuous Culture System for Assessing Microbial Activities in the Piezosphere

Continuous culture under elevated pressures is an important technique for expanding the exploration of microbial growth and survival in extreme environments associated with the deep biosphere. Here we present a benchtop stirred continuous culture bioreactor capable of withstanding temperatures ranging from 25 to 120°C and pressures as high as 69 MPa. The system is configured to allow the employment of media enriched in dissolved gases, under oxic or anoxic conditions, while permitting periodic sampling of the incubated organisms with minimal physical/chemical disturbance inside the reactor. In a pilot experiment, the fermentative growth of the thermopiezophilic bacterium Marinitoga piezophila was investigated continuously for 382 h at 65°C and at pressures ranging from 0.1 to 40 MPa while the medium flow rate was varied from 2 to 0.025 ml/min. The enhanced growth observed at 30 and 40 MPa and 0.025 ml/min supports the pressure preferences of M. piezophila when grown fermentatively. This assay successfully demonstrates the capabilities of the bioreactor for continuous culturing at a variety of dilution rates, pressures, and temperatures. We anticipate that this technology will accelerate our understanding of the physiological and metabolic status of microorganisms under temperature, pressure, and energy regimes resembling those of the Earth''s piezosphere.     

具有变消耗率微生物连续培养模型的定性分析

研究了一类具有变消耗率的微生物连续培养系统,当消耗率是线性函数时得到了正平衡点全局渐近稳定的充要条件,当消耗率是二次函数时得到了系统存在极限环的充分条件,同时利用分支理论研究系统存在Hopf分支的条件,判定了极限环的稳定性．     

Method for Measuring Microbial Growth in Rumen Content

Radioactive sodium sulfide was used to label the sulfide pool of rumen contents in vitro. Microbial protein synthesis was calculated from the size and rate of dilution of label in the sulfide pool, and from the radioactivity incorporated into protein together with a conversion factor specifying the nitrogen-sulfur ratio determined for microbial protein. The microbial cell yield, calculated on the basis of the adenosine triphosphate (ATP) available from fermentation, was 13 to 14 g (dry weight) per mole of ATP, which is in good agreement with the values obtained for pure cultures of bacteria. Calculation of microbial protein yield per kilogram of ration agreed fairly well with previous estimates for similar rations.     

Rumen Bacterial Competition in Continuous Culture: Streptococcus bovis Versus Megasphaera elsdenii

Streptococcus bovis and Megasphaera elsdenii were grown in continuous culture with maltose as the limiting substrate at dilution rates of 0.36, 0.22, and 0.12 h. After each steady-state turnover, the pH was decreased by adding concentrated HCl to the medium reservoir. Relative counts of the two species at each dilution rate indicated that when the pH was high (6.6 to 6.0), higher dilution rates selected for a higher ratio of S. bovis to M. elsdenii. At intermediate pH (6.0 to 5.4), higher dilution rates once again selected for greater numbers of S. bovis in relation to M. elsdenii, but the increase in S. bovis numbers was much less at the 0.36-h dilution rate. Decreasing the pH below 5.4 caused the ratio of S. bovis to M. elsdenii to increase greatly, and no M. elsdenii cells were seen below pH 5.1. The ratio of the two species could be explained by their relative affinities for maltose if pH was greater than 6.0, but the lower relative numbers of S. bovis in the 0.36-h, intermediate-pH (6.0 to 5.4) incubations could not. Analysis of lactate production by S. bovis in pure culture showed that l-lactate was produced only if the pH was less than 5.2 at dilution rates of 0.22 and 0.12 h and less than 6.0 at a rate of 0.36 h. The lower numbers of S. bovis relative to M. elsdenii in the incubations with a dilution rate of 0.36 h and intermediate pH thus could be explained by utilization of l-lactate by M. elsdenii. The very high numbers of S. bovis at pH less than 5.4 were consistent with the greater tolerance of this organism to low pH.     

Rumen Microbial Ecology in Mule Deer

Mule deer rumen microbial populations from animals in the natural habitat in Utah and from captive deer fed various rations were studied. The microorganisms were characterized on the basis of morphology and Gram reaction. Rumen samples contained 13 identifiable types of bacteria and one genus of ciliate protozoa (Entodinium). Highest rumen bacterial populations were produced on rations containing barley. No differences in proportions of ruminal bacteria in the various morphological groups could be detected when animals were fed either natural browse plants or alfalfa hay. The total numbers of bacteria were similar for animals feeding on controlled diets of browse or hay and those in the natural habitat. Numbers of some bacterial types were directly related to ciliate protozoal numbers, whereas others were inversely related. Highest rumen ciliate protozoal populations were observed on rations containing barley. No differences in protozoal populations were noted between diets containing only browse or hay. Seasonal variations were noted in ciliate protozoal numbers from deer feeding in the natural habitat. The total number of ciliate protozoa decreased in the fall and winter and remained low until spring. There were indications that salt in the deer diet favorably affected rumen ciliate protozoa. Rather than revealing direct deer management applications, this study serves to stimulate and illuminate new approaches to research in range and wildlife nutrition.     

Automatic pH Control and Soluble and Insoluble Substrate Input for Continuous Culture of Rumen Microorganisms

An artifical rumen continuous culture with pH control, automated input of water-soluble and water-insoluble substrates, controlled mixing of contents, and a collection system for gas is described.     

A Comparison of pH-controlled and Dissolved Oxygen-controlled Nutrient Addition for the Maintenance of Steady-state in a Mixed Continuous Culture

Controlled substrate addition was used to maintain mixed microbial cultures in fermenters at either pH 7.0 or 70% dissolved oxygen saturation. Control of pH permitted a greater volume of substrate to be processed. Ammonium nitrogen concentrations were similar for both fermenters but concentrations of oxidized nitrogen varied. Nitrification/denitrification sequences appeared to be initiated by unscheduled changes in dissolved oxygen concentration. It was possible to maintain a steady state with respect to a controlled parameter and end-product quality but other parameters fluctuated.     

微生物连续发酵模型及正解的存在性与稳定性分析

本文引入一个新的非线性动力系统,描述Klebsiella pneumoniae以甘油为底物发酵生成1,3-丙二醇的过程.此模型考虑了细胞内物质的浓度,研究甘油主动运输及1,3-丙二醇被动扩散的跨膜方式.证明模型解的存在唯一性,通过引入微分包含和上哈密尔顿函数证明正解的存在性.此外,以稀释速率0.1为例计算平衡点,并对系统的稳定性做出分析.     

Consequences to the Host of Changes in Rumen Microbial Activity

BOTH the microbial species in the rumen and the composition of the short chain fatty acids produced there as waste products of fermentation are influenced by the chemical composition, physical form and, in some circumstances, the amount of the diet. Thus, in cattle receiving barley cubes to 80% of the limit of appetite there was a high proportion of butyric acid in the mixture of short chain fatty acids associated with a large population of ciliate protozoa. With barley cubes given ad libitum there was a high proportion of propionic acid, a reduction in the number of ciliates and an increase in the bacterioides-type rods and certain curved Gram negative rods¹.