A model of clostridial dominance in ensilage

A simulation model of the growth and fermentation of lactic acid bacteria and clostridia in silage is described. The model is used to predict the occurrence of a well-preserved lactate silage or a poorly-preserved clostridial silage. Results of the model are compared with published experimental studies not used in the model's development. The results are summarized with maps showing the combinations of initial water-soluble-carbohydrate content, crop buffering capacity, dry matter content, temperature, initial pH, and initial population of lactic acid bacteria which result in a clostridial silage. The specific processes modelled include the growth and death of lactic acid bacteria and clostridia; the use of substrate and accumulation of fermentation end products; release of ammonia; and change in silage pH.     

A model of aerobic fungal growth in silage.

The components of a simulation model of mesophilic and thermophilic yeast and mould growth in aerobically deteriorating silage are presented. The purpose of the model is to examine the relative roles of yeasts and moulds in deterioration and the factors affecting aerobic stability. Information for the model was based on literature studies. Growth of the fungi was assumed to be affected by temperature, pH, water activity, and lactic and acetic acid concentrations. The substrates utilized in the model, in order of preference, were water-soluble carbohydrates, ethanol, lactic acid and acetic acid. Gas movement and heat transfer were not considered. Consequently, oxygen and carbon dioxide concentrations were assumed to be those for open air, and the heat of respiration retained in the silage was set as a constant percentage of that released by fungal growth.
Based on the relationships developed for the model, pH affects yeast growth minimally and does not affect mould growth at all. Water activity over the range in silage affects yeast growth more than mould growth, but affects neither one strongly. Undissociated lactic and acetic acids decrease growth rate and may be important factors in silage stability. Compared with moulds, yeast growth rates are less affected by suboptimum temperatures but are more sensitive to temperatures in excess of the optimum.
A succeeding study compares the model with published aerobic deterioration studies and examines the predicted succession of microbial groups and the variation in silage stability as affected by silage parameters, initial temperature, and initial microbial populations.     

Growth performance, appetite and plasma ammonia concentrations in heifers fed inoculated lucerne prior to ensilage, then supplemented with sodium bicarbonate

A study was conducted to explore the possibility of a synergistic effect of an inoculum of lactic acid bacteria and sodium bicarbonate in improving the nutritive value of lucerne silage. Twenty Holstein heifers (live weight 183 ± 9·4 kg) were assigned to four dietary treatments consisting of: lucerne uninoculated or inoculated prior to ensilage and then either unsupplemented or supplemented with NaHCO₃ (40 g kg⁻¹ dry matter (DM)). The lucerne silage contained 320 g kg⁻¹ DM and constituted 600 kg⁻¹ diet DM. The animals were fed once daily for 90 d and samples of jugular blood were analysed for plasma ammonia and indices of blood acid-base status. Inoculation of Lucerne reduced ( P <0·01) silage pH but had no other effect on silage composition. In the absence of NaHCO₃, liveweight gain and organic matter intake were improved by inoculating lucerne prior to ensilage; however, in the presence of NaHCO₃, inoculation reduced growth rate but had no major effect on intake. Dietary treatment did not (P>0·05) affect the concentration of plasma ammonia or measures bf blood acid-base status. The results suggest that NaHCO₃ may have nullifled any potential benefit of inoculating lucerne silage to improve its nutritive value for heifers.     

The effect of addition of sugar and inoculation with two commercial inoculants on the fermentation of lucerne silage in laboratory silos

Lucerne (DM 236 g kg^-1, WSC 49 g (kg DM)^-1) was ensiled in test-tube silos with or without either glucose or fructose and with or without one of two commercial inoculants. The WSC content of the forage as ensiled was too low to obtain a well preserved untreated silage. By day 4 the pH values of the silages with added sugar or inoculant were significantly lower (P< 0·001) than the control silage. A satisfactory fermentation was attained only in the silages to which sugar and an inoculant had been added. These silages had a lower pH, more protein-N (P< 0·001), less ammonia-N (P<0·001), a faster increase in counts of lactic acid bacteria, and decrease in counts of coliforms than the other silages. Homo-fermentative lactic acid bacteria dominated the fermentation in the inoculated silages while leuconostocs dominated the early stages of fermentation in the control silages. The results indicate that if there is insufficient sugar in the original crop, then the bacteria in an inoculant will not be able to produce enough lactic acid to lower the pH to an acceptable level. This has important implications for the ensilage of lucerne and other highly buffered low sugar crops.     

Fermentation characteristics and aerobic stability of grass silage inoculated with Lactobacillus buchneri, with or without homofermentative lactic acid bacteria

Aerobic spoilage by yeasts and moulds is a major cause of reduced nutritional value of silage and increases the risk of potential pathogenic microorganisms. Recent studies have shown that inoculation with Lactobacillus buchneri inhibits yeast growth and reduces the susceptibility to aerobic spoilage of various ensiled forages. The aim of this study was to determine whether these effects are retained when L. buchneri is added in combination with homofermentative lactic acid bacteria. In three experiments, silages were produced from perennial ryegrass [240–421 g kg⁻¹ dry matter (DM)] inoculated with L. buchneri or L. buchneri plus a mixture of Pediococcus pentosaceus and Lactobacillus plantarum (inoculant PL). Uninoculated silage and silage inoculated with PL alone served as controls. Silages were examined for pH and DM loss in the course of ensilage and chemical and microbiological composition and aerobic stability after 3–4 months. L. buchneri plus PL and PL alone increased the initial rate of pH decline. L. buchneri alone and L. buchneri plus PL enhanced aerobic stability and, in general, reduced yeast and mould counts. In addition, these inoculants increased the final pH and DM loss and the concentrations of acetic acid and 1,2-propanediol (or propionic acid and 1-propanol instead of 1,2-propanediol), and decreased the concentration of lactic acid. The effects of L. buchneri on fermentation products increased with decreasing DM content. In silages of less than 270 g kg⁻¹ DM, L. buchneri increased the ammonia-N concentration. It is suggested that this was associated with the relatively high final pH resulting from the high metabolic activity of L. buchneri in these silages.     

A model of the anaerobic phase of ensiling

A model of ensiling is described which incorporates the interactions of lactic acid bacteria and Clostridia with water soluble carbohydrates. The processes modelled include bacterial growth, the production of lactic and butyric acids from water soluble carbohydrates, and the conversion of lactic acid to butyric acid. The calculation of pH, and the regulation of bacterial activity by pH are important components of the model. The model predicts time courses for pH and the five State variables, which are the concentrations of water soluble carbohydrates, lactic acid, butyric acid, lactic acid bacteria and Clostridia. There is good qualitative agreement between the predictions of the model and experimental data on ensiling.     

A mathematical model of the aerobic deterioration of silage

A mathematical model of the aerobic deterioration of silage is described that includes the physical processes of gas and heat Rows, as well as an established model of microbial growth and the associated chemical changes. Yeasts and acetic acid bacteria are included in the growth model. The version of the model described relates to a cylinder of silage subject to forced aeration, a common experimental configuration. The model was very sensitive to variations in insulation thickness and yeast growth rate, and to initial pH, especially when maize was the forage. Great care is needed when selecting input data for use in the model to be sure that the physical and chemical properties are accurate. The model solution was sensitive to grid size, but with 361 cells, the maximum grid resolution that could be used on a 486 series PC, solutions were achieved with an acceptably small loss of accuracy. The model predicted the results of an experiment on silage deterioration quite accurately. However, the model also predicted that considerable spatial gradients of parameters, such as temperature, pH, and lactic acid, would occur. This indicates the need for care when taking samples or making temperature measurements in deterioration experiments. The model is potentially a useful tool in predicting the optimal time and position to take samples for analysis in a time-course experiment.     

Proteolysis and amino acid degradation during ensilage of untreated or formic acid-treated lucerne and maize

Lucerne ( Medicago sativa ), field wilted to 330 g dry matter (DM) kg⁻¹, and whole-plant maize ( Zea mays ), 383 g DM kg⁻¹, were treated with formic acid (FA) at the rate of 4-5 1 t⁻¹ fresh forage. The effects of FA treatment on proteolysis and amino acid degradation was investigated at several intervals over a 90-d period of ensilage in laboratory silos. Formic acid treatment produced different patterns of pH decline during ensilage of the two forages, suggesting some degree of crop specificity in response to FA treatment. After 7 d of ensilage of lucerne there were increases of 81% in nonprotein nitrogen (NPN), and 104% in free amino acid nitrogen (FAA-N), but with FA-treated lucerne the increases were 39% and 31%, respectively. FA treatment resulted in a reduction in the concentration of most free amino acids in lucerne silage, with the exception of glutamic acid and serine; the branched chain amino acids as well as glycine, tyrosine and proline were significantly (P<0·05) reduced after 3 d of ensilage. By contrast, FA treatment of maize did not significantly ( p <0·05) affect the NPN content nor was there a consistent pattern in the changes in FAA-N. Formic acid treatment of maize did produce a significant reduction ( P <0·05) in the concentration of most individual free amino acids at 90 d of ensilage. Overall, FA treatment had a more predictable effect on proteolysis and amino acid degradation in ensiled lucerne than in ensiled whole-plant maize.     

PLANT JUICES IN RELATION TO SILAGE FERMENTATION

Using ryegrass and lucerne silages, investigations were made into the relationship between plant-cell breakdown, as evidenced by a collapse of the silage mass and by an increase in its electrical conductivity, and the initiation of lactic-acid production. Changes in the water activity of the juices available as a medium for the silage bacteria were also examined. It has been shown that cell breakdown and the resultant release of the plant juices Is a necessary pre-requisite for the production of significant amounts of lactic acid during ensilage. The investigation also confirms that the complete exclusion of fresh air from the silage mass can usually be expected to result in cell breakdown within a few hours.     

The effects of lactic acid bacteria strains isolated from various substrates on the fermentation quality of common vetch (Vicia sativa L.) in Tibet

Eight lactic acid bacteria (LAB) strains isolated from five naturally fermented silages of different ensilage materials on the Tibetan Plateau were characterized, and their effects on the silage quality of common vetch (Vicia sativa L.) were studied. These LAB isolates were evaluated using morphological, physiological and biochemical tests. The eight isolated strains and one commercial inoculant (Lactobacillus plantarum MTD‐1) were subsequently added to common vetch for ensiling 60 days. All the isolated strains (LW4, M1, WG27, O30, I2, LCG3, LCG9, CG35) could grow normally at 5–20°C, pH 3.5–6.0 and NaCl (3.0%, 6.5%), and they were identified as Lactobacillus plantarum and Pediococcus pentosaceus by sequencing 16S rDNA. All the LAB inoculants improved the silage quality of common vetch, indicated by significantly (p < .05) higher lactic acid (LA) contents and ratios of lactic acid to acetic acid (LA/AA), and significantly (p < .05) lower pH and ammonia nitrogen (NH₃–N) contents. Strain LCG3 performed best among all LAB inoculants, indicated by the highest (p < .05) LA content and ratio of LA/AA, and the lowest (p < .05) pH and NH₃–N content. Strain LCG3 is recommended as starter culture for common vetch silage.     

Microscopic and chemical changes during the first 22 days in Italian ryegrass and cocksfoot silages made in laboratory silos

Although many aspects of grass silage have received intensive study, the changes that take place within the grass blades during ensilage have received little attention.
In two factorial experiments Italian ryegrass (cv. Lemtal) and cocksfoot (indigenous) were ensiled in laboratory silos (capacity 0·2 kg) with the grass under pressure (700 Pa) and subjected to two treatments (with or without 3·31 t^-1 85% formic acid) and two silos from each treatment opened on eight sampling dates(1,2,3,4,7,10,14 and 21 d).
For each sampling date transverse sections of grass laminae were examined and changes in the cross sections of the protoplasts and of cells, as defined by the cell walls, were recorded. These data were used to calculate the ratios of protoplast to cell volumes at each sampling date.
Each silage was analysed for dry matter, pH, lactic acid, VFA, ammonia and glucose. The distance settled by the silage (a measure of decrease in silage volume) and the silage conductivity were also recorded.
Both of the untreated grasses yielded silages with typical lactic acid levels and pH values. In all of the silages there was a marked shrinkage of the plast within the space defined by the cell wall. The ratio of protoplast to cell volume eventually stabilized at 0·4. The formic acid-treated grasses reached this level by day 1 but a longer period was required by untreated grasses.
It is suggested that the decrease in silage volume is related to the lactic acid content of the liquid phase due to their close relationship in silages produced from untreated grasses ( r = 0·97***).     

Influence of bacterial inoculant and substrate addition to lucerne ensiled at different dry matter contents

Field-wilted lucerne was chopped with a forage harvester at 33 ± 1·5, 43 ± 2·0 and 54 ± 1·8% dry matter, treated and ensiled in laboratory silos during four cuttings in each of two years. Treatments were control (C), sugar addition at 2% of fresh weight (S), inoculum applied at 3 × 10⁵ bacteria g⁻¹ herbage (I), and sugar and inoculum combined (IS). Duplicate silos were opened and analysed after 1, 2, 3, (4 or 5), 7, 14 and 60 d of fermentation. The initial rate of proteolysis of lucerne protein decreased with increasing dry matter (DM) content of the lucerne, and was not influenced by the year, cutting or silage treatment. Inoculation increased ( P <0·05) the rate of pH decline for all silage dry matters, and shortened the lag time prior to pH decline with 33 and 43% dry matter silages. Sugar addition had no effect on rate of pH decline or lag time. Inoculation and sugar addition both lowered final pH, acetic acid, ammonia (NH₃), free amino acids (FAA) and soluble non-protein N (NPN) in silages ( P <0·01) and increased lactic acid content with 33 and 43% dry matter silages. Only inoculation was beneficial at 54% DM with no difference between I and IS. The influence of forage characteristics (epiphytic lactic acid bacteria, buffer capacity and sugar:buffer capacity ratio) on treatment effectiveness varied with dry matter content.     

Changes in the distribution of nitrogen and plant enzymatic activity during ensilage of lucerne treated with different additives

Effects of formic acid, formaldehyde and two levels of tannic acid on changes in the distribution of nitrogen (N) and plant enzymatic activity during ensilage of lucerne (Medicago sativa) were studied. Lucerne [300 g dry matter (DM) kg^?1 forage] silages were prepared untreated (control) and with formic acid (4 g kg^?1 DM), formaldehyde (1 g kg^?1 DM) and two levels of tannic acid (20 and 50 g kg^?1 DM) as additives. Inhibition of proteolysis by formic acid was more effective than the other additives during the first 7 d of ensiling. Tannic acid was as effective at inhibiting production of non‐protein‐N, ammonia‐N and free amino acid‐N as formic acid and formaldehyde. However, increased concentrations of non‐protein‐N and free amino acid‐N in silage from day 1 to 35 of ensiling were less with the higher level of tannic acid than that in the control and other additive‐treated silages. Carboxypeptidase lost its activity slowly with increasing time of ensiling. At day 2, it still had 0·79 of the original activity in the control silage. After 21 d of ensiling, high levels of carboxypeptidase activity, proportionately 0·41, 0·49, 0·10, 0·35 and 0·30 of the original activity, remained in the control silage, and silages made with formic acid, formaldehyde, and low and high levels of tannic acid respectively. There were higher levels of activity of acid proteinase in formic acid‐treated silage than in the control silage until day 2 of ensilage indicating that the reduction of proteolysis by formic acid was probably due to acidifying the forage below the pH optima of plant protease. Aminopeptidase activity in all silages declined rapidly after ensiling.     

The effect of an additive containing non-protein nitrogen on some fermentation characteristics of maize silage

A non-protein nitrogen additive containing ammonia, molasses and minerals was applied to precision-chopped forage maize to raise the crude protein content of the ensiled crop from approximately 8% to 10, 14 or 16%. The dry matter content, total nitrogen, water-insoluble nitrogen, and water-soluble nitrogen concentrations were measured at ensiling and 2 and 8 weeks later. The pH and lactic, acetic, propionic and butyric acid contents of the resultant silage were determined 2 and 8 weeks after ensiling. The levels of crude protein achieved were 10.8, 11.5 and 14.2%, which are equivalent to recoveries in the silages of 108, 82 and 89% of the applied nitrogen. After 8 weeks the water-insoluble nitrogen content of the treated silages had increased while the water-soluble nitrogen content had decreased. The increase in water-insoluble nitrogen may have been due to either decreased proteolysis or increased levels of microbial protein. There was a significant increase of 0.6 units in the pH of the silage when comparing the control to the highest rate of additive application. The additive increased the levels of lactic and acetic acid in the maize silage.     

Effects of Lactobacillus buchneri isolated from tropical maize silage on fermentation and aerobic stability of maize and sugarcane silages

This study was aimed to perform a screening of Lactobacillus buchneri strains from maize silage and use them as inoculant in maize and sugarcane silages. In all, 151 lactic acid bacteria (LAB) strains were isolated from whole‐plant maize silage, and their identification was based on the sequence analysis of 16S rDNA. In total, 15 strains were categorized to the L. buchneri group and eight of these were selected based on growth rate and fermentation pattern. The selected strains were evaluated on fermentation and aerobic stability of maize and sugarcane silages. For maize, the inoculated silages had lower pH and higher LAB population, but lower acetic acid concentration in comparison with the untreated control silage. For sugarcane silage, the strains 56.1, 56.4 and 40788 resulted in highest dry‐matter (DM) content and lowest DM losses. However, only the strain 40788 showed lowest counts of yeasts and moulds. Sugarcane silages inoculated with the strains 56.9, 56.26 and the untreated control silage showed highest concentrations of lactic acid and ethanol, besides the great DM losses. Even so, for both crops, the aerobic stability was not affected by inoculation. After air exposure, all silages increased temperature and had high population of yeast and moulds. Nevertheless, the strains 56.1 and 56.4 are promising for use as a silage inoculant.     

A simulation model of the microbiological and chemical changes accompanying the initial stage of aerobic deterioration of silage

A mathematical model is presented that predicts the time-course of aerobic deterioration in grass and whole-crop maize silages. The model predicts the stability of the silage taking into account the buffering capacity of the silage, the initial contents of organic acids and ethanol, pH, the initial temperature and the initial populations of the microorganisms. The specific processes simulated include the growth of yeast and acetic acid bacteria, the oxidation of fermentation products, the consumption of oxygen and the production of carbon dioxide, the rise in temperature. and the increase in pH.
The deterioration of silage is seen to be initiated by acetic acid bacteria or by yeast, or by both groups together. The factors that determine which groups will prevail are the dry matter contents and the chemical composition of the silage. The output of the model is validated by comparison of the simulated data with data from published work on the deterioration of silage.     

不同添加剂对柱花草青贮品质的影响

以热研2号柱花草为原料， 通过直接青贮（CK）、 添加山梨酸（0.2％）、 蔗糖（2％）、 青贮宝（0.1％）等处理， 30 d后测定了柱花草青贮饲料pH值、 乳酸、 乙酸、 丙酸、丁酸等含量并分析其主要营养成分含量。结果表明： 柱花草直接青贮品质较差， 添加山梨酸、 蔗糖、 青贮宝等处理能降低pH值和提高乳酸含量， 添加2％蔗糖能极显著降低pH值(p<0.01)、 提高乳酸含量(p<0.05)， 改善青贮品质， 提高营养价值。     

Silage fermentation characteristics of three common grassland species in response to advancing stage of maturity and additive application

This study investigated the effects of advancing stage of maturity and additive treatment on the fermentation characteristics of three common grassland species. Perennial ryegrass (PRG; Lolium perenne L., var. Gandalf), timothy (Phleum pratense L., var. Erecta) and red clover (Trifolium pratense L., var. Merviot) were harvested at three dates in the primary growth (11 May, 8 June and 6 July; Harvests 1–3) and ensiled with the following additive treatments: (1) control (i.e. no treatment), (2) formic acid‐based additive, (3) homofermentative lactic acid bacterial (LAB) inoculant and (4) heterofermentative LAB inoculant. Additive application to the PRG (irrespective of harvest date), the late‐harvest timothy (Harvest 3) and the red clover (Harvests 2 and 3) herbages had little impact on silage fermentation characteristics, where a lactic acid dominant fermentation already prevailed. However, the application of the formic acid‐based additive to the Harvest 1 and 2 timothy and Harvest 1 red clover herbages, where the silage fermentation characteristics were poorer, resulted in an increase in the proportion of lactic acid in total fermentation products and a decrease in pH. In contrast, the heterofermentative LAB inoculant had a negative impact on silage fermentation characteristics where a poor preservation prevailed in the corresponding control silage.     

The effect of additives on the quality of white lupin–wheat silage assessed by fermentation pattern and qPCR quantification of clostridia

The efficacy of different silage additives on different mixtures of white lupin and spring wheat was investigated in four separate trials. The bicrop was harvested 96 days (trials 1 and 2) and 110 days (trials 3 and 4) after sowing. For each maturity stage, two mixtures of white lupin and spring wheat were reformed in the ratios of 1:2 and 2:1 on fresh matter (FM) basis respectively. The crops were treated with formic acid (FA), sodium nitrite–hexamine mixture (NaHe) or homofermentative lactic acid bacteria (LAB). The control silage was made without additive. Additives were not able to improve the quality of white lupin–wheat silage in all trials, compared with untreated silage. The treatment with LAB showed good results only at the first stage of crop maturity with sufficient amounts of water‐soluble carbohydrate in the pre‐ensiling crops. The FA treatment showed elevated butyric acid levels in all trials, which suggests that the FA application level used (4 L t^?1 FM, 100% FA) was insufficient to decrease pH enough for preventing the growth of clostridia and butyric acid fermentation. NaHe was the only additive that was able to inhibit the activity of clostridia in all trials.     

Lactic acid bacteria strains for enhancing the fermentation quality and aerobic stability of Leymus chinensis silage

Leymus chinensis is an important grass in China and Russia. Six lactic acid bacteria (LAB) strains (LB, LPL1, LPL2, LPL3, LCL and WH) from L. chinensis silage were screened and identified and their effects on fermentation quality were investigated. All six strains were grown at 6·5% NaCl and pH 4·00. Strains LPL1, LPL2 and LPL3 were identified as Lactobacillus plantarum, and LB, WH and LCL were classified as Lactobacillus brevis, Weissella hellenica and Lactobacillus casei respectively. The six isolated strains and a commercial inoculant (Lactobacillus buchneri) were added to L. chinensis for ensiling at densities of 500 and 600 kg m^?3. The control was sprayed with the same volume of distilled water. The effects of the strains on fermentation quality after 45 d ensiling and aerobic stability during 8 d of exposure to air were evaluated. The 600 kg m^?3 silage had lower pH, butyric acid, ammonia nitrogen content and coliform bacteria counts than the 500 kg m^?3 density silage (P < 0·05). The six isolated strains decreased pH, butyric acid content and increased lactic acid content, and all inoculants increased L. chinensis silage aerobic stability except LCL (P < 0·05). The fermentation quality of L. chinensis silage increased with higher ensiling density. The LAB strains improved the fermentation quality, and high‐quality silage could be obtained at low ensiling density with the addition of the LAB strains. The strains improved the aerobic stability; Lb. buchneri and Lb. brevis showed the best performance.