Synergism in Degradation and Utilization of Intact Forage Cellulose, Hemicellulose, and Pectin by Three Pure Cultures of Ruminal Bacteria

Pure cultures of ruminal bacteria characterized as using only a single forage polysaccharide (Fibrobacter succinogenes A3c, cellulolytic; Bacteroides ruminicola H2b, hemicellulolytic; Lachnospira multiparus D15d, pectinolytic) were inoculated separately and in all possible combinations into fermentation tubes containing orchard grass as the sole substrate. Fermentations were run to completion, and then cultures were analyzed for digestion of cellulose plus degradation and utilization of hemicellulose and pectin. Addition of the noncellulolytic organisms, in any combination, to the cellulolytic organism F. succinogenes had little effect on overall cellulose utilization. F. succinogenes degraded but could not utilize hemicellulose; however, when it was combined with B. ruminicola, total utilization of hemicellulose increased markedly over that by B. ruminicola alone. L. multiparus was inactive in hemicellulose digestion, alone or in any combination. Although unable to degrade and utilize purified pectin, B. ruminicola degraded and utilized considerable quantities of the forage pectin. In contrast, L. multiparus was very active against purified pectin, but had extremely limited ability to degrade and utilize pectin from the intact forage. Both degradation and utilization of forage pectin increased when F. succinogenes was combined with B. ruminicola. Sequential addition of two cultures, allowing one to complete its fermentation before adding the second, was used to study synergism between cultures on forage pectin digestion. In general, synergistic effects did not appear to be related to a particular sequence of utilization. The ability of F. succinogenes to degrade and B. ruminicola to degrade and utilize forage pectin contradicts both previous and present data obtained with purified pectin. Thus, isolation and characterization of ruminal bacteria on purified substrates may be misleading with regard to their role in the overall ruminal fermentation.     

Degradation and Utilization of Hemicellulose from Intact Forages by Pure Cultures of Rumen Bacteria

Several pure strains of rumen bacteria have previously been shown to degrade isolated hemicelluloses from a form insoluble in 80% acidified ethanol to a soluble form, regardless of the eventual ability of the organism to utilize the end products as energy sources. This study was undertaken to determine whether similar hemicellulose degradation or utilization, or both, occurs from intact forages. Fermentations by pure cultures were run to completion by using three maturity stages of alfalfa and two maturity stages of bromegrass as individual substrates. Organisms capable of utilizing xylan or isolated hemicelluloses could degrade and utilize intact forage hemicellulose, with the exception of two strains of Bacteroides ruminicola which were unable to degrade or utilize hemicellulose from grass hays. Intact forage hemicelluloses were extensively degraded by three cellulolytic strains that were unable to use the end products; in general, these strains degraded a considerably greater amount of hemicelluloses than the hemicellulolytic organisms. Hemicellulose degradation or utilization, or both, varied markedly with the different species and strains of bacteria, as well as with the type and maturity stage of the forage. Definite synergism was observed when a degrading nonutilizer was combined with either one of two hemicellulolytic strains on the bromegrass substrates. One hemicellulolytic strain, which could not degrade or utilize any of the intact bromegrass hemicellulose alone, almost completely utilized the end products solubilized by the nonutilizer. Similar synergism, although of lesser magnitude, was observed when alfalfa was used as a substrate.     

Rate of isolated hemicellulose degradation and utilization by pure cultures of rumen bacteria

Rate studies on the utilization or degradation (or both) of isolated hemicelluloses were conducted with six strains of rumen cellulolytic bacteria. Utilization was estimated by total pentose loss, and degradation values were based on solubilization of the hemicellulose in acidified 80% ethyl alcohol. With the various strains of ruminococci, degradation of flax and fescue grass hemicellulose was near the maximum within the first 12 hr of incubation. However, where applicable, the rates of utilization were considerably slower. Both degradation and utilization of corn hull hemicellulose occurred at much slower rates than observed with the other two substrates. With flax and fescue grass hemicellulose, the rates of degradation did not appear to be influenced by the organism's ability, or inability, to utilize the substrate as an energy source. The rates and extent of isolated hemicellulose degradation and utilization were compared between the cellulolytic ruminococci and three strains of bacteria isolated from the rumen with a xylan medium. Similar values were obtained with both types of bacteria. These observations would suggest that the cellulolytic ruminococci may be important in the overall fermentation of forage hemicelluloses in the rumen. The acidified 80% ethyl alcohol supernatant fluids, obtained from fermentations of isolated fescue grass hemicellulose by two strains of Ruminococcus flavefaciens, of which only one was able eventually to utilize the substrate, were investigated by thin-layer chromatography. Results indicated that soluble oligosaccharides were produced, which were observed to disappear gradually with time in fermentations with the utilizing strain and to accumulate in fermentations with the nonutilizing strain. Examination of the acidified 80% ethyl alcohol-insoluble residue hydrolysates, obtained from fermentations with the utilizing strain, revealed that the concentration of all the constituent sugars decreased uniformly.     

Selection strategies for developing smooth bromegrass cell wall ideotypes

Summary A random sample of 80 families of the B8HD smooth bromegrass (Bromus inermis Leyss.) population were tested in three environments for forage yield and cell wall constituents. Expected progress from one cycle of family selection was computed for single-trait selection and multiple-trait restricted selection. Expected gains were compared to desired goals and actual results from one cycle of phenotypic selection. Desired goals were: Model I = reduced lignin and cellulose, with increased hemicellulose, resulting in no change in cell wall content; Model II = reduced lignin and cellulose with no change in hemicellulose; or Model III = reduced lignin, cellulose, and hemicellulose. Single-trait selection for high hemicellulose in first harvest or low cellulose in second harvest had the best expected responses, of any single trait, for Model I. Possible undesirable effects of selection for low cellulose would be a reduction in forage yield potential. Multiple-trait restricted selection was judged to be more effective, with responses all in the desired direction, by specifying increased hemicellulose in index development. Selection in second harvest was expected to have similar responses as first harvest, except for a greater increase in forage yield. Development of Models II or III is expected to be difficult due to a negative correlation estimate between first and second harvest cell wall concentration.     

Single cell protein from various chemically pretreated wood substrates using Chaetomium cellulolyticum

The growth behavior of Chaetomium cellulolyticum, a new cellulolytic fungus, has been examined in slurry fermentation systems using various chemically pretreated sawdusts from hardwoods as substrates. Both acid- and alkali-pretreatment methods were used and the fermentation media included the spent pretreatment liquor in an attempt to concurrently maximize substrate utilization and minimize the biological oxygen demand (BOD) level in the process effluent. Diauxic growth patterns were found in the three cases studied, suggesting an initial utilization of soluble hemicellulose sugars followed by utilization of the insoluble cellulose. This behavior patterns was supported by separate growth experiments using the major sugars of hemicellulose as carbon sources. The organism was found to be a good convertor of both cellulose and hemicelluloses into single cell protein (SCP). In terms of rate and extent of protein production in the insoluble biomass product, acid pretreatment appears to be better than alkali pretreatment if the product is intended as ruminant feed.     

Fermentation of Isolated Pectin and Pectin from Intact Forages by Pure Cultures of Rumen Bacteria

Studies on the rate and extent of galacturonic acid and isolated pectin digestion were carried out with nine strains of rumen bacteria (Butyrivibrio fibrisolvens H10b and D16f, Bacteroides ruminicola 23 and D31d, Lachnospira multiparus D15d, Peptostreptococcus sp. D43e, B. succinogenes A3c, Ruminococcus flavefaciens B34b, and R. albus 7). Only three strains, 23, D16f, and D31d, utilized galacturonic acid as a sole energy source, whereas all strains except A3c and H10b degraded (solubilized) and utilized purified pectin. Nutrient composition of the basal medium and separate sterilization of the substrate affected the rate and extent of fermentation for both substrates. Pectin degradation and utilization were measured with two maturity stages each of intact bromegrass and alfalfa. For bromegrass I, all strains tested (B34b, 23, D16f, D31d, D15d, and D43e) degraded a considerable amount of pectin and, with the exception of B34b, utilized most of what was degraded. Similar, but lower, results were obtained with bromegrass II, except for the two strains of B. ruminicola, 23 and D31d, which were unable to degrade and utilize pectin from this forage. All strains were able to degrade and utilize pectin from both maturity stages of alfalfa; however, values were considerably lower for strains 23 and D31d. Synergism studies, in which a limited utilizing strain, B34b, was combined with the limited degrading strain, D31d, resulted in a slight increase in degradation and a very marked increase in utilization of the pectin in all four forages. Similar results were obtained on both alfalfa substrates with a combination of strains B34b and D16f; however, no increases were observed with this combination on bromegrass.     

Resistance of Bacillus Spores to Combined Sporicidal Treatments

S ummary . Moist heat at 82° (100° for Bacillus stearothermophilus ) and solutions of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride at 24° separately showed no sporicidal activity against B. pumilis, B. stearothermophilus, B. subtilis and B. subtilis var. niger . Spores of the last organism were the most sensitive to γ radiation, the D value being 0.16 Mrad. Prior irradiation with a dose of 0.16 Mrad brought about only a slight increase in the sensitivity of the spores to moist heat. The presence of bactericide during irradiation did not affect radiation resistance. Inactivation rates were greater when the spores were heated in the presence of a bactericide than in aqueous suspension and benzalkonium chloride was more active than chlorocresol. Chlorocresol enhanced the heat activation of B. stearothermophilus at 100°. Irradiation in the presence of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride had no effect on the subsequent resistance of the spores when heated in the presence of these bactericides. It is concluded that it is unlikely that combinations of moist heat, radiation and bactericides, each less severe than when used in an accepted sterilization process, will lead to an alternative process which, while less damaging to the materials being sterilized, would still maintain the accepted standards of freedom from contamination.     

Extraction of hemicellulose from ryegrass straw for the production of glucose isomerase and use of the resulting straw residue for animal feed

The hemicellulose fraction of ryegrass straw was extracted with NaOH and used for the production of glucose isomerase by Streptomyces flavogriseus. The level of hemicellulose extracted increased proportionately with increasing NaOH concentration up to about 4%, then the rate of increase slowed down. Hemicellulose extraction was facilitated by the combined application of heat and NaOH. Approximately 15% hemicellulose (12% as pentosan) could be obtained by treating straw with 4% NaOH for either 3 hr at 90°C or 24 hr at room temperature. The highest level (3.04 units/ml culture) of intracellular glucose isomerase was obtained when the organism was grown at 30°C for two days on 2% straw hemicellulose. The organism also produced a high yield of glucose isomerase on xylose or xylan. The NaOH-treated straw residue, after removal of hemicellulose, had approximately 75% higher digestibility and 20% higher feed efficiency for weanling meadow voles than untreated straw. Thus, the residue could be used as animal feed. A process for the production of glucose isomerase and animal feed from ryegrass straw was also proposed.     

Mechanism of Isolated Hemicellulose and Xylan Degradation by Cellulolytic Rumen Bacteria

Although certain strains of cellulolytic rumen bacteria cannot utilize isolated hemicelluloses or xylan as a source of energy, all strains examined can degrade or solubilize these materials from an 80% ethyl alcohol insoluble to a soluble form. Centrifugation and washing of the cellobiose-grown bacterial cells did not affect the rate or extent of utilization or degradation or both. When the level of a nonutilizing culture inoculum (either normal or washed) was doubled, a corresponding increase in the initial rate of degradation was observed. With a nitrogen-free medium, utilization of xylan was almost completely inhibited for a utilizing strain, whereas degradation by either type of organism was not markedly affected. Cellobiose medium cell-free culture filtrates from a nonutilizing strain were able to degrade or solubilize xylan. The percentage of degradation increased with the volume of cell-free filtrate, and all activity was lost when the filtrate was boiled. No utilization (loss in total pentose) was observed with cell-free filtrates from utilizing or nonutilizing strains. The release of free hexose from insoluble cellulose by culture filtrates from a nonutilizing strain was very limited. On the other hand, carboxymethylcellulose (CMC-70L) and cellulodextrins were degraded to an 80% ethyl alcohol soluble form by filtrates from both types of organisms. Similar enzyme activity was obtained in cell-free culture filtrates from four additional strains of cellulolytic rumen bacteria (one xylan utilizer and three nonutilizers). When the assays were carried out aerobically, CMC-70L solubilization was reduced to a much greater extent than xylan or cellulodextrin solubilization. The enzyme or enzymes responsible for the degradation of hemicellulose by cellololytic rumen bacteria unable to utilize the hemicellulose as an energy source appear to be constitutive in nature, and this activity may be a nonspecific action of a beta-1, 4-glucosidase or -cellulase.     

Ferric hydroxamate transport without subsequent iron utilization in Bacillus megaterium.

Iron transport and utilization were examined in Bacillus megaterium Ard1, a mutant that is resistant to the hydroxymate antibiotic A22765 and whose growth is inhibited by the structurally similar hydroxamate Desferal. Rapid, low-level uptake of Desferal-50Fe was observed; such uptake was temperature and energy independent. Gel filtration chromatography of the cytoplasmic fraction of protoplasts labeled with Desferal-55Fe for 30 to 120 s demonstrated only unchanged esferal-55Fe in the cytoplasm. Although B. megaterium Ard1 showed transport of Desferal-59Fe by a process that resembles facilitated diffusion, this organism was unable to transfer iron from this chelate to cellular macromolecules for metabolic use. High-level transport of the ferric hydroxamate schizokinen-59Fe by B. megaterium Ard1 was both temperature and energy dependent. Within 30 s, protoplasts labeled with schizokinen-55Fe contained iron associated with certain macromolecules and in an apparent "pool" of schizokinen-55Fe in the cytoplasmic fraction. Prior transport of Dseferal-55Fe by protoplasts of strain Ard1 did not interfere with subsequent transport and utilization of schizokinen-59Fe. These studies suggest that transport of ferric hydroxamates may occur by a facilitated diffusion-type process; transfer of iron to cellular macromolecules may drive high-level transport of the chelate and may be the step at which energy is required in the iron transport-assimilation process.     

基于产量效应的间作紫花苜蓿/禾本科牧草光能利用特征

为探究紫花苜蓿/禾本科牧草间作下光能利用特性、光能利用诸因素的产量效应及其调控机理,通过2017—2019年3年田间试验,以紫花苜蓿、饲用小黑麦(C3植物)、饲用玉米(C4植物)3种单作模式为对照,研究了紫花苜蓿/小黑麦和紫花苜蓿/玉米两种间作模式下的产量效应、光能利用各因子对产量形成的影响、光能利用特征差异及机理。结果表明: 两种间作模式的土地当量比均大于1,表明两种间作模式的土地利用率都高于单作,均有高于单作的产量效益,且增产潜力较大的是紫花苜蓿/小黑麦间作模式。光能利用各因子对产量的贡献依次是: 叶面积指数(1.531)＞净光合速率(0.882)＞胞间CO₂浓度(0.282)＞蒸腾速率(-0.229)＞冠层开度(-0.291)＞光合有效辐射截获率(-0.681)＞气孔导度(-0.751)。其中,叶面积指数不仅是表征光合能力的重要指标之一,更是以收获营养体为目标的牧草作物产量的重要构成因子,光合特性诸因素中净光合速率是影响产量的主要因子。与单作相比,间作下紫花苜蓿、小黑麦、玉米的净光合速率均存在差异,且表现为相同的规律。间作下净光合速率提高的主要途径为: 小黑麦和玉米通过增强CO₂的羧化固定能力,提高对强光的利用能力,从而提高净光合速率,促进产量增加;而紫花苜蓿则是通过提高功能叶的叶绿素b含量,改变叶绿素构成,增强对光能的收集和传递,从而提高净光合速率,促进其在弱光下光合能力的提高和正常生长。     

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C₅ and C₆ sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of β-xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.     

On the Nature of the Heat Stabilizing Effect of Inorganic Ions on Escherichia coli

The growth lag of Escherichia coli at 45°C was reduced by the addition of sodium, potassium, magnesium and calcium ions to the growth medium. A method to quantitatively determine the lag-reducing effect of these ions was developed. The results obtained showed that equivalent amounts of the ions produced the same reduction of the growth lag. According to the results of plasmolysis experiments cells of E. coli suspended in peptone broth were permeable to all four ions. The course of plasmolysis and subsequent deplasmolysis was registered as changes in the cells' ability to scatter light. The heat stability of catalase from E. coli was increased by addition of the four ions. This was observed in experiments with intact cells and with a crude cell-free preparation of catalase. The results of our experiments are most easily explained by assuming a stabilizing effect of the ions tested on the intracellular bacterial proteins.     

Evaluation of Processing Technology for Triarrhena sacchariflora (Maxim.) Nakai for Ethanol Production

The effects of dilute H₂SO₄ concentration, forage:sulfuric acid ratio, digestion time, and digestion temperature were evaluated to determine effects on ethanol yield of Triarrhena sacchariflora (Maxim.) Nakai. Twenty single factor experiments were conducted to evaluate H₂SO₄ concentration (0.5, 1.0, 1.5, 2.0, and 2.5%, w/w), forage:sulfuric acid ratio (1∶6, 1∶8, 1∶10, 1∶12, and 1∶14, g/ml), digestion time (15, 30, 45, 60, and 90, min), digestion temperature (80, 100, 110, 120, and 125 °C) for 3 replicates of the 5 levels of each factor. Based on results of the single factor experiments, an incomplete factorial was designed to evaluate ethanol yield from the best combinations of single factors. Finally, the best combination was tested by enzymatic hydrolysis and fermentation experiment in selected combinations according to pretreatment results. Percentage cellulose, hemicellulose, and lignin contents of forage residue after pretreatment, and glucose and xylose concentrations of the filtrate were analyzed prior to enzymatic hydrolysis, and percentage crystallinity was observed in untreated grass and pretreated residue. In addition, the solid residues were then hydrolysed and fermented by cellulase and yeast, the concentrations of glucose and ethanol being monitored for 96 h. Results showed that the order of the effect of main effect factors was as follows: digestion temperature > dilute H₂SO₄ concentration > digestion time > forage:sulfuric acid ratio. The best process parameters evaluated were sulfuric acid concentration of 1.5%, forage:sulfuric acid ratio of 1∶6, digestion time of 15 min, and digestion temperature of 120°C. With this combination of factors, 80% of the cellulose was hydrolysed in 96 h, and 78% converted to ethanol. The findings identified that hemicelluloses were the key deconstruction barrier for pretreatment of Triarrhena sacchariflora (Maxim.) Nakai for ethanol production. The results of this research provide evidence of appropriate combinations of processing factors for production of ethanol from Triarrhena sacchariflora (Maxim.) Nakai.     

The Relationship between Chiasmata and Crossing over in TRITICUM AESTIVUM

Telocentrics for the β arm of chromosome 4A and the long arm of 6B were used as cytological markers for the determination of chiasma frequency. In concomitant studies of recombination, terminal segments of rye and T. umbellulatum chromatin carrying Hp (Hairy peduncle) and Lr9 (Leaf-rust resistance), respectively, marked 4A and 6B. Two temperatures, 21° and 32°, were used for both the 4A and 6B experiments.—Only one chiasma was observed in each heteromorphic bivalent. Because there was a substantial reduction in pairing between diakinesis and metaphase I, all determinations of chiasma frequency were made at diakinesis. In the 21° experiments, agreement was good between genetic recombination and cytological prediction on the basis of the partial chiasmatypy hypothesis that each chiasma represents a crossover. At 32° both chiasma frequency and crossing over, but particularly the latter, were strongly reduced. The fewer crossovers than expected are explained in part by stickiness of chromosomes at the high temperature, sometimes resulting in adjacent chromosomes being wrongly scored as having a chiasma, and in part by premetaphase disjunction of some recombined bivalents and subsequent independent behavior of the two resulting univalents.—Male transmission of the 4A telocentric from the heteromorphic bivalent was unusually high: 51% at 21° and 31% at 32°.     

Alkali solubility of hemicelluloses in relation to delignification

The polysaccharide composition of bark from Pinus radiata, Salix fragilis, and Populus euramericana has been determined. All the barks contained lower levels of cellulose and hemicellulose than the corresponding woods; cellulose: hemicellulose ratios were also lower in the barks. Alkali extracted all of the hemicellulose-A but only half of the hemicellulose-B from P. radiata bark without prior delignification. Similar alkaline extraction removed almost all of the hemicellulose (A + B) from ryegrass leaves without delignification. With the other samples tested only a part of the hemicellulose A and B is extracted without delignification. It is suggested that the polysaccharide so extracted represents wall hemicellulose which is not linked to lignin or other wall constituents by alkali-stable links.     

THE INCIDENCE AND THERMAL RESISTANCE OF MESOPHILIC SPORES FOUND IN MILK AND RELATED ENVIRONMENTS

SUMMARY: A spore 'spectrum' is described of aerobic mesophiles capable of resisting different heat treatments. It is shown that B. licheniformis is the most common spore former found in bulk milk but since its spores are rapidly destroyed at 100°, the more heat resistant B. subtilis is the dominant surviving spore former in commercial sterilized milk. The thermal resistance of strains of B. subtilis and B. licheniformis isolated from different sources has been investigated and the strains of B. subtilis typed according to the behaviour of their spores when heated at 100°. All strains of B. licheniformis were destroyed more rapidly by boiling for 2 min than strains of B. subtilis but only those strains of the latter which showed some degree of heat activation were more resistant than B. licheniformis . The 'resistant' and heat activated strains of B. subtilis appear to be sparsely distributed in nature and were only isolated from sterilized milk where the heat treatment applied would tend to eliminate other strains. The spore content of bovine faeces was similar to that in bulk milk and the total spore content varied seasonally, the spore content of faeces being on the average a hundred times greater during indoor feeding than during the period when the cattle were fed outside. A faecal infection of the milk in the ratio of 1:10⁴ would infect the milk with spores at about the same concentration as they are found in bulk raw milk, and it is suggested that bovine faeces could be a primary source of spore formers in milk supplies.     

Butanediol production from lignocellulosic substrates by Klebsiella pneumoniae grown in sequential co-culture with Clostridium thermocellum

Summary A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to butanediol and ethanol. Growth of Clostridium thermocellum on solka floc and aspenwood xylan resulted in the release of extracellular endoglucanase and xylanase enzymes into the culture medium. Low levels of fermentation products were formed and unutilized sugars accumulated in the medium. Inoculation of Klebsiella pneumoniae as a sequential culture resulted in the rapid utilization of the accumulated sugars and the formation of additional fermentation products, including butanediol, ethanol, and acetoin. This approach was applicable to the use of mixed cellulose and hemicellulose substrates, including steam-exploded aspenwood. Further improvement in solvent production from steam-exploded substrates could be obtained by using a fed-batch approach to circumvent the problem of inhibitors associated with the natural substrates.     

Plow down effects of different forage legume species,cultivars, cutting strategies and seeding rates on the yields of subsequent crops

Summary Under some conditions the plow down of forage legumes increases the yield of subsequent crops, which is usually caused by improved soil N. However, better soil structure is also a contributing factor. Three experiments were conducted to measure the effect of legume plow down on the yield of subsequent corn crops grown at the Ottawa Research Station (ORS), Ottawa, Canada. In all experiments, corn yields were not affected by legume species, legume cultivars, and/or planting methods. Corn yields from barley plots receiving 0, 60, or 120 kg N ha⁻¹ did not differ until two years after establishment in one experiment and three years in another. The data from these experiments indicated that soil N was high at the ORS, which may inhibit N₂-fixation by forage legumès in the establishment year. Therefore, legume plow down was not beneficial to subsequent crops under these conditions. Two other experiments were conducted to measure the effect of legume plow down on the yield of subsequent barley crops. In both experiments, barley yields in the field were not affected by legume type or legume seeding density. Greenhouse and field data indicated that the two cuts with removal strategy benefitted the most to succeeding crops. Data from the greenhouse test indicated that soil N levels were not low in the establishment year, and that some cultivars improved soil fertility more than others.     

Impact of Inoculum Preparation and Storage Conditions on the Response of Escherichia coli O157:H7 Populations to Undercooking and Simulated Exposure to Gastric Fluid

This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75°C) water followed by lactic acid (2%, 55°C), vacuum packaged, stored at 4 (28 days) or 12°C (16 days), and periodically transferred to aerobic storage (7°C for 5 days). During storage, samples were exposed to sequential heat (55°C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4°C was lower than that of cells on nondecontaminated meat stored at 12°C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12°C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4°C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12°C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.