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1 ENSILING PROCESS AND ITS BIOCHEMISTRY Ensiling is a common preservation method for forages, based on anaerobic fermentation by lactic acid bacteria that produce organic acids, reduce pH, and prevent growth of yeasts, fungi and competing bacteria (Kreuger,2011).

The major processes involved can be divided into four phases: (1) aerobic phase, (2) active fermentation, (3) stable phase, (4) feedout phase. Each phase has distinctive characteristics that must be monitored in order to maintain silage quality (Kreuger,2011)..

2.1.a Aerobic Phase

Once a plant is cut and cells lose their structures, they continue to consume oxygen. As the chopped forage enters the silo, two important plant enzyme activities occur: (1) respiration, (2) proteolysis (Rooke,2011).

Respiration is the complete breakdown of plant sugars to carbon dioxide and water, using oxygen and releasing heat according to the equation

Figure 2.1. Aerobic Digestion of Glucose

This equation indicates respiration converts sugar into carbon dioxide, water, and heat. Thus respiration results in loss of plant nutrients in the presence of oxygen with in one to two days (Michel Wattiaux, 2007). Oxygen also reduced and NADH2 is oxidized.

Simultaneously, plant proteases degrade proteins to primarily amino acids and ammonia and, to a lesser extent, peptides and amides (i.e., asparagine and glutamine).

The loss of sugar is crucial from the standpoint of silage preservation. Sugars are the principal substrate for the lactic acid bacteria (LAB) to produce the acids to preserve the crop. Excessive heat production (i.e., temperatures above 42 to 44C) can result in Maillard or browning reactions, which reduce the digestibility of both protein and fiber constituents. The main aerobic phase losses occur during exposure to air before a given layer of forage is covered by a sufficient quantity of additional forage to separate it from the atmosphere or before an impermeable cover (i.e., polyethylene sheeting) is applied (Chen, et. al, 1994).

2.1.b Active Fermentation Processes during this phase occur under anaerobic conditions and should be dominated by growth of lactic acid bacteria. The species of lactic acid bacteria are usually separated into homofermentative and heterofermentative bacteria based on their type of hexose fermentation (Rooke,2011). Homofermentative bacteria transform nearly all of the sugars they use, especially glucose into lactic acid. The homofermentative pathway includes a first phase of all the reactions of glycolysis that lead from hexose to pyruvate. The terminal electron acceptor in this pathway is pyruvate which is reduced to lactic acid. See Figure 2.2. In fermentation, pyruvate is decarboxylated to ethanal, which is the terminal electron acceptor, being reduced to ethanol.

Figure 2.2. The homofermentative pathway of lactic acid bacteria. Adapted from McDonald et al. (1991). Bacteria using the heterofermentative pathway use the pentose phosphate pathway. In this pathway, NADPH is generated as glucose is oxidized to ribose 5-phosphate. This fivecarbon sugar and its derivatives are components of important biomolecules such as ATP, CoA, NAD+, FAD, RNA and DNA. NADPH is the currency of readily available reducing power in cells (NADH is used in the respiratory chain). This pathway occurs in the cytosol (Sluiter, 2012).

Figure 2.3. The heterofermentative pathway of lactic acid bacteria. Adapted from McDonald et al. (1991).

The homofermenters are the most desirable because their activity does not cause dry matter loss as do heterofermenters. When oxygen is present, heterofermenting bacteria produce less lactic and more acetic acid and, in the absence of sugar, convert lactic to acetic acid (Rooke,2011). This period lasts for one to four weeks and is characterized by a pH decline to around 4.0. During the first days of ensiling, however, plant enzymes and acetic acid producing bacteria compete with lactic acid bacteria for sugars and proteins. Plant enzymes break down proteins to soluble nonprotein nitrogen (NPN). Protein breakdown is highest during the first day after sealing and decreases rapidly as oxygen is used up, with very little protein breakdown occurring after one week of proper ensiling. Maximum protein breakdown occurs at a pH of 5.5 to 6.0, the typical pH of a freshly chopped crop at ensiling. Most lactic acid bacteria grow well above 60F with fastest growth rates at temperatures between 80 and 100F (Kung, 2003). Other biochemical pathways that occurs during this phase include the following: Homofermentative Glucose 2 Lactic acid Fructose 2 Lactic acid Pentose Lactic acid + acetic acid Hetero fermentative Glucose Lactic acid+ Ethanol + CO2 3 Fructos Lactic acid + 2Mannitol + acetic acid + CO2 3 Fructose + glucose Lactic acid + 2Mannitol + acetic acid + CO2 Pentose Lactic acid + acetic acid Clostridia Saccharolitic

2 Lactic acid Butyric acid + 2 CO2 + 2H2 Proteolitic Deamination Leucine Isivaleric acid +NH3 + CO2 Lysine acetic acid + Butyric acid + 2NH3 Serine Pyruvic acid + NH3 Tryptophan Indolepropionic acid + NH3

2.1.c Stable Phase When lactic acid bacteria have used up all the sugar in the crop or when pH gets low enough to stop their growth (4.0 4.2 or lower), the stable phase begins. The presence of lactic acid inhibits further degradation for the remaining time of preservation period. (Limin Kung1995, Marshal 1997). Two other undesirable phases can also take place and cause important loss of dry matter and forage quality in a silo: (1) Butyric acid fermentation by clostridia or butyric acid bacteria (See Figure 2.4) , which occurs if lactic acid fermentation (Phase 3) fails to produce enough lactic acid to stabilize the silage (Rooke,2011).

Figure 2.4. Butyric Acid Fermentation

(2) Aerobic deterioration by molds and yeast that develop rapidly when well preserved silage is exposed to oxygen after opening a silo.

2.1.d Feedout Phase

When the silo is opened, oxygen usually has unrestricted access to the silage at the face. During this phase, the largest losses of DM and nutrients can occur because of aerobic microorganisms consuming sugars, fermentation products (i.e., lactic and acetic acids) and other soluble nutrients in the silage. These soluble components are respired to carbon dioxide and water, producing heat (Palmqvist,1999).

Yeasts and molds are the most common microorganisms involved in the aerobic deterioration of the silage, but bacteria such as Enterobacteriaceae and Bacillus spp., also have been shown to be important in some circumstances. Besides the loss of highly digestible nutrients in the silage, some species of molds can produce mycotoxins or other toxic compounds that can affect livestock and human health (Rooke,2011).

The microbial activity in the feedout phase is the same as that occurring because of oxygen infiltration during the stable phase. The major difference is the amount of oxygen available to the microorganisms.

2.2 POTENTIAL EFFECTS OF ENSILING IN BIOGAS PRODUCTION

Ensiling may function as a beneficial pretreatment for lignocellulosic materials before further processing to methane or ethanol. The process results to a decrease in pH by lactic acid bacteria and it has been found out that acidic pretreatments enhance the production of biogas by decreasing the crystallinity of the cellulosic material (Mosier et. al, 2005), by increasing the accessible surface area of plant substrates, and by altering lignin structure (Hendricks and Zeeman,2009).

The formed acids in the ensiled material clearly contributed to the subsequent biogas yield, as bacteria in the inoculum first hydrolyse the carbohydrate polymers into sugars that are further fermented to carboxylic acids and finally to carbon dioxide, hydrogen and acetate, the intermediates of methane production(Mosier et. al, 2005).

Decomposition of cell walls during ensiling could improve the availability of nutrients and carbohydrates for the methanogens and thus enhance methane production (Amon et. al., 2007). A study conducted by Pakarinen et. al. showed that ensiling with formic acid was a particularly efficient method for storing maize and faba bean, and methane yields in these species were fully retained or even increased up to 50%. Ensiling of maize and hemp proved to be viable options for biogas production, as the biogas yields were remarkably increased in all treatments in their study. Due to the increased formation of lactic and acetic acids in ensiling, higher methane yields have been obtained (Neureiter et. al., 2005).

REFERENCES Mosier N, Wyman C, Dale B, Elander R, Lee Y, Holtzappe M, Ladisch M: Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 2005, 96:673-686. Hendricks ATWM, Zeeman G: Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 2009, 100:10-18. Amon T, Amon B, Kyvoruchko V, Zollitsch W, Mayer K, Gruber L: Biogas production from maize and dairy cattle manure - influence of biomass composition on the methane yield. Agric Ecosyst Environ 2007, 118:173-182. Neureiter M, dos Santos JTP, Lopez CP, Pichler H, Kirchmayr R, Braun R: Effect of silage preparation on methane yields from whole crop maize silages. In In Proceedings of the 4th International Symposium on Anaerobic Digestion of Solid Waste: 31 August-2 September 2005; Water Sci Techn. Volume 53. Edited by: Ahring BK, Hartmann H. Copenhagen, Denmark; 2005:109-115. Palmqvist E, Grage H, Meinander NQ, Hahn-Hgerdal B. (1999) Main and interaction effects of acetic acid, furfural, and p- hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnol Bioeng, 63(1):46-55. Kreuger E, Nges I, Bjrnsson L: Ensiling of crops for biogas production: Effects on methane yield and total solids determination. Biotechnology for Biofuels 2011., 4(44) Rooke JA, Hatfield RD: (2011) Biochemistry of ensiling. In Silage science and technology. Edited by Buxton DR, Muck RE, Harrison JH, Madison , Wisconsin . USA: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America; 2003::95-140 Kung LJ, Stokes MR, Lin CJ (2003): Silage additives. In Silage science and technology. USA: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America;:305-360. Applied Environmental Microbiology. (2003) January; 69(1): 562567. Copyright 2003, American Society for Microbiology) Chen, J., M.R. Stokes, and C.R. Wallace. (1994). Effects of enzyme-inoculant systems on preservation and nutritive value of haycrop and corn silages. J. Dairy Sci. 77:501. Ensiling process www.agric.gov.ab.ca/$department/deptdocs.nsf/all/for4911 Michel Wattiaux Introduction to Silage- Making Dairy Updates Feeding No. 502 University of Wisconsin http://babcock.cals.wisc.edu Sheperd, A.C. (1994). Effect of an additive containing plant cell wall degrading enzymes

on the nutritive value of corn silage for ruminants. M.S. Thesis. University of Delaware. Pakarinen A, Maijala P, Stoddard F, Santanen A, Kymalainen M, Viikari L: Evaluation of annual bioenergy crops in the Boreal zone for biogas and ethanol production. Biomass Bioenergy 2011, 35:3071-3078. Faulkner JS: A comparison of faba beans and peas as whole crop forages. Grass Forage Sci 1985, 40:161-169. Sluiter A, Hamnes B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2012) : Determination of structural carbohydrates and lignin in biomass. Laboratory analytical procedure.[http://www.nrel.gov/biomass/ analytical_procedures.html].

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