Lactobacilli in Ensiled High-Moisture Corn

Aerobic mesophilic bacteria, molds, yeasts, and Lactobacillus species were enumerated at various intervals of the ensiling process of high-moisture corn (HMC). A total of 466 isolates of Lactobacillus were identified to determine the species of Lactobacillus associated with ensiled HMC and remoistened corn. The numbers of aerobic bacteria in the ensiled HMC increased to 10(8) per gram within 1 week and remained at this level throughout the storage period. The numbers of aerobic bacteria in the remoistened corn increased to 10(9) per gram and remained at this level during the 60-day storage period, after which they increased rapidly. Yeasts were the predominant microflora of the ensiled HMC. Mold numbers were low in the silos, but were very high in the remoistened corn, especially toward the end of the 60-day storage period. The species of Lactobacillus most frequently isolated from the ensiled HMC were L. plantarum, 52% of the isolates; L. brevis, 19% of the isolates; L. brevis (atypical), 15%; L. fermenti, 11%; and L. buchneri, 3%. The lactic acid bacteria, therefore, are the predominant bacterial flora of ensiled HMC, as they are of other types of plant forages that undergo similar fermentations.     

The effect of temperature on the ensiling process of corn and wheat

AIMS: The purpose of this work was to study the effect of temperature on the ensiling process and aerobic stability of corn and wheat silages. METHODS AND RESULTS: The crops were ensiled in 1.5 l anaerobic jars, with and without an inoculant, at room or elevated temperatures (37-41 degrees C). After two months of ensiling, the silages were subjected to an aerobic stability test at room and elevated (33 degrees C) temperature. The results indicate that ensiling at elevated temperatures resulted in higher pH values, less lactic acid and higher losses. The silages which were stored at elevated temperatures were more susceptible to aerobic spoilage than those stored at room temperature, especially when the test was performed at elevated temperature. CONCLUSION: High temperatures are detrimental to both the ensiling process and the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the current study suggest that in a warm climate, special care should be taken during silage making and storage in order to avoid heating as much as possible. In addition, in a warm climate, silages are more susceptible to aerobic deterioration and therefore, special care should be taken during unloading.     

The effect of temperature and Lactobacillus amylovorus and Lact. plantarum, applied at ensiling, on wheat silage

The effects of applying Lactobacillus plantarum and Lact. amylovorus at ensiling on wheat silage stored at 25 and41 °C was studied under laboratory conditions. The inoculants were applied at 10⁶ cfu g⁻¹.Silages with no additives served as controls. Three jars per treatment were sampled on days 2, 8 and 60 after ensiling, for chemical and microbiological analyses. After the ensiling period, the silages were subjected to an aerobic stability test. The control and Lact. plantarum inoculated wheat fermented faster at 25 than at 41 °C, whereas silages inoculated with Lact. amylovorus fermented faster at 41 °C. This was apparent from the rate of pH decrease and from the contents of residual sugars and lactic acid in the final silages. The numbers of lactobacilli in the control and Lact. plantarum silages at 41 °C after 2 and 8 days of ensiling were lower than in the corresponding silages at 25 °C. For the Lact. amylovorus silage the opposite held true. The control silages at both temperatures and the Lact. plantarum silage at 41 °C were the most stable silages under aerobic exposure.     

The effect of applying lactic acid bacteria at ensiling on the aerobic stability of silages

The effect of applying commercial lactic acid bacteria inoculants at ensiling on the aerobic stability of silages was studied under laboratory conditions. The silages used were wheat, hedysarum, corn and various sorghum cultivars at various stages of maturity. Three inoculants were used, two containing Lactobacillus plantarum, Enterococcus faecium and Pediococcus acidilactici (H/M F, Medipharm, USA and Sil-All, Alltech, UK) and one containing Ent. faecium (Lacticil, M74, Medipharm, Sweden). The inoculants were applied at 0.5 times 10⁶ cfu g^-1. Silages with no additives served as controls. After treatment, the chopped forages were ensiled in 1.5 1 anaerobic jars; there were six jars per treatment. After ensiling for 45 d, the silages were tested for aerobic stability in a test in which CO₂ production was measured along with chemical and microbiological parameters.
The inoculated silages that spoiled upon aerobic exposure faster than the controls were those of wheat and of the sorghum cultivar FS5 at the milk stage of maturity. This was evident from intensive CO₂ production and development of yeasts and moulds. Regression analysis indicated that aerobic deterioration of inoculated silages was associated with high levels of residual water-soluble carbohydrates and lactic acid and lack of volatile fatty acids. Aerobic spoilage of inoculated silages was attributed mainly to yeast activity.     

Fate of Escherichia coli during ensiling of wheat and corn

A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 x 10(8) CFU/g into direct-cut wheat (348 g of dry matter kg(-1)), wilted wheat (450 g of dry matter kg(-1)), and corn (375 g of dry matter kg(-1)). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.     

The effects of Propionibacterium acidipropionici and Lactobacillus plantarum, applied at ensiling, on the fermentation and aerobic stability of low dry matter corn and sorghum silages

The aim of this work was to study the effects of applying a strain of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability characteristics of low dry matter (DM) corn (Zea mays L.) and sorghum (Sorghum bicolor L.) silages. Corn at the dent stage and sorghum at the flowering stage were harvested. Treatments comprised control (no additives), P. acidipropionici, L. plantarum and a combination of P. acidipropionici and L. plantarum. Fresh forages were sampled prior to ensiling. Bacterial inoculants were applied to the fresh forage at 1.0×10⁶ colony-forming units per gram. After treatment, the chopped fresh materials were ensiled in 1.5-l anaerobic glass jars equipped with a lid that enabled gas release only. Three jars per treatment were sampled on days 2, 4, 8, 16 and 60 after ensiling, for chemical and microbiological analysis. At the end of the ensiling period, 60 days, the silages were subjected to an aerobic stability test. The L. plantarum inoculated silages had significantly higher levels of lactic acid than the controls, P. acidipropionici and combination of P. acidipropionici and L. plantarum inoculated silages (P<0.05). The P. acidipropionici did not increase propionic and acetic acid levels of the silages. After the aerobic exposure test, the L. plantarum and combination of P. acidipropionici and L. plantarum had produced more CO₂ than the controls and the silages inoculated with P. acidipropionici (P<0.05). All silages had high levels of CO₂ and high numbers of yeasts and molds in the experiment. Therefore, all silages were deteriorated under aerobic conditions. The P. acidipropionici and combination of P. acidipropionici and L. plantarum were not able to improve the aerobic stability of fast-fermenting silages, because they could not work well in this acidic environment. The results showed that P. acidipropionici and combination of P. acidipropionici and L. plantarum did not improve the aerobic stability of low DM corn and sorghum silages, which are prone to aerobic deterioration.     

Bioconversion of corn straw by coupling ensiling and solid-state fermentation

A two-stage process that combined solid-state fermentation (SSF) and ensiling was used for bioconversion of corn straw, in order to increase nutritional value and palatability for animal feed. SSF of corn straw increased the level of protein from 6.7% to 14.7% and decreased the cellulose by 38.0% and hemicellulose by 21.2%. Cellulase and xylanase were produced during SSF. After SSF, the fermented substrate was directly ensiled by inoculating with lactic acid bacteria (LAB). In situ produced enzymes and bacterial inoculation resulted in a rapid drop in pH, a high level of lactic acid production, partial degradation of cell wall components and generation of reducing sugars (RSs). Efficiency of ensiling at 25 degrees C, 30 degrees C, 35 degrees C, 40 degrees C was evaluated. Temperature influenced the effect of ensiling; the higher the temperature, the shorter the ensiling period. The combined fermentation upgraded the nutritional value, enhanced the efficiency of ensiling and reduced bioprocessing costs.     

Yeasts in Ensiled High-Moisture Corn

A total of 1,365 yeasts were selected from ensiled high-moisture corn at various stages in the ensiling process to determine the sequence and relative numbers of yeast species. The yeast species most frequently isolated from freshly harvested corn were Candida parapsilosis and C. intermedia; these two species were isolated infrequently after the third week of storage. Species of yeasts that predominate after the 12th day of storage were Hansenula anomala (66% of the isolates studied) and C. krusei (26% of the isolates studied). The preponderance of H. anomala and C. krusei in ensiled corn is believed to be associated with the ability of these two species to assimilate lactic acid.     

秸秆覆盖免耕对土壤氮素转化细菌区系的影响

不同耕作方法影响土壤N素转化细菌的组成和数量.多年连续秸秆覆盖免耕试验结果表明,好氧固氮菌和厌氧固氮菌数量在铁茬处理土壤中较少,而在免耕与翻耕土壤中较多,免耕厌氧固氮菌数量明显地高于翻耕和铁茬,而铁茬最低,这种趋势特别在0～0.1m和0.1～0.2m土层中表现明显.土壤反硝化细菌的数量在表层土壤中较少,三种耕作方法相差较小,但在0.1～0.2m,0.2～0.3m土层中,翻耕反硝化细菌数量比免耕和铁茬多,而铁茬最少.不同耕作方法间氨氧化细菌数量相差不明显,免耕土壤中氨氧化细菌数量较高,动态变化趋势不规则.0～0.1m土层中免耕亚硝酸氧化细菌数量高于翻耕和铁茬,0.1～0.2m土层免耕与翻耕亚硝酸氧化菌数较多,而铁茬较少.     

玉米青贮过程中乳酸菌动态变化

为了揭示青贮玉米在发酵过程中乳酸菌数量及种群的动态变化,以及微生物添加剂对乳酸菌种群变化的影响,采用了培养方法计数发酵过程中乳酸菌数目的变化,利用16S rRNA基因序列比对方法分析青贮玉米中乳酸菌的多样性及种群变化趋势。经过对15d时间内青贮玉米中乳酸菌变化趋势的分析显示:一周后对照组乳酸菌数最高达到2.1×106CFU/g,两处理组中处理组Ⅱ乳酸菌数达到最高5.5×107CFU/g;利用MRS平板分离、培养出典型乳酸菌菌落152株,经16S rRNA基因序列比对分析为乳杆菌属和片球菌属,其中86%的菌株属于乳杆菌属。此研究表明微生物添加剂有利于青贮玉米发酵过程中乳酸菌的快速增殖,乳杆菌属和片球菌属都是青贮玉米发酵的启动菌之一,在发酵前期一直存在,但发酵后期乳杆菌属是玉米青贮过程中乳酸菌的主要菌群。     

Effects of Bacillus subtilis and its metabolites on corn silage quality

Cellulolytic micro-organisms are potent silage inoculants that decrease the fibrous content in silage and increase the fibre digestibility and nutritional value of silage. This study aimed to evaluate the effects of Bacillus subtilis CCMA 0087 and its enzyme β-glucosidase on the nutritional value and aerobic stability of corn silage after 30 and 60 days of storage. We compared the results among silage without inoculant (SC) and silages inoculated with B. subtilis 8 log₁₀ CFU per kg forage (SB8), 9 log₁₀ CFU per kg forage (SB9) and 9·84 log₁₀ CFU per kg forage + β-glucosidase enzyme (SBE). No differences were observed in the levels of dry matter, crude protein and neutral detergent fibre due to the different treatments or storage times of the silos. Notably, the population of spore-forming bacteria increased in the SB9-treated silage. At 60 days of ensiling, the largest populations of lactic acid bacteria were found in silages treated with SB8 and SBE. Yeast populations were low for all silages, irrespective of the different treatments, and the presence of filamentous fungi was observed only in the SBE-treated silage. Among all silage treatments, SB9 treatment resulted in the highest aerobic stability.     

Nitrate-removal activity of a biofilm attached to a perlite carrier under continuous aeration conditions

The nitrate-removal activity of a biofilm attached to a perlite carrier from an aerobic bioreactor used for treating dairy farm wastewater was examined by batch experiments under continuous aeration conditions. Despite aeration, the biofilm removed nitrate at a rate of 114.4 mg-N/kg-perlite/h from wastewater containing cow milk and manure. In a clone library analysis of the biofilm, bacteria showing high similarity to the denitrifying bacteria Thauera spp. were detected.     

微生物对油田污水-聚丙烯酰胺体系粘度的影响

比较了厌氧和曝气处理后的油田采出水配制聚丙烯酰胺溶液(采出水-聚丙烯酰胺体系)粘度及该体系中腐生菌,铁细菌和硫酸盐还原菌的数量变化。结果表明,曝气有利于聚合物溶液粘度的保持,粘度损失率明显低于厌氧采出水-聚合物体系。总体上,腐生菌或其代谢产物对聚合物粘度的影响很小;铁细菌可以利用聚合物生长,是破坏聚合物粘度的主要微生物类群;硫酸盐还原菌不能直接利用大分子量聚合物,可以利用小分子或分子链断裂的聚合物生长。     

The bacterial community and metabolome dynamics and their interactions modulate fermentation process of whole crop corn silage prepared with or without inoculants

Multi-omics approach was adopted to investigate the modulation of bacterial microbiota and metabolome as well as their interactions in whole crop corn ensiling systems by inoculating homofermentative Lactobacillus plantarum or heterofermentative Lactobacillus buchneri. Inoculations of the two different inoculants resulted in substantial differences in microbial community and metabolic composition as well as their dynamics in ensiled corn. Inoculants also altered the correlations of microbiota in different manners, and various keystone species were identified in corn silages with different treatments. Many metabolites with biofunctional activities like bacteriostatic, antioxidant, central nervous system inhibitory and anti-inflammatory were found in the present silage. A constitutive difference in microbiota dynamics was found for several pathways, which were upregulated by specific taxa in middle stage of fermentation, and widespread associations between metabolites with biofunctions and the species of lactic acid bacteria dominated in silage were observed. Multiple microbial and metabolic structures and dynamics were correlated and affected the fermentation process of the corn ensiling systems. Results of the current study improve our understanding of the complicated biological process underlying silage fermentation and provide a framework to re-evaluate silages with biofunctions, which may contribute to target-based regulation methods to produce functional silage for animal production.     

Dynamics in the microbiology of maize silage during whole‐season storage

Aims: To monitor seasonal variations in the microbiology of maize silage and to determine whether the risk of fungal spoilage varies during whole‐year storage. Methods and Results: A continuous survey of 20 maize silage stacks was conducted over a period from three to 11 months after ensiling. Filamentous fungi, yeasts and lactic acid bacteria (LAB) were enumerated at five time‐points, and cultivable species of filamentous fungi were identified. Significant differences in the numbers of filamentous fungi, yeast and LAB were detected. The highest numbers of fungi were five to seven and the lowest 11 months after ensiling, while the LAB decreased in numbers during the study. Filamentous fungi were isolated from all stacks at all time‐points. The most abundant toxigenic mould species were Penicillium roqueforti, Penicillium paneum and Aspergillus fumigatus. Conclusions: There are significant variations in the microbiology of maize silage over a whole storage season. The risk of fungal spoilage was highest 5–7 months after ensiling and lowest after 11 months. Significance and Impact of the Study: This information is valuable in the assessment of health risks connected with spoiled maize silage and may be useful in the management of maize silage stacks, when whole‐season storage is applied.     

Fate of Escherichia coli O26 in corn silage experimentally contaminated at ensiling, at silo opening, or after aerobic exposure, and protective effect of various bacterial inoculants

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for human illness. Ruminants are recognized as a major reservoir of STEC, and animal feeds, such as silages, have been pointed out as a possible vehicle for the spread of STEC. The present study aimed to monitor the fate of pathogenic E. coli O26 strains in corn material experimentally inoculated (10⁵ CFU/g) during ensiling, just after silo opening, and after several days of aerobic exposure. The addition of 3 bacterial inoculants, Propionibacterium sp., Lactobacillus buchneri, and Leuconostoc mesenteroides (10⁶ CFU/g), was evaluated for their abilities to control these pathogens. The results showed that E. coli O26 could not survive in corn silage 5 days postensiling, and the 3 inoculants tested did not modify the fate of pathogen survival during ensiling. In the case of direct contamination at silo opening, E. coli O26 could be totally eradicated from corn silage previously inoculated with Leuconostoc mesenteroides. The combination of proper ensiling techniques and the utilization of selected bacterial inoculants appears to represent a good strategy to guarantee nutritional qualities of cattle feed while at the same time limiting the entry of pathogenic E. coli into the epidemiological cycle to improve the microbial safety of the food chain.     

Nitrate-Removal Activity of a Biofilm Attached to a Perlite Carrier under Continuous Aeration Conditions

The nitrate-removal activity of a biofilm attached to a perlite carrier from an aerobic bioreactor used for treating dairy farm wastewater was examined by batch experiments under continuous aeration conditions. Despite aeration, the biofilm removed nitrate at a rate of 114.4 mg-N/kg-perlite/h from wastewater containing cow milk and manure. In a clone library analysis of the biofilm, bacteria showing high similarity to the denitrifying bacteria Thauera spp. were detected.     

Occurrence of Phosphate-dissolving Micro-organisms in the Rhizosphere of Rice Plants and in Submerged Soils

S ummary . The incidence of phosphate-dissolving micro-organisms in two submerged tropical rice soils, and in the rhizosphere of the rice plant growing in one of these soils at different stages of plant development was studied. Large numbers of anaerobic and aerobic phosphate-dissolving bacteria were found in both soils after 11 weeks of submergence. Phosphate-dissolving fungi were found in only one of the soils, a latosolic soil. Both anaerobic and aerobic phosphate-dissolving bacteria were stimulated in the rice rhizosphere. However, the rhizosphere effect was greater in the case of the anaerobic phosphate-dissolving bacteria. The rhizosphere effect was greatest at the transplanting stage of rice plant growth but declined with age of the plants. The greatest decline in R/S ratio occurred between transplanting and initial tillering.     

Determination of some rheological parameters for the characterization of activated sludge

The rheological characterization of activated sludge originating from different aeration tanks and from a lab-scale plant was carried out using rotation tests. Two rheological parameters were used: limit viscosity (mu(infinity)) and reduced hysteresis area (rHa). Mu(infinity) was strongly influenced by the total suspended solids (TSS) content of sludge and an exponential relation was found between TSS and the rheological parameter mu(infinity). Significant differences in sludge viscosity were found for sludge of different origins, but all of 10 g/l TSS content, indicating ability of viscosity to show different sludge qualities. Marked changes in activated sludge microorganisms communities were shown to have an influence on rheological parameters. During aerobic digestion of sludge, variations in mu(infinity) were noticed, indicating change of sludge quality. An over-growth of filamentous bacteria species was shown to induce a particular rheological behavior which could be characterized by rHa. This parameter was proposed as an index of filamentous bacteria proliferation in activated sludge aeration tanks.     

Effect of NaCl-tolerant lactic acid bacteria and NaCl on the fermentation characteristics and aerobic stability of silage

NaCl-tolerant lactic acid bacteria (LAB) strains LC-10 ( Lactobacillus casei ) and LP-15 ( Lact. plantarum ) and NaCl were used as additives to sorghun ( Sorghum bicolor ). Numbers of LAB were significantly ( P < 0·05) higher in all the additive-treated silages than in the control silage at an early stage of ensiling. During the fermentation process, addition of NaCl or LAB effectively inhibited the growth of aerobic bacteria and clostridia, but not yeasts. All the additive-treated silages had significantly ( P < 0·05) lower pH, ammonia nitrogen content, dry matter loss and gas production but significantly ( P < 0·05) higher lactic acid content and residual water soluble carbohydrates compared with the control silage. The improvement in silage quality was in the order : LAB > NaCl > control. Yeast counts were high in all additive-based silages and they increased during the exposure of the silages to air. As a result, these silages suffered aerobic deterioration, whereas the control silage was stable. The results confirmed that the NaCl or LAB improved fermentation quality but did not prevent aerobic deterioration of the silage.