Gilad Ashbell

Technology of Forage and By-Products Preservation

16. The microbiology and chemistry of silage

The chemistry and microbiology of silage fermentation are linked, but the microorganisms are the dominant factor in the fermentation process, and they have the main influence on the quality of the end product. Many species of microorganisms are involved in the fermentation process, and their relative importance varies according to the prevailing conditions. As these conditions change during the fermentation process, the microorganism population mixes changes accordingly. The following discussion focuses only on the two most important groups of microorganisms involved in fermentation in the ensiling industry: LAB and clostridia.

Lactic acid bacteria (LAB) Because forages are preserved by lactic acid (LA), the microorganisms which are most important to the ensiling process are the LAB. They primarily ferment sugars to lactic acid, but they also produce some acetic acid, ethanol, carbon dioxide and other minor products. The LAB comprise a rather large group of bacteria, encompassing species belonging to six genera. They are divided into two categories: Homofermentative and Heterofermentative. Under anaerobic conditions the homofermentative LAB produce only LA from hexoses, such as glucose and other six-carbon sugars, whereas heterofermentative LAB, in addition to LA, also produce ethanol, acetic acid, and carbon dioxide. Homofermentative fermentation, which is the more desirable, yields lactic acid, which is a stronger acid than acetic acid, and does not produce carbon dioxide, which represents a loss of DM. Thus, homofermentative fermentation results in little or no loss of DM and only a small energy loss. However, even a totally heterofermentative process will rarely result in more than a 5% loss of DM, and the gross energy loss is negligible.

The following table summarizes pathways of importance in the ensiling process
Pathway: Substrate recovery %
Substrate End product Dry matter Energy
Homofermentative
1 glucose → 2 lactic acid 100 97
Heterofermentative
1 glucose → 1 lactic acid
+ 1 ethanol
+ 1 CO2 		76 97
3 fructose → 1 lactic acid
+ 2 mannitol
+ 1 acetic acid
+ 1 CO2 		95 98
Clostridial
2 lactic acid → 1 butyric acid
+ 2 CO2
+ 2 H2 		49 81
2 alanine → 2 propionic acid
+ 1 acetic acid
+ 2 NH3
+ 1 CO2 		78 81
Yeast
1 glucose → 2 ethanol
+ 2 CO2 		51 97

Source: McDonald (1981)

The stronger acid (LA) lowers the pH more quickly, so reducing the activity of proteolytic enzymes, and conserving protein. The low pH also stops the growth of other undesirable anaerobic bacteria, such as enterobacteria, clostridia and listeria.

Fermentation of LAB:

1. Homofermentative fermentation.
C6 H12O6 → 2(C3H6O3)
(sugar) (lactic acid)

 

 

2. Heterofermentative fermentation.
C5H10O5 → C3H6O3 + C2H4O2
(sugar) (lactic acid) (acetic acid)

 

C6H12O6 → C3H6O3 + C2H5OH + CO2
(sugar) (lactic acid) (ethanol) (carbon dioxide)

 

3C6H12O6 + H2O → C3H6O3 + C2 H4O2 + 2C6H14O6 + CO2
(sugar) (water) (lactic acid) (acetic acid) (mannitol) (carbon dioxide)

 

Some LAB of importance during ensiling:

From the genus Lactobacillus: homofermentative LAB are L. acidophilus, L casei, L. coryniformis, L. currvatus, L. plantarum and L. salvirus; heterofermentative LAB are L. brevis, L. buchneri, L. fermentum and L. viridescens.

From the genus Pediococcus: homofermentative LAB are P. acidilacti, P. damnosus and P. pentosaceus.

From the genus Enterococcus: homofermentative LAB are E. faecalis and E. faecium.

From the genus Lactococcus: a homofermentative LAB is L. lactis.

From the genus Streptococcus: a homofermentative LAB is S. bovis.

From the genus Leuconostoc: a heterofermentative LAB is L. mesenteroides.
The names of some of the homofermentative LAB are often displayed on the labels of the commercial inoculant packages.

 

Clostridial spoilage

Clostridia are a group of undesirable bacteria that grow in silage, especially in moist silage (below 30% DM) under anaerobic conditions. They are spore-forming bacteria, which enables them to survive for a long time, even under bad conditions. Soil and manure are the most common sources of clostridia, therefore great care should be taken to avoid contamination of the forage with soil during harvesting.

Clostridia can be divided into two groups. The first group comprises saccharolytic species such as C. butyricum, that ferment sugars and LA to produce butyric acid, gaseous carbon dioxide and hydrogen, via the following pathways:
C6H12O6 → C3H7COOH + 2CO2 + 2H2
(sugar) (butyric acid) (carbon dioxide) (hydrogen)

 

2C3H6O3 → C3H7COOH + 2CO2 + 2H2
(lactic acid) (butyric acid) (carbon dioxide) (hydrogen)

 

The second group comprises proteolytic species such as C. perfingens, that ferment amino acids to a variety of products, including acetic acid, ammonia, amines and volatile fatty acids, via pathways such as:

CH3CHNH2COOH + 2CH2NH2COOH + 2H2O → 3C2H4O2 + 3NH3 + CO2
(amino acid, alanine) (amino acid, glycine) (acetic acid) (ammonia)

 

Production of carbon dioxide and hydrogen gas represent losses of digestible DM and energy, and production of ammonia is a loss of protein. Also, butyric acid is a weaker acid than LA, and clostridia produce only one molecule of butyric acid from two molecules of LA or sugar. Such fermentation reduces the DM content by about 50%, and the energy content by almost 20%; it is referred to as secondary fermentation, and is accompanied by increasing pH. The weakness of the acid and the production of ammonia both raise the pH, and high levels of ammonia also depress the animals’ intake.

 

Clostridial fermentation

Characterized by a foul smell, indicative of the production of components such as putrescine and cadaverine; its many other negative effects include poor preservation of forage and silage, high pH, high DM losses, high ammonia–N production, low intake, and butyric acid production. Thus, it is important to prevent clostridial bacterial activity, which is encouraged mainly by low DM content and low pH, and can therefore be limited by ensiling forage with DM of at least 30%, and by rapidly achieving a low pH in the silage. All factors that contribute to these two aims, such as type of crop, wilting rate, buffer capacity, sugar content, and soil contamination, will also influence the activity of clostridia. C. botulinum will be discussed in the chapter on forage health.

 

Chemistry of silage

The main chemical components that undergo changes during silage fermentation are WSCs (sugars), organic acids, and nitrogen compounds. Fructose, glucose, sucrose and fructans are the principal sugars in forage crops, and both sucrose and fructans are rapidly hydrolyzed to monomeric components during ensiling. The WSCs are converted mainly to mixtures of organic acids, whose composition depends on the types of microorganisms present and on the prevailing conditions. The predominant organic acids in plants are citric and malic acids; they constitute 2-6% of the DM in grasses and 6-8% of that in legumes. The salts of these acids have a buffering action during the fermentation process, but the plant enzymes quickly metabolize them during the ensiling and wilting. In the case of the nitrogen compounds, rapid and extensive proteolysis occurs directly after cutting, mainly through the action of plant enzymes. This process continues during the ensiling, and its duration depends strongly on the ensilability of the forage, and conditions in the silage. It can be expected that over 50% of the true protein present in the forage will have disappeared from the silage when the fermentation process is over.

 

 

 

 

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