Chemical Changes in Phosphoric Acid Silage EDOUARD PAGE AND L. A. MAYNARD Cornell University, Ithaca, N. Y. Phosphoric acid (68 per cent, food grade) was added in amounts varying between 0 and 24 pounds per ton to different layers in a silo filled with a crop of the following composition: clovers, 62.2 per cent; alfalfa, 19.8; grasses, 16.2; and weeds, 1.8. These layers were separated by waterproof rubber sheets. The following determinations were carried out in addition to routine feed analyses: pH, volatile bases, amino acids, butyric and acetic acids, total and water-soluble phosphorus and total titratable acidity. The position of the layer influenced markedly the quality of the final product. This shortcoming of the layer method as an experimental procedure
was partially counteracted by repeating in the upper half of the silo the treatments applied to the lower layers. Although all layers had a good appearance and odor, chemical analyses revealed that the presence of lactic acid as well as of phosphoric acid was responsible for the higher quality of some samples. On the basis of the chemical evidence it is concluded that, while phosphoric acid is of definite value as a preservative, its action must be supplemented by a strong lactic acid production for best results. Full consideration must therefore be given at the time of ensiling to all factors likely to affect the course of fermentation.
HE quality of grass silage is dependent upon a number Using concrete pits, Watson and Ferguson (19) tried out the following acid treatments: Penthesta green; the Defu of factors, chief of which are the moisture content of the solution (HC1+ HaPo,) with the addition of a little molasses, crop, the amount of readily fermentable carbohydrates a t a rate sufficient to give a pH of 4.5 in the mass; a phospresent, and the acidity of the ensiled material. Literature on this subject is abundant, and the reader is referred for a phoric acid monoammonium phosphate solution; and a general discussion to the review by Bender and Bosshardt ( 2 ) . solution of phosphorus oxychloride (P0Cl3) to give a pH value of under 4.0. The average collective analyses for Although the exclusion of air from the material curtaiIs detrimental aerobic activities, a high acidity has a further these various treatments were reported as follows: pH, 3.96; volatile bases as crude protein, 0.33 per cent; amino acids as effect in t h a t i t represses undesirable fermentations, whether crude protein, 0.93 per cent; volatile acids as acetic acid, they be of the aerobic or the anaerobic type. It has, there0.51 per cent (all percentages on a fresh basis). These values, fore, been the aim of various ensiling processes to promote the acidification of the silage by the addition of either lactic as well as those reported by Nebelsiek, indicate a silage of good quality as may be seen by comparison with the values acid cultures, mineral acids, readily fermentable carbolisted in Table IV. hydrates, or a combination of them. We felt t h a t these data on various phosphorus compounds The addition of inorganic acids has gained prominence should be supplemented by further and more extensive studthrough the A. I. V. process developed by Virtanen (16). ies, particularly with the acid itself. We therefore underThis process involves the addition of a sulfuric-hydrochloric took the present investigation of the chemical changes taking acid solution to the forage, in order to bring the p H value of place in silage preserved with different amounts of phosphoric the latter between 3.0 and 4.0. The efficiency of other acids acid. as preservatives has also been investigated. Wilson and Webb (20) called attention to the special advantages of Material and Methods phosphoric acid as follows: “If phosphoric acid is employed, These studies were made on silage produced by Lepard and it produces preservation conditions for the fodder, increases Savage (11) in a comprehensive experiment designed to asthe quantity of phosphorus available for animal consumption certain the amount of phosphoric acid (68 per cent, food grade) and results in a higher phosphatic fertilizer for spreading on required to produce silage of satisfactory quality, and to study the land. This makes the phosphoric acid do triple duty and the losses in dry matter involved. T o accomplish this obspreads the cost of preserving the silage to more than one ject i t was essential that the material to be ensiled with the farm undertaking. It would seem that phosphoric acid different amounts of acid be as nearly identical as possible might be recommended for this purpose because many of our in other respects. Each silo was filled with a particular crop soils and the forage crops grown on them are deficient in this which was divided into layers by waterproof rubber sheets. constituent .” A definite amount of acid, varying from 0 to 24 pounds per Relatively few reports, however, have been published on ton of fresh fodder, was added to each layer. The influence the chemical changes of silage prepared with phosphorus of other factors, such as the composition of the crop, its moiscompounds. Nebelsiek (12) reports on the use of Penthesta ture content, and bacterial flora, was thus made fairly unigreen or phosphorus pentachloride, a proprietary solid comform throughout the silo so that differences between layers pound. I n three diff erept silos t h e p H value ranged between in the final product could be attributed to the acid treatment. 4.0 and 4.1, and the acetic acid content between 0.47 and It is apparent, however, that pressure cannot be made uni0.63 per cent of the fresh silage. 1140
T
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INDUSTRlAL AND ENGINEERING CHEMISTRY
form for all layers. As a consequence, expulsion of air is achieved more quickly in the lower layers, which gives them a distinct advantage. Therein lies a limitation of the layer method. This limitation was partly overcome by repeating in the upper half of the silo the treatments applied to the lower layers. Three silos were filled, one with medium- to well-matured soybeans, another with timothy and other grasses, and the third with mixed grasses, clover, and alfalfa. Chemical studies were made on the contents of all three silos, but data are reported for the third one only. These data were chosen in preference t o those of the other silos because of the greater uniformity of the crop and the greater reliability of the analytical work. They also revealed more fully the action of the phosphoric acid, although none of the results obtained tl-ith the first two silos contradict in any way those presented here. The mixed crop silage came from an excellent stand of new seeding which was cut and ensiled, allowing no wilting, in a 12 x 23 foot wooden silo on June 10 and 11, 1938. The crop was composed of the following plants : clovers, before bloom, 62.2 per cent; alfalfa, one tenth t o one half bloom, 19.8 per cent; grasses, immature, 16.2 per cent; and weeds, 1.8 per cent. The data in Table I show the position of each layer, its thickness after settling, the amount of acid added, and the appearance of each layer upon opening the silo. The layers were numbered in the order in which they were put in. TABLE I. EXSLINGOF MIXEDCROPBY Hap04
Layers
a
per T o n Founds
T o p (not analyzed) Experimental 7 Experimental 6 Experimental 6 Experimental 4 Experimental 3 Experimental 2 Experimental 1 Odor a n d appearance of each layer on
THE
1141
The bottom layer was somewhat higher and layer 2 was lower than had been assumed. Comparison of the total phosphorus in the ingoing crop and the outgoing silage gives a measure of the amount of acid lost through drainage. As might be expected, these losses are highest in the layers having received the most acid. A slight gain in layer 4, to which no acid had been added, shows that some seepage took place in the course of the considerable settling which occurred. This situation, however, does not) seem to have affected the results markedly. Table I1 shows, that layers which received a similar acid treatment also retained substantially the same amount of acid. Comparison between these paired layers thus remains justified. I
114
LAYERMETHOD
Depth of Layer on Removal= Inches
20 114 20 24 8 25 16 25 0 13 8 25 16 27 20 27 removal was excellent.
The top layer was removed on February 8, 1939, and the bottom layer on March 13, 1939. When a layer was opened, a sample was taken about halfway in from the side of the silo and in a vertical section extending from the top of the layer to the bottom. Aliquots from this sample yere used for the determination of the pH, volatile constituents, soluble phosphorus, and other acidic bodies. A representative sample from the whole layer was taken for routine analyses and total phosphorus determinations. The pH was determined by means of the quinhydrone electrode on a sample of juice pressed out of the silage. The total titratable acidity, amino acids, and volatile acids and bases were determined accordin to Woodman’s modification of Foreman’s method as describe$ by Watson (18) Butyric and acetic acids were determined by Wiegner’s method also described by Watson. The sum of these last two determinations was found to agree very well with the total volatile acids as determined by the Foreman method. Volatile constituents were also determined on the oven-dried samples in order t o estimate the losses incurred in the process of drying. Suitable corrections corresponding to the total losses in volatile constituents were then applied to the dry matter. The losses in volatile acids were calculated as acetic acid and credited to the ether extract fraction of the silage. The losses in volatile bases were expressed as crude protein and added to the value for crude protein. The phosphorus was determined according to the Fiske and Subharm method ( 6 ) , using a photoelectric colorimeter.
Results The results are summarized in Table 11. An approximation of the amount of acid added was attempted by subtracting the plant phosphorus from the total phosphorus in the ingoing silage and expressing the difference as 68 per cent phosphoric acid. The values thus obtained are listed in Table I1 under “H8P04,as analyzed”. They agree fairly well with the amounts of acid measured a t the time of ensiling. These amounts are recorded under “H3P04,as added”.
7
5
3
7
5
3
I
a
LAYER
3.7
I
NO.
FIGURE1 (above). Amioma PRODUCTION IN RELATION TO AMOUNTOF ACID ADDED TO EACH LAYER OF SILAGE FIGURE 2 (below). PRODUCTION OF VOLATILE ACIDS I N RELATIONTO FIXAL PH OF SILAGE The lactic acid was not determined directly but-was assumed to correspond approximately to the following formula
+
volatile Lactic acid = total titratable acidity - (amino acids acids water-soluble phosphorus taken as phosphoric acid)
+
By including the water-soluble phosphorus as phosphoric acid, it was hoped to cut down the discrepancy often reported between the values for residual acidity and for true lactic acid when mineral acids have been added to the silage. The residual acidity is the lactic acid as above calculated plus the phosphoric acid. The residual acidity was the value used in calculating the ratio of nonvolatile to volatile acids. Examination of the data as a whole reveals a number of facts which may be briefly stated prior to a more detailed discussion:
TABLE 11. Layer KO. Dry matter, 5% Outgoing Ingoing silage silage p H , outgoing silage Hap04 (68R), lb./ton As added As analyzed Outgoing silage Phosphorus, kg. Ingoing silage Outgoing silage
SLXlAIARY O F A S A L Y S E S O F
MIXEDC R O P
7 6 5 Dry hlatter a n d Acidity
4
g::;!
iy:;! 4.30 4.46 Phosphorus 8 9 7
20 20 17
16 15 12
3
4.23
4.10
0 0 2
8 7 7
2.5 5.0
8.5 8.8
3.61 16.86
4.03 18.57
4.39 18.56
4.06 16.29
4.10 15.61
0.84 23.35
0.86 20.75
0.92 1.24 2 0 . 4 3 30.61
1.12 27.48
0.41 11.41 2.05
0.49 11 81 1.76
0.46 o 54 1 0 . 3 3 13.17 1.96 2.32
0.46 10.90 2.52
Volatile Acids (Outgoing Silage)
fresh silage Acetic acid Butyric acid Total volatile acids Total volatile acids as matter
1.20 0.98 0.99 0.77 0.08 o i o 0.12 0.06 0.s3 70 of d r y 1.28 6.96 4.88 4.58 3.30 Residual Acidity (Outgoing Silage) Residual acidity, a s lactic acid, 1.15 1.30 2.07 5% of fresh silage 1.19 Lactic acid 2.00 0.93 0.97 % of fresh silage 0.72 70of d r y matter 3.36 4.20 4.01 8.03 1.72 0.63 0.73 0.81 Ratio, nonvolatile : volatile acids a Calculated as crude protein-i. e., N X 6.25.
p H 4 a t least, so that no decomposition of amino acids takes place below this value”. 2 1 Following the disappearance of the available oxygen in the plant material, anaerobic :;:$ activities set in. Under these conditions spore4.05 3.73 forming anaerobes, if numerous and unhampered by a low pH, will cause extensive protein 16 20 breakdown. However, if mineral acids have 11 24 been added or if lactic acid fermentation has 10 18 progressed sufficiently, such detrimental proc13.7 35.7 12.6 16.7 esses will be reduced to a minimum or may be completely inhibited. Butyric acid production 4.16 4.58 will likewise be held in check, and therefore the 15.67 1 6 . 4 3 determinationof this constituentgivesagood clue 0.55 to the extent of anaerobic protein breakdown. 0.69 16.64 1 2 . 0 3 It is generally agreed that a critical acidity 0.39 0.28 exists, approximately a t p H 4, below which 9.35 6.11 1.78 1.97 ammonification and butSrric acid fermentation are practically inhibited. This has been shown by a number of workers, including Virtanen (16), 0.68 0.65 0.10 0.00 Watson and Ferguson ( I @ , and Cunningham 0 , 7 8 O.” and Smith (S). With respect to the formation 2.95 1.98 of acetic acid, Cunningham and Smith report good agreement between i t and the pH value, 2.79 3.14 but no point* I n the present experiment, we likewise find 2.43 2.57 9.16 9.21 a gradual decrease in acetic acid with an in2.47 3.80 creasing acidity and no evidence of a critical point. On the other hand, in the one layer (KO. 1) with a p H below 4.0, no butyric acid is found. The fact that ammonia production bears a definite relation to the amount of acid added and not so much to the final p H values indicates that phosphoric acid has played a major part in curtailing proteolysis, and also that ammonification occurs for the most part in the early stages of fermentation. These findings bear out Virtanen’s contention that, when the pH of the fodder is not brought below 4.0 a t the time of ensiling, the determination of ammonia is a better criterion of the quality than the final p H value. Of two layers which received the same amount of acid, the lower one of each pair has a lower p H value and contains less ammonia and more lactic acid (Table 111). The position of the layer thus affected markedly the quality of the silage. The lower layers, being under higher pressure, presumably achieved an anaerobic condition more rapidly than the upper one, and thus cut short detrimental aerobic activities and favored lactic acid production.
SILAGE
; ;E:
4.20
23.5 12.0 15.2 15.4 8.7 10.6 Nitrogen (Outgoing Silage)
Crude 70ofprotrin fresh silage 70of dry matter Amino acid@ 70of fresh silage 5% of crude protein Volatile bases&, 70of fresh silage 70of crude protein Ratio, amino acids ; volatile bases
r0of
VOL. 32, NO. 8
INDUSTRIAL AND ENGINEERING CHEMISTRY
1142
0.74 0.13 0,87 3.31
2.03 1.89 7.18 1.65
1. The ammonia production (volatile bases), as percentage of the crude protein, is more closely related t o the amount of acid added at the start than to the final pH value. This is further brought out in Figure 1. 2. The ratio, amino acids to volatile bases, varies little from one layer to another, which indicates a nearly parallel increase in both these constituents. 3. The volatile acids show a gradual decline from the top to the bottom of the silo and little relation to the amount of acid added. This decline is accompanied by an increasing total acidity as measured by the pH value, further shown in Figure 2, and by the lactic acid content. As a consequence, the ratio of nonvolatile to volatile acids increases from top t o bottom. 4. The butyric acid content is uniformly low in the upper six layers, and no butyric acid is found in the bottom layer which has a pH value below 4.0. The fact that all layers were reported as excellent on the basis of appearance and odor illustrates the shortcoming of this method in the appreciation of the quality of silage. This is not surprising in view of the fact that, despite variations between layers, all of them were relatively low in butyric acid content.
Discussion The fermentation processes in silage may be arbitrarily divided into the aerobic and the anaerobic phases. I n the early stages of fermentation, before the oxygen has been completely utilized or expelled, the development of acidity occurs simultaneously with the degradation of protein. According to Allen and Watson ( I ) , lactobacilli are largely responsible for acidification while various groups of aerobic bacteria are concerned with proteolysis and deaminization. The part played by plant enzymes in the latter respect has not yet been clearly demonstrated. C. A. Hunter (8) showed that hydrolysis of protein to amino acids is due chiefly to plant enzymes, whereas bacteria participate equally in ammonification. Lamb (10) likewise states that protein decomposition is caused by enzymes first and later by microorganisms. 0. W. Hunter (9),however, failed to find enzymatic proteolysis of any magnitude. More recent studies of siIage fermentation have been concerned mainly with the bacterial flora. Virtanen (17) merely states that “proteoclastic enzymes of green plants become inactivated below
OF POSITION OF LAYERS ON FERMESTATABLE111. INFLUENCE TION ISTENSITY
Layer No. 7 1 5 2 6 3 4
HaPO4, Lb. per Ton 20 24 15 11 9 7 0
70 of Total N as NHs 11.41 6.11 10.33 9.35 11.81 10.90 13.17
Lactic Acid a s 70 of Dry Matter 3.36 9.21 4.01 9.16 4.20 7.18 8.03
pH 4.30 3.73 4.20 4.05 4.46 4.10 4.23
Total Volatile Acids as % of Dry Matter 5.96 1.98 4.58 2.95 4.88 3.31 3.30
Layer 4,to which no acid mas added, has the highest amount of ammonia in spite of its high lactic acid content. This affords further evidence that ammonification took place to a large extent before lactic acid production had proceeded extensively. It also demonstrates the beneficial effect of phosphoric acid in cutting down proteolysis.
AUGUST, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
1143
Comparison of layer 7 with layer 1 shows that lactic acid fermentation was largely responsible for the low p H value which was reached in the bottom layer as well as for the latter's loa. content of volatile constituents. Although all layers can be considered as satisfactory, layer 1 is the only one which really measures up to the best samples of acid silage reported in the literature with respect to the amount of volatile constituents present. A few of these reported values are recorded in Table IV. Layer l is definitely higher in lactic acid content.
The phosphoric acid method is in a sense a compromise between the A. I. V. and the molasses methods. The latter relies entirely on lactic acid fermentation by adding a sufficient supply of readily fermentable carbohydrates. Since the desired degree of acidity is not reached immediately, more proteolysis is bound to occur than in the A. I. V. procedure and probably more than occurs in the phosphoric acid method. This disadvantage appears to be minor, however. The addition of an adequate amount of molasses ensures a proper acidity in a tightly packed silo, and this seems to be a somewhat more satisfactory procedure than the use of an amount of mineral TABLE Ihr. AN.kLYsEs OF ACIDSILAGE WITH PH VALUE BELOW 4.0 acid which must be supplemented by lactic acid ----P ,o of Fresh Silage-Amino Am- -% of Total h-7 fermentation to produce the needed acidity. Authors Acetic acid Lactic acid acidsm moniaa NHzS KH3N 6,1 Watson and Ferguson (19) and Fagan and Page and hfaynard, layer 1 0 . 5 5 ( 2 . 0 ) " 2.57 (9.21). 0.55 0.28 12 Ashton (5) stated that the addition of moderate Watson and Ferguson ( 1 9 ) , phosphoric acid silage 0.51 ..... 0.93 0.33 ... ... amounts of acid (such as is represented by the Sebelsiek ( 1 2 ) . Penthesta green 0.47-0.63 .... . .. ,. 5-21 ... i,b, phosphoric acid procedure of the present investiCunningham and Smith (3) 0.4-2.1C .. .. Virtanenb 0.2-0.3 o.oili:o5 .. .. 13 1.9 gation), with or without molasses, has not been 0.07-0.5 0.28-1.44 .. .. ,.. 1.4-7.3 Steinera so satisfactory as the addition of molasses alone. \'on Beynun and Petteb 0.12-0.63 0.52-1.43 .. .. ... ... Mertinsb 0.06-0.60 0.05-1.31 ., .. .. ... ... Based upon these considerations, as well as on Peterb 0.20-0.60 0.60-2.2 ,. Watson and Fergusonb 1.4-2.6c 0.30-1,li .. , . i61is 6.016.5 the findings in the present paper, it appears that Peterson ef a1.b 2.8-4,O C 2.3C .. .. 14-30 1.2-6.7 , , , 3.5-5 from the standpoint of chemical changes the Hegsted et al. (7) 1.32-1.89C 0.71-3.9SC Davies et al. (4) 0.43 ..... 0:3i o:io ... ... phosphoric acid procedure may not prove so a Based on volatile base and amino nitrogen calculated as crude protein-i. e., Ii X 6.23. generally satisfactory as the use of molasses. b According to Cunningham and Smith ( 3 ) . c Percentage of dry matter. Further studies on this point are needed, however. It should also be borne in mind that several othersfactors, such as cost of material, ease of handling, and fertility value, must be considered in addiI n the present instance, one is led to the conclusion that tion to the chemical evidence here presented. the addition of phosphoric acid resulted in high-quality silage, in part a t least because i t was supplemented by a strong lactic acid fermentation. This fact represents a different situaAcknowledgment tion from that where the A. I. V. method is used. I n the The authors are indebted to 0. L. ~~~~~dand E. Savage, latter procedure One can the added acid to of the Department of Animal Husbandry, Cornell University, Produce a satisfactory ~ r o d ~without ct regard to other condifor making the presentstudy possible, particularly to 0. .,J tions. Lepard who collected the samples analyzed and supplied the There is evidence from theoretical considerations that a data as to the appearance and condition of the crop before larger amount of phosphoric acid would be required as the and after ensiling. sole acidifying agent than of the A. I. V. mixture. This was shown to be true experimentally by Virtanen (16) and is Literature Cited borne out by studies in this laboratory. Of the nine silage (1) Allen, L. A., m a t s o n , S. J., a n d Ferguson, W. S., J . Agr. Sci., 27, samples treated with 16 to 24 pounds of phosphoric acid,
s.
Only had a pH 49 and in each it was shown that lactic acid fermentation contributed significantly to the final acidity. I n a lengthy review on silage Ruschmann (14) pointed out that, according to the amount of acid added, one may obtain a true silage, as in the present case, or a chemically "stabilized" fodder, as in the A. I. V. process. The former type, in his opinion, is preferable because the procedure both restricts the initial protein breakdown and favors lactic acid production, a self-adjusting process. The danger of overacidifying is thus avoided, and there is less upset of the acid-base balance from a nutritive point of view. The handling and application of the A. I. V. acid presents some special problems not met with phosphoric acid. On the other hand, the use of phosphoric acid in the amounts customarily recommended must be supplemented by a substantial lactic acid fermentation for best results, requiring more attention to the condition and carbohydrate content of the crop as ensiled than is the case for the A. I. V. method' This need for carbohydrate fermentation is recognized in the Defu method designed to bring down the pH to 4.5 by adding a little molasses to the mixture of hydrochloric and phosphoric acid. Both Edin, according to Virtanen ( 1 6 ) , and Wiegner, according to Piraux et al. (IS), reported that this procedure does not give such good results as the A. I. V. method.
294 (1937). (2) B e n d e r , C. B., and B o s s h a r d t , D. I
