Ensiling of Manured Crops—Does Repeated Spreading of Slurry Increase the Hygienic Risk?

The influence of cattle slurry applied to a silage crop was studied in three successive years. Each year, 40 t slurry ha^-1 were applied in two equal applications. Unfertilised and mineral-fertilised field plots were used as controls. Before spreading, the slurry was contaminated with spores of Clostridium tyrobutyricum (10³–10⁴ g^-1 FM). The fields were harvested two times a year, the yields were measured and the chemical composition and bacterial content of the crop were determined. The fresh material was chopped, ensiled without additives, and stored for 100 days before opening. The bacterial content of the soil surface and available N, P and K in soil were measured. Slurry did not seem to have any significance in a short-term perspective. Yield increase as an effect of slurry application was slight and inconsistent in comparison with the yield from unfertilised plots. The number of Clostridium or Bacillus spores in the soil surface was not significantly affected by the slurry application, nor was the number of detrimental organisms in harvested crop at ensiling. Nonetheless, in three of the four silages in which clostridial spores were estimated, numbers were higher in silage made from slurry-treated crops. © 1997 SCI.     

Growth of Clostridium tyrobutyricum during fermentation and aerobic deterioration of grass silage

Grass experimentally infected with Clostridium tyrobutyricum, Hansenula anomala and Candida lambica was inoculated with lactic acid bacteria or formic acid, ensiled at 20°C, 28°C and 37°C and stored for 100 days. Chemical and microbial compositions were determined after 4, 11, 40 and 100 days of storage. According to the chemical and microbial criteria, all the silages were of good quality. The silage were exposed to air for 14 days after 40 and 100 days of anaerobic storage, which stimulated clostridial growth. High concentrations of spores of Clostridium tyrobutyricum and a high content of butyric acid were found in deteriorated parts of the silages, both on the surface and at 7-cm depth. Increased levels of ATP were registered in deteriorated parts. The deteriorated parts had high concentrations of the bacteria Bacillus polymyxa, B licheniformis and B sphaericus and of the mould Monascus ruber while yeast counts were low. High levels of the initially added Hansenula anomala and Candida lambica were only observed in formic acid treated silages after the air exposure.     

High production of butyric acid by Clostridium tyrobutyricum mutant? ?

The objective of this study was to improve the production of butyric acid by process optimization using the metabolically engineered mutant of Clostridium tyrobutyricum (PAK-Em). First, the free-cell fermentation at pH 6.0 produced butyric acid with concentration of 38.44 g/L and yield of 0.42 g/g. Second, the immobilized-cell fermentations using fibrous-bed bioreactor (FBB) were run at pHs of 5.0, 5.5, 6.0, 6.5 and 7.0 to optimize fermentation process and improve the butyric acid production. It was found that the highest titer of butyric acid, 63.02 g/L, was achieved at pH 6.5. Finally, the metabolic flux balance analysis was performed to investigate the carbon rebalance in C. tyrobutyricum. The results show both gene manipulation and fermentation pH change redistribute carbon between biomass, acetic acid and butyric acid. This study demonstrated that high butyric acid production could be obtained by integrating metabolic engineering and fermentation process optimization.     

Effect of a nisin-producing lactococcal starter on the late blowing defect of cheese caused by Clostridium tyrobutyricum

Clostridium tyrobutyricum causes swelling, cracks and off-flavours of cheeses (late blowing defect, LBD) due to butyric acid fermentation. To control this spoilage bacterium, we investigated the use of nisinogenic Lactococcus lactis subsp. lactis INIA 415 as starter in cheeses contaminated with C. tyrobutyricum spores. Control cheese made with spores showed LBD after 14 days of ripening. However, in cheese made with the bacteriocin producer and spores, in which (unlike control cheese) bacteriocin activity was detected throughout ripening, LBD occurred after 21 days. At this stage, level of lactic acid was 1.22-fold higher (P < 0.01) and concentrations of propionic and butyric acids were 2.15- and 2.32-fold, respectively, lower (P < 0.01) in cheese made with the nisin producer than in control cheese, according to the less pronounced spoilage symptoms showed by the former cheese. The bacteriocin producer delayed the appearance of LBD, although it cannot arrest completely C. tyrobutyricum growth.     

以废弃毕赤酵母为高效氮源强化丁酸发酵生产

为有效利用固态废弃毕赤酵母,高效发酵生产丁酸,建立了以80 g/L玉米淀粉为基础培养基、以废弃毕赤酵母处理液为高效有机氮源,连续补加葡萄糖的丁酸发酵工艺。7 L厌氧发酵罐下,发酵20h,先以1∶4的比例添加250~400 g/L规格的废弃酵母处理液替代昂贵的发酵用复合培养基,再连续补加葡萄糖浓缩液。最终的丁酸质量浓度为45 g/L、得率为40%,丁酸占总酸的比率(butyric acid ratio over total organic acids,B/TA)≥0.90的较高水平。该工艺可以有效地利用来自于废弃酵母处理液中的氨基酸,提高细胞生长速度和浓度,增强丁酸合成所依赖的NADH再生速度,间接改善了丁酸发酵性能。与此同时,实现了废弃生物质的有效利用和资源化。     

Influence of farmyard manure on the quality of grass silage

The influence of adding small aggregates (5 g) of farmyard manure (FYM) and/or a bacteria suspension (BS), containing Escherichia coli, and spores of Bacillus cereus and Clostridium tyrobutyricum, to silage was studied in two successive years. Direct cut (approximately 200 g DM kg⁻¹), precision-chopped grass herbage was ensiled in 1·6 l experimental silos. At ensiling the BS was either well distributed in the crop or added directly to 5 g of grass or FYM, which was placed in the centre of the green material when the silos were filled. Silage was further treated with formic acid (4 kg t⁻¹ FM), or an inoculant of lactic acid bacteria (10⁶ cfu g⁻¹ FM), and compared with no additive. Only minor influences on silage quality resulted from the addition of BS, although the number of clostridial spores increased slightly. Silages given additions of small aggregates of FYM were of poor quality. The inferior silage with a high number of clostridial spores (⩾3·9 cfu g⁻¹) and a high concentration of butyric acid (⩾2·8 g kg⁻¹) was found in the immediate vicinity of the addition. Application of silage additives did not improve silage quality. The poor quality of ‘FYM-silages’ could not solely be explained by the high number of organisms, but other factors in the manure and/or crops may contribute to the poor silage fermentation. © 1997 SCI.