Chromatografija, kompostas, humusas

CONTENTS Chapter 1 Chapter 2 Chapter 3

CHROMATOGRAPHY APPLIED TO QUALITY TESTING Ehrenfried E. Pfeiffer The Art and Science of Composting (Recent Observations and Testing Methods) It is obvious that the chemical analysis of a compost with regard to its nitrogen, phosphate and potash content (NPK) gives only limited and incomplete information as to its biological value. Nor does it really tell how good a compost is. There are plants, the roots of which are very sensitive to raw, crude, decaying materials, which demand the very best humus — for instance, legumes and fine grasses; there are others — such as corn, tomatoes, grapevines — which thrive well on crude matter.

There is not just one compost for everything, nor is all the organic material or waste (from the moment it arrives in the dump or compost yard, and on through all stages of fermentation and decay) yet to be defined as compost.

To know, to properly judge and to evaluate the biological quality of a compost is not only important for the farmer, gardener and orchardist, so that he can select the proper type and amount to be applied with the optimal result and least expense or waste, but it is also important for the compostmaker in the small garden or the manufacturer in a big way. He has to have such knowledge in order to produce the best material with the least expense and the least losses of organic matter, nitrogen or other ingredients of value.

The problem has been complicated by the fact that, because of the

difference in their biological or intrinsic value, composts with almost the same NPK content in pot and field tests have given quite different results with regard to growth, yield and state of health of plants. One experimental station in Germany recently compared various composts from municipal garbage compost operators and found that one compost from the pilot plant in Erlangen produced in test plots of spinach and carrots a far better yield, even though this compost was applied at a rate of one-fourth the others which were used for comparison.

Experiments performed by the Portuguese Experimental Station in Ponta Delgada, Azores, with compost also made with the B.D. Compost Starter, showed that an application rate of compost one-seventh that of the best farmyard manure produced equal yields. This, in turn, led to the fact that the government granted a

20% subsidy (20% of the retail sales price) to the compost manufacturer to make this compost available to the farmer. In terms of value, it was estimated that the intrinsic value of such composts is equivalent, if not a bit higher, than the NPK value because of the presence of organic matter and trace minerals, and in view of the increased availability of all minerals due to certain bacterial action and the quality of the humus, that is, fully digested organic matter (not crude organic matter). All these factors we will call biological value.

While it is easy to make biological tests for NPK following the officially recognized methods (AOAC) (1), it has been thus far a difficult problem to determine the biological value. One problem has been what to look for; another has been the development of adequate analytical methods.

The Biochemical Research

Laboratory of the Bio-Dynamic Farming and Gardening Association, Inc.1 has devoted much study to these problems over the last ten years. It has worked on setting up proper composting methods with the highest degree of efficiency, and has developed testing methods which make it possible to judge the chemical as well as the biological value of compost. Some of the methods will be described in this article. Before we can do this, however, the processes of composting and the happenings during the fermentation must be described.

The author of this paper has incorporated his knowledge and observations in a manual which discusses our knowledge in detail with a view to large-scale waste composting (2). This article will serve as a supplement to this manual, elaborating further on the problem.

In the literature, we find various

methods described for the determination of so-called “humus”.

Humus is not a definite chemical substance but a state of matter, an integration of mineral with organic matter. There is, for instance, the acid humus in forest soils which is entirely different from a neutral, colloidal humus; there is the alkali-soluble humus; there is Professor Springer’s concept of “effective organic substance.” (3) To determine these, good analytical methods have been developed (3,4). All of these methods are valuable but still do not give a complete picture of the biological situation.

It is in the nature of composting that one deals first with waste materials, garbage, plant refuse from gardens, lawns, stubble, old hay or silage, seed cleanings, cotton burr, slaughterhouse refuse, field (crop) refuse, sludge, pea vines or other refuse from processing

of agricultural products, pomace, oil press cake, manures of all kinds, leaves, conifer needles, wood shavings, sawdust, etc. These are not compost yet, nor are they humus.

Between these raw and source materials and the final product is a long chain of reactions and transformations.

All of these substances are called organic — not so much because they contain strictly “organic matter� (that is because they are compounds of nitrogen, carbon, oxygen, plus sulphur and phosphorus in the organic molecule), but because of their origin from an organic process; i.e., the growth and life to which they owe their existence.

The final product is compost, humus in various degrees of decay or fermentation. At the very end of all this, in touch with the soil, compost earth and humus soil will result under favorable conditions.

Under

unfavorable conditions, just “earth” will result with a rather low content of organic matter; that is, the product has become mineralized and the goal of creating “organic” matter for the soil has been overshot. Nothing of the structure and nature of the source materials has been left.

In the manual (2), three phases of composting have been discussed: First: The breakdown phase, which effects the primary change of the crude source material; the original proteins, amino acids, proteins, cellulose, starches, sugars, lignin, are broken down. This could happen by ordinary decay without the interaction of micro-organisms (bacterial, for example), but usually microorganisms, bacteria, fungi and microscopic animal organisms, which digest the raw materials, are present.

Second:

The building up phase; microorganisms take over and transform the source materials, which they use as food, building up their own bodies.

Now it becomes very important which type of microbial activity is present: that which results only in the release of carbon dioxide, ammonia, nitrites or hydrogen disulphides, or the other type, which stabilizes the breakdown and produces either a stable or an unstable humus, a lasting or a perishable humus. The stable humus will build up a soil; the unstable, perishable humus will provide plant food but will “burn up� in the soil quite rapidly and will not have any lasting effects. With regard to the application of such composts, it is quite important what kind of soil is to be fertilized. Sandy soil, with its greater access to air and its faster warming up in spring, needs more stable, lasting humus, while heavy

clay and loamy soil will benefit more from the unstable, quicklydecomposing humus, even though a certain amount of stable humus is necessary in order to build up these soils.

Third: Gradually the organic matter will be more and more decomposed, get lost as carbon dioxide, and lose nitrogen via ammonia and nitrites; that is, the original proteins and amino acids are completely broken down into the simplest chemical compounds and therefore the compost mineralizes. The result can be favorable. Then we have the rich compost earth which in older days was called “compost� and was produced mainly by the home gardener and farmer, but the organic value is much reduced, below the potential of the source materials.

In practice we find, therefore,

composts with as much as 40%, 30%, 20%, 10% or less of “organic matter” (determined by the combustion or oxidation method) no matter whether the original content was 60%, 50%, 20% or less. Most old-fashioned garden composts contain 8% to 16% organic matter.

Very few manufactured composts contain as much as 30% organic matter, and still fewer more than 40% organic matter.

To say that a compost is 100% organic is misleading. It can be 100% organic because of its origin (made entirely from refuse of material exclusively produced by the life processes of plants and animals), but it certainly does not contain 100% organic matter. This is due to the nature of living tissue (or cells) which consist of 70% to 90% water, 15% to 20% strictly “organic” substance (that is, proteins, amino acids, carbohydrates — in short, carbon

compounds), and of 2% to 10% mineral, inorganic matter — phosphate, potash, calcium, magnesium, major and minor (trace) inorganic elements. Nitrogen is part of the organic substance; part of the inorganic matter can be present in organic compounds but also can be present as salts or otherwise. Mineral compounds present in organic matter are less doomed to be washed out than those present in strictly inorganic states — salts, for instance, and, above all, potassium. This is true as long as the organic compounds are stabilized.

Now there is in addition quite a difference whether these organic compounds are preserved — that is to say, locked up in the living bodies of micro-organisms, or are “floating” free in some phase of breakdown.

Only if they are locked up will they be stabilized. As soon as conditions

of moisture, warmth or aeration favor them, the breakdown and loss will continue. While the home gardener or farmer has only a limited control of these processes, the industrial manufacturer would do well, if only for economic reasons, to make himself familiar with the scientific background of composting. He needs this for production control, maximum efficiency (the saving rather than the loss of valuable substances) and, finally, for the production of crops with high quality and biological value. It is, of course, true that the mere NPK concept applies correctly once the completely mineralized state of compost has been reached, for then all the intrinsic biological values have been lost.

It is necessary to follow all these processes carefully, with experience, know-how, and where possible with analytical methods. The compost-

maker of large quantities needs this knowledge. In fact, the farmer and gardener should also know and appreciate these differences, for they are the key to the application rates, even though the NPK content may not vary widely, and the efficiency in application consequently also is different.

The farmer can save himself a lot of money if he uses compost with a higher biological value. He needs to use a much smaller quantity per acre.

Think only of the difference in loading and spreading time and the lessened compacting of the soil due to the equipment's traveling less frequently over the field. To the producer it also means dollars and cents, for the cost of handling is about the same whether he produces a high or low quality of compost.

In our laboratory we have analyzed hundreds of composts. We have seen

few with 40% or more organic matter and a high availability of minerals.

The difficulty so far has been that no one single method of analysis has existed for determining the biological value of compost. We shall now discuss the various methods of analysis and evaluate them. To many of our readers, this may sound foreign, but they would do well to read it nevertheless, for this knowledge is to their benefit. pH: This determines the degree of acidity or alkalinity. 7.0 is neutral, 8.0 or more is strictly alkaline. Below 7.0 is acid, 5.5 would be very acid.

Decaying materials, because of the release of organic acids, are acid — between 5.0 and 6.5. Such acid composts, therefore, belong mostly to phase 1. All natural, biological, aerobic bacterial action proceeding toward humus formation tends to produce an alkaline reaction, in

general from 7.1 to 8.0. When a compost moves from acid to neutral, it indicates that the second phase has taken over. 7.1 to 8.0 indicates the beginning of stabilization. A few exceptions may exist, which we will leave to the experts to understand. pH meters, or, for quick orientation, colorimetric indicators, are used.

Different methods give somewhat different values.

Total Organic Matter: This is determined by combustion or oxidation of the carbon compounds.

This determination does not tell the state of matter in which the organic matter is present. A piece of coal and a plant protein could not be differentiated. However, this testing is very valuable for following the gradual vanishing of organic matter during fermentation, downward from the original content. During phase 1, this downward trend is greatest

because many microorganisms which effect the breakdown consume organic matter and produce carbon dioxide.

During phase 2, a stabilization takes place.

Under very favorable conditions, many organisms can grow in such a way as to produce a slight increase of organic matter, as plants do on a field, so as to compensate for the earlier losses or even improve on them. It is, therefore, important to go through phase 1 as fast as possible and to arrest excessive losses in phase 2. On this rests the success or failure of economic and efficient composting.

We have seen many composts where 50% and more of the original organic matter was lost. The higher the original content, the greater the danger of losses, and the more carefully one must work. The danger of losses is greatest with hot and fast fermentation. This method, which we

favor because of its greater economy, needs considerably more skill and knowledge than the cold, wet fermentation. The latter is the oldfashioned garden pile method, with 50% moisture or more. The entirely different anaerobic sludge fermentation shall not be discussed here. The fast, hot method of fermentation works with a moisture content of 30%-40%, although a dry product below 20% shows no appreciable action. This method, therefore, needs much knowledge and continuous control.

We repeat: the knowledge of the kind of organic matter one has is important in order to introduce and judge the process to be decided upon.

The organic matter test alone will not give the answer to this question.

Whether, in the laboratory, ashing in a crucible or a combustion train, or the wet oxidation method gives more

reliable results, is a matter for discussion, involving circumstances and details which need not be gone into here.

The final breakdown product of the carbon in organic compounds is CO2 — carbon dioxide. Many organisms of phase 1 produce CO2. The more active they are, the more CO2 escapes. This explains in part the losses of organic matter. It is quite essential that natural processes or biochemical shifts take over before the carbon dioxide is formed, and that the materials are used by other organisms prior to the final breakdown into CO2, ammonia or free nitrogen. In our laboratory,2 we use in fermentation tubes measured samples of compost and determine the amount of CO2 which is produced over a set period of time — two, three, five, seven and fourteen days. This gives a

measure of the intensity of breakdown and organic matter losses. In the composition of the B.D. Compost Starter, we have reduced the presence of C02-producing organisms to the absolute minimum, with the hope that the other, more economical organisms grow fast enough to outgrow or to crowd out such wasting organisms which are naturally present in waste materials.

Inorganic Matter: The ashes of the combustion test, again, give only the sum total and in no way inform one as to whether the origin is from minerals or from formerly living cells and tissues. To analyze these ashes for their composition involves complicated analytical procedures.

Spectographic tests may serve as an initial orientation but are unreliable, not so much because of the method used as because of sampling errors.

The heterogenous materials of small

samples would need many repeated tests to arrive at an acceptable average. This is a matter for the skilled chemist.

Moisture: The determination of moisture is important, not only for the final product but throughout the fermentation. Micro-organisms are very specialized, are adapted to specific moisture levels. The moisture content, therefore, will indicate which organisms under certain given conditions will survive, or “go to town� or remain inactive.

The study of soils gives much information on the subject of the ideal moisture content for fermentation, which is quite different from that needed for arresting and keeping finished material.

As soon as the cell structure and walls of the source material are destroyed, mechanically or biologically, moisture will ooze out.

In the beginning we will, therefore, frequently have too high a moisture content. This is the case especially with swill, fresh sludge, pomace and cow or hog manure. Excessive moisture predisposes to anaerobic fermentation, especially if the compost piles are packed, compacted and of great height and width, or set up on poorly-drained soil where a lot of outflow from the piles and rain water collect.

A Russian paper of 1911 (the original has been lost and the source cannot be quoted) explained that the ideal conditions in neutral black soils in the Ukraine, which belong to the most fertile humus soils, were maintained when the precipitation rate and the evaporation rate of water were balanced. This soil would absorb moisture and hold it but never looked drenched or wet. We compare this condition with that of a wrung-

out, not dripping, sponge. This condition secures optimal aeration in a soil. Air is important, for most of the humus-forming micro-organisms need air for breathing. Also, nitrogen fixation takes place best in the topsoil, for these organisms acquire nitrogen from the air. If they are excluded from air, they will consume nitrogen from the organic waste and thus cause considerable nitrogen losses. Fast, hot, fermentation works best with a moisture content ranging from 30%-40%; a slow, cold aerobic fermentation works best with a moisture content between 40%-50%; the upper limit for a favorable and economic aerobic fermentation is 65%. A higher degree of moisture will lead to considerable problems; it will favor decay or putrĂŠfaction for a while until the heat of a compost pile and the thirst of the micro-organisms have reduced the moisture to an

optimal level. Frequent turning, or flat piles, may speed the drying-out process, provided it does not rain; if it does, then matters get worse.

The control of moisture in industrial composting is considered so important that we recommend a moisture tester for such operations and would correct excessive, as well as too little, moisture, at once. Too low a moisture is corrected exactly as per calculation; that is, we do not pour on water but measure it out.

Here the home gardener or farmer is at a disadvantage and has to take things as they come. Eventually, he too will get humus or compost soil, for this is the final state of all things: the return to soil.

Total Nitrogen: The analytical method for nitrogen determination is strictly a laboratory method and needs skill. It tells only the amount of nitrogen and not where the nitrogen

comes from. Within the reach of the chemist are also methods for the determination of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, as well as quick extraction methods for the latter — as in the case of soil tests, which in the field are helpful for quick orientation.

For exact determination, the AOAC methods are the only ones permissible.

For control of compost fermentation in the compost yard, the quick extraction methods are useful to find out how much ammonia, nitrite and nitrate a pile develops. Ammonia is the breakdown product of bacterial action or decaying nitrogen compounds and may be considered as an “end� product of breakdown. (In living animal tissue this would correspond to the N-metabolism and the formation of urea.) In the field, the smell of ammonia indicates the degree to which the source materials are

broken down and nitrogen eventually lost. It is in the interest of economical composting to interrupt the production of ammonia before it is too late, or to direct a fermentation in such a way that the bacteria take over and rebuild more stable nitrogen compounds — for instance, in bacterial protein. To establish this balance is really the “secret� of successful and scientific composting. The means are: control of moisture, aeration, proper mixture of original ingredients, turning a pile at the right moment and interrupting prolonged, excessive heat by turning.

The value of nitrogen saved will more than compensate for the expense of turning with suitable equipment. In large-scale operations, the advantage is that excellent machines for this purpose are available which are expensive to buy but which pay for themselves by cheapest operating cost. With one Barber Green

overhead loader we were able to turn and mix piles at a rate of 60-80 tons/hour at a cost of 20¢ per ton (everything included — labor, maintenance, depreciation, etc.). The small compost-maker is again at a disadvantage here. However, by turning and mixing a pile at the right moment one is able to save a lot of N losses and to convert the compost more quickly into humus. The human nose is very sensitive to the odor of ammonia. A so-called strong smell might mean a concentration of only 0.1%, which is negligible.

If a pile develops too much heat and ammonia, we turn it immediately, together with another pile which has cooled down already and does not smell of ammonia. With the use of the Barber Green, this is no problem whatsoever. Maybe in the near future we will have smaller, inexpensive machines for medium-sized

operations; by this is meant 500-3,000 tons/year. What must be remembered is that ammonia is always the indicator of the breakdown process and of the presence of prevalent ammonia-producing micro-organisms.

Less noticeable is the breakdown into nitrite by other microorganisms.

This form of nitrogen will fall apart very easily and release free nitrogen, usually a total loss. These bacteria, therefore, are not desirable at all, though they are very helpful in the initial phase of breakdown (phase 1).

The problem is to interrupt their activity at the earliest possible moment, by the same methods as given for ammonia, and to introduce, as fast as possible, phase 2 of the fermentation.

Aeration also will help to foster the third group of organisms which either form nitrates or fix nitrogen into more complicated compounds — for

instance, bacterial protein and amino acids. Nitrites and nitrates in the field can be determined by quick extraction methods, such as are used for soil. In fact, they must be determined in this way unless a laboratory is nearby, for a sample taken and shipped will change very rapidly, so that the ammonia and nitrite situation is obscured, while nitrates in a dry sample have more stability.

In the laboratory we determine the presence of ammonifiers, nitrifiers or nitrate-formers by using fermentation tubes as for bacteriological research.

The compost sample or the pure culture, as the case may be, is kept in such fermentation tubes in the incubator at controlled temperatures (we use 29°C) and the amount of ammonia, nitrite and nitrate produced by a weighed sample is measured after three, five, seven and fourteen days. In this way, we gain a deeper

insight into the actual processes present in any given sample and corresponding to the situation in the pile.

One could also arrive at an answer by separating and isolating the organisms on culture media and determining the species, etc. But this is a long, drawn-out affair which needs skilled personnel specifically trained in the bacteriology of compost fermentation, which is not taught as yet, and for which no textbook exists.

If it is said that one can leave all this to nature, that proper conditions will establish themselves, this cannot be denied. This is what has happened ever since there has been soil. But the economy of compost-making demands better methods than that; it requires time-saving and material-conserving methods. This is a necessity if one wants to do business. Since the proper conditions for the many

organisms involved can be studied, it is not too difficult to introduce proper procedures. Those who deny this, prove only that they don’t as yet know the facts.

The appearance of nitrate comes at a later stage of fermentation and is a sign of beginning stabilization and mineralization. In fact, a finished compost should contain only stable organic nitrogen and a small amount of nitrates. With regard to soils, it is said that about 5% of the organic matter of a soil should be present in the form of nitrogen. This, of course, does not include undecomposed matter or green manuring.

As a rule of thumb, this applies to many composts, too. The point should not be pressed too hard but serves for swift orientation.

During the breakdown phase, the presence of free amino acids can be demonstrated by chromatographic

methods. We use one method which has been published elsewhere (5, 6).

Likewise, the presence of free amino acids can be demonstrated in humus soils, especially those on which legumes have been growing over longer periods, while rundown, mineralized soils show less variety of amino acids and less of each amino acid. No doubt, organic nitrogen in well-rotted, stabilized compost is bound to proteins and amino acids.

When the fermentation is completed towards the formation of humus, none, or very few, amino acids show up because all nitrogen is fixed to higher forms of life. The same is true of simpler organic acids — acetic, citric, maleic acid, sometimes oxalic acid — that is, acids with no nitrogen, which also appear in the free state only during the breakdown. Our readers may now begin to understand why the term “total nitrogen” cannot

cover the differentiation as to its origin, biological value, stability or instability, availability and quick or slow release in the soil, all of which are important factors for the efficiency and application rate of compost — in short, factors of quality and value.

The problem of the C:N (carbon:nitrogen) relationship has recently been discussed and thoroughly investigated (3, 7), so that it is not necessary to go into detail here. As long as the relationship is excessively in favor of carbon — for instance, 44:1 as in straw or sawdust — as much organic matter must be broken down and eventually wasted as is necessary to reduce the C:N relationship to a favorable bracket.

The ideal figure is said to be 11:1 but, in practice, we found that at a rate of 20-22:1 one already gets a good and economical fermentation. If it is not

possible to mix high nitrogencontaining source material with low nitrogenous materials, a nitrogen correction must be applied by the addition of specific sources, such as hoof, horn, hair, castor bean pomace, fish, blood meal, or poultry manure.

Of mineral nitrogen compounds, calcium nitrate has been found most beneficial, while urea and ammonium sulphate have to be added with great care because of their easy breakdown and quick release. Ammonium nitrate is dangerous, for with dry compost it may form an explosive mixture.

The narrow C:N relationship of 8:1 or less is highly exposed to too quick a release of nitrogen and very unfavorable for stabilized composting. It is here that greater problems exist if one wants to break down, without losses, such materials as we have mentioned above as corrective additions.

Theoretically, this can be expressed also in other terms. A content of 1.5% nitrogen in compost or manure is the upper borderline of stabilization (6). Above that limit, all materials will tend to produce ammonia and lose nitrogen. It is much better to adjust the formula of mixtures right from the beginning so that one does not exceed the critical concentration; then one has the best chance of not encountering carbon and nitrogen losses. Once the breakdown has gained momentum, it is quite difficult to arrest the downward movement. Within a few days, one will observe that a 2% N content has been reduced to 1%. Many manures have 1.5-2.5% N when they leave the animal. At the time these manures are applied to the field and worked into the soil, they have run down to 0.5% N. No wonder many agronomists say that manuring is a wasteful procedure.

The U.S. Department of Agriculture recommends the immediate plowing under of manures. This, alas, is no solution to the problem, for the devaluation takes place in the soil.

The composting and stabilization of manure is a much more conserving practice, provided that one knows how to do it.

Ammonia is preserved (that is, fixed) in acid solution but liberated in alkaline media. Since the biological process tends towards the alkaline, the ammonia-producing phase must be overcome, the earlier the better. For nitrogen fixation and such organisms as azotobacter and nitrosomonas, the presence of lime (calcium), even in small amounts, is beneficial.

Unless one deals with very acid and wet materials, we feel that lime should be added only to the second phase of fermentation; then, however, it is very beneficial. One needs only

fifty pounds of limestone, finely ground, per ton of compost. In areas of limestone formation, with the water and plants containing more calcium, this may not be necessary. In fact, in some such areas calcium damage to acid-loving plants has been observed when ordinary garbage was composted.

Quick release of ammonia and calcium damage are part of the reason why “reinforced” composts cause burns on lawns, flowers and shrubs; they are too “rich”. Undecomposed poultry manure is known to cause such burns.

While it is relatively easy to get composting underway, because it is in the nature of the materials involved to get started with some kind of breakdown (decay will always be the result), it is more difficult to direct this decay into definite channels of fermentation and to correct an

undesirable trend. The knowledge of all the factors mentioned so far is of help, but the home gardener, farmer or even the manager of an organic composting plant will ask: How do I get to know about all this without a laboratory? Our advice in the past has been (a) to use as many different materials as possible for composting, for then you will have the best chance of getting a little from every process; (b) in order to brake violent, onesided action, to use soil as addition, which acts as a kind of buffer and also favors the development of the phase 2 organisms; (3) to control your moisture; (d) in case anything goes wrong, to turn your pile. Excessive heat is as undesirable as a cold, wet, “lame-duck�. Excess of anything is undesirable.

We could, of course, give composting classes, showing the different types of fermentation and

behavior of source materials and explaining why and how the pile looks and smells as it does and has its particular structure. We could show, for comparison, controlled experiments with most or even all of the contributing factors known. In this way the layman could learn by comparison and orient himself by the examples. I have on occasion given such instructions and demonstrations.

Usually, people in the audience begin to tell how they make it, assuming that their method is gospel. To me it is a puzzle why in this field we have on the one hand so much dilettantism, and from the side of science, on the other hand, so much prejudice.

The plant manager in charge of large-scale composting, in order to make the operation efficient and economical, needs some basic information; knowing the temperature, moisture, organic matter, nitrogen,

structure and degree of aeration are all musts. It is important for him to know when to turn a pile, when to interrupt fermentation, when and how much water to add. It must be realized that some of the tests we have mentioned above, especially isolation of micro-organisms and fermentation tests, take time and the compost is probably stockpiled or sold by the time the results come in.

Temperature can be read in a few minutes; a moisture test with suitable equipment takes up to ž to 1 hour; a micro-Kjeldahl test for nitrogen about 1 hour, a macro-test longer; organic matter by combustion, several hours.

(These need analytical equipment, exact scales and some experience.)

All these tests indicate details. We have been looking out for a simple method to check the overall pattern.

We have tried the sensitive crystallization method developed by

this author which, in the checking of other biological materials, has performed quite satisfactorily. With regard to compost it may give some information, but the method is too cumbersome for practical surveys.

For the last three years, we have been using a method of circular filter paper chromatography which has performed quite satisfactorily and is rather simple, requiring very little equipment. This method is based on the property of filter paper to separate fractions in a solution by way of capillarity. It then needs a reagent to “fix� the fractions and make them visible.

Chromatographic Determination of Humus Extracts from Compost Fractions Principle: The test described is a qualitative one in order to separate different fractions of humus extracts by means of the capillarity of suitable filter papers. The filter paper is prepared with a photo-reactive substance (for instance, silver nitrate) which also reacts with the extraction substances.

The precipitation of this reaction occurs at various distances from the point of application of the substance to be tested. The distance, the pattern, the color and the shape of the reaction area are significant for an interpretation of the substances contained in the extracts.

In using this method, no attempt is

made to identify the chemical nature of the reacting substance, since the pattern obtained can itself be used as a diagnostic means. However, identification is possible. Of the different possible techniques for chromatography, the circular method of chromatography was selected since it gives easily obtainable results with simple equipment and is easy to interpret.

Method: 1. Preparatory Steps: (a) Use circular filter paper discs of Whatman No. l, or No. 4, paper, 5⅞ inch (15 cm.) in diameter. Punch a hole in the center, about 1/16 inch diameter. (b) Prepare a wick of the same filter paper by cutting strips ž inch square and rolling them tightly into a cylinder. Place this wick into the center hole, protruding on either side

of the disc but so that it touches the bottom of the porcelain dish underneath (cf. e). It is important that the wick touch evenly on the circumference of the center hole in the filter paper disc in order to make a good, homogeneous contact.

(c) Make pencil marks at 1½ inch and 2â…œ inch distances from the center to indicate how far the solution should penetrate or be absorbed (see sketch).

Depending on the run of the fiber in the filter paper, the solutions sometimes spread more quickly in one

direction than in the other — hence the double pencil marks in two different directions at a right angle (see sketch). (d) Have a 0.5% solution of nitrate of silver, Ag N03, tested purity, in aqua dest., available. The solution should be kept in a green or brown bottle, not exposed to direct sunlight (no cork stopper); it can be made once a week. In any case, it should not be too old and not show any dark rings or deposits. (e) Place the filter paper disc with the wick on a petri dish of 3½ inch diameter, center over center; the height of the petri dish is ⅝ inch.

Inside the petri dish, center over center, place a porcelain dish of 1¾ inch diameter and 7/16 inch height (see sketch). (f) Pour 3 to 5 cc of the 0.5% Ag NO3 solution into the porcelain dish;

remove the filter paper disc before pouring and replace it afterward. This amount of Ag NO3 solution can be used for the preparation of 3 filter paper discs in succession. (g) The solution of 0.5% Ag NO3 will, by capillarity, spread over the disc, radiating in all directions. When it has reached the first pencil mark at 1½ inch distance (a1 or a2 — see sketch), remove the disc immediately from the petri dish and take out the wick at once, very carefully in order not to tear the hole. Then place the disc on another petri dish for drying.

Protect from direct light and dust (no darkroom is necessary). As soon as the disc is dry, put it into a dark box to keep it in a dry atmosphere. The discs will last for about 3-5 hours before the Ag NO3 reacts with the paper and causes discoloration. No disc showing even a slight

discoloration should be used. It is, therefore, best to prepare the disc immediately prior to use. Needless to say, all filter paper should be handled with care; no fingerprints or smudges should be retained by the filter paper; these will cause undesirable reactions.

2. Testing Procedure: Have the petri dishes and the porcelain dishes ready in a box (cf. apparatus). Pour the humus extract, or whatever you want to test, into the porcelain dish. Use 5 ml of the extract to be tested (see No. 6). Put the prepared filter paper with a new wick over the solution on the petri dish, so that the wick just touches the bottom of the porcelain dish. Let spread until the solution reaches the first 2⅜ inch pencil mark (b1 or b2). This will take about 20-60 minutes. Don’t let it run over the mark. Remove the disc and wick and place disc again on a petri

dish for drying.

3. Developing Phase: The development of the pattern should take place in diffuse daylight (not direct sun).

In order to obtain comparable results, the developing should be done in the same intensity of light. Unless you use measured artificial light (for instance, fluorescent lights), you need a certain judgment as to the intensity of development — that is, when to evaluate the pattern. Just do not expose to direct sunlight.

4. Evaluation: Keep discs in a dark (any folder) and dry place. Put a dry paper in between each sheet in order to avoid reactions between discs. Fingerprints will still show on finished discs.

5. Apparatus: The test is carried out in a box, the top of which consists of a glass plate,

subdivided into three removeable sections for observation purposes.

The size of the box is arbitrary. We use one 36″ long, 17″ wide and 4″ high. Twelve discs can be processed in it. The box is painted with aluminum paint. It should be aired and cleaned after each use. It is wise not to crowd too many discs into one box; you should have several boxes if you want to run more tests at a time. The atmosphere in the box should be moist, close to the saturation or dew point. Therefore, we place shallow, small dishes (crystallization dishes or beakers) with distilled water at equal distances here and there in the box.

This water will evaporate and maintain an even moisture. The temperature is not too essential but should be between 65° and 85°F. Needed glassware, etc.: Petri

dishes, 90mm (3½″)

diam., 15 mm (⅝″) high Porcelain dishes, 40mm (1¾″) diam., 12 mm (⅞″) high Filter paper, Whatman No. 1 or No. 4, circular, 15 cm. diam.

5 cc graduated cylinders or test tubes marked at the 5 cc level

Standards for Preparation and Extraction of Samples Many different methods of extraction and degrees of concentration of samples have been tried out in the Biochemical Research Laboratory. In the following we report only those standards which have given us the most satisfactory results and which can be used for comparison with our test materials and for our way of interpretation. It must be realized that any change of extraction, or of concentration, changes the observable pattern, so that chromatograms deriving from the differences in the preparatory steps cannot be compared. Only those which have been prepared completely alike can be compared. For all other cases, new standards of interpretation must be developed. Concentrations and extraction times may also neèd

occasional variation.

Preparation of Soil and Compost Extracts Put 5 grams of compost or soil into a 125 cc Erlenmayer flask. Add 50 cc of a 1% sodium hydroxide solution (prepared from sodium hydroxide pellets). Mix thoroughly by twirling the flask, let stand 15 minutes, then repeat the twirling. After an hour, mix thoroughly once more by twirling the flask. Then allow the flask to stand undisturbed for five more hours. After the sixth hour, carefully pour the supernatant liquid into a small beaker.

Use 5 cc of this extract for each porcelain dish.

Extracts of Grains, such as Wheat, Rye, Oats, or Barley Grains are ground, as received, in a laboratory grinder, such as the Wiley Multicut Mill (for materials

with a low oil content). This mill contains shearing plates and shreds. A nut mill can also be used. It is important that the material pass through the mill in the shortest possible time, without heating up. The sample should be processed immediately after grinding. Ground samples should not be stored for any length of time. We usually use 12 grams of sample in order to obtain a good average sampling and have enough material on hand.

Weigh 2.25 grams of the ground material, place in an Erlenmayer flask of 125 cc capacity, add 50 cc of the 0.1% sodium hydroxide solution. Mix thoroughly by twirling the flask, repeat after 15 minutes and then let stand for 14 hours. Cover the flask with an inverted glass beaker. The extraction should be started in the late afternoon to be ready for the next morning. Carefully decant the

supernatant liquid into a glass beaker of about 50 cc capacity and measure 5 cc with a small measuring cylinder for each flat-bottomed, capsule-form, porcelain crucible (10 cc capacity).

We also run a test with 1.5 grams of ground sample in 50 cc of the 0.1% NaOH solution, proceeding in the same way as described for the 2.5 gram sample. This method gives additional information if needed. It does not always show such drastic differences as the other.

Small Seeds Grind the seeds cautiously in a small nut mill; avoid heating up. From the ground material weigh 1 gram into the flask and add 50 cc of the 0.1% NaOH solution. Mix by twirling the flask at the start, after 15 minutes and again after 30 minutes. Total extraction time: 4 hours.

Flours and Dough

Flour and dough are used as received from the outside, or as freshly prepared in the laboratory.

Again we use 2.25 gram and 1.5 gram samples and proceed exactly as described above.

Breads of All Kinds The measured amount of bread is mortared for a few minutes in a porcelain or glass mortar with a small amount of the 50 cc of the 0.1% NaOH solution (to be used for the extraction) to make a homogeneous paste. 2.5 grams of bread are used for our standard determination and 1.5 grams for supplementary information.

Proceed as with the grains. In the case of bread samples, however, the extraction time is 4 hours.

Fresh Green Leaves (All Kinds), Vegetables, Fruit, Nuts The incoming material should be as

fresh as possible. It is first cut as finely as possible with clean (not rusty) scissors and then mortared. Use 2.5 grams of the finely-cut material to 50 cc of the 0.1% NaOH solution and continue as described previously. The time interval between cutting, mortaring and adding to the extracting solution should be kept as short as possible. The extraction time for all of these products is 4 hours. Incoming material can be kept in a refrigerator at about 40°F. Special storage and research on keeping quality may demand different handling of the source material, but the method of extraction, etc. remains the same.

Roots, Tubers, and Bulbs These are finely grated prior to mortaring; then one proceeds as with leaves, vegetables, etc. Use 2.5 grams of material to 50 cc of the 0.1% NaOH solution. Extraction time is 4

hours.

Green Herbs Fresh green material is finely cut, mortared and processed as with fresh green leaves; that is, no tea or infusion is brewed. Our standard is 2.5 grams of material; because of grade variations in strength, 1.5 grams may be needed for added information.

Dried Herbs and Teas Two paths of investigation are open: (a) to proceed as with any other dried or dehydrated material, or (b) to test the material in the form of a tea.

In the latter case, an infusion of the dry herb or tea is made by putting 2.5 grams in 50 cc of distilled water, bringing it up to the boiling point without losing fragrance. Then immediately add 50 cc of the 0.1% NaOH solution, let stand for 2 hours, then proceed as usual. A cold

extraction might be used, as well.

In the case of very strong teas (black) containing lots of extractives, a direct chromatogram of the tea alone could be tried. In that case, 5 grams may be used in 100 cc of water.

Juices, Drinks

Pressed

Extracts,

Soft

The juices are not filtered but used as they are received. 5cc are added to 5cc of the 0.1% NaOH solution.

Proceed as usual but here the extraction time is only 1 hour.

Enzyme Preparations Of the pure enzyme preparation in dry powder form, 0.1 gram is used.

10 cc of the 0.1% NaOH solution are used for all enzymes which are active in alkaline media, while 10 cc of the 0.1% HCL solution are used for enzymes active in acid solution.

Extraction time is 1 hour.

Yeast Preparations 0.1 gram of the dried yeast powder or yeast cake is extracted in 10 cc of the 0.1% NaOH solution. Extraction time is 1 hour.

Vitamin Preparations, Single, Pure, or in Mixture (i.e., Complex) The concentration depends somewhat on the strength of the preparation. To begin with, we use 0.1 gram in 10 cc of 0.1% NaOH solution. Extraction time is 1 hour. It might be necessary to modify this concentration. The spread is between 0.5 gram and 0.01 gram. For pure crystalline vitamins, one can either reduce the amount of substance or increase the amount of the NaOH solution. Then, too, if different vitamin preparations are to be compared, it will be necessary to calculate the actual formula to have the same amount of strength of each

vitamin, especially if comparison with pure vitamins is desired.

Dried Foods, such as Macaroni, Egg Noodles, etc.

Use 2.5 grams in 50 cc of 0.1% NaOH solution. Extraction time is 4 hours.

Sugars, Honey, Molasses, Maple Syrup In general, 2.5 grams in 50 cc of 0.1% NaOH solution are used.

Extraction time is 1 hour.

Milk For fluid milk, 2.5 grams in 50 cc of 0.1% NaOH solution are used.

Extraction time is 1 hour.

Dried, Dehydrated Milk Powder 0.1 gram in 10 cc of 0.1% NaOH solution is used. Extraction time is 1 hour.

References 1. Official Methods of Analysis of the Association of Official Agricultural Chemists, 8th ed.

Washington, DC, 1955.

2. Ehrenfried E. Pfeiffer. The Compost Manufacturer’s Manual. Philadelphia: The Pfeiffer Foundation, Inc., 1956.

3. U. Springer. Methodenbuch, Band II. VII. “Die Untersuchung von Humushandelsduengern.”

Berlin: Newman Verlag, 1954.

4. C. S. Piper. Soil and Plant Analysis.

New York: Interscience Publishers, Inc., 1950.

5. Ehrenfried E. Pfeiffer. “A Study of Amino Acid Metabolism with Urine from Tuberculosis Patients.” The American Review of Tuberculosis and Pulmonary

Diseases, 76, No. 5 (1957).

6. Ehrenfried E. Pfeiffer. “The Amino Acid Metabolism and a New Test for Amino Acids in the Urine.� Journal of Applied Nutrition, 11, No. 3 (1958).

7. Firman E. Bear. Soils and Fertilizers, 3rd ed. New York: John Wiley & Sons, 1949.

1 This laboratory, founded by Dr. Pfeiffer in 1946, was closed in 1975.

2 The Biochemical Research Laboratory mentioned earlier.

A QUALITATIVE CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF BIOLOGICAL FACTORS I. Differences in Humus and Compost Quality Method: Since 1953, a simple chromatographic method has been used in my laboratory for the purpose of determining differences in the formation of humus in soils, as well as in compost differences which cannot be determined by chemical analysis. There are soils which have almost identical values of available mineral substances; their biological

efficiency, however, as well as the yield and quality of the crops grown on such soils, differs widely. Quite a few years ago, we published the analyses of two soils which were nearly identical. The one field produced maximum yields, the other far below average, medium yields.

The difference between the two lay in the soil structure and the humus condition. In a similar way, composts containing the same mineral substances, the same NPK, the same pH, even the same amount of organic substance, may have a widely varying effect on soil structure, humus formation, humus condition, yield, germinating quality of the seeds, and protein quality.

In order to differentiate between these qualitative and biological values, the method to be described here was developed. It rests upon the property of certain specially

manufactured filter papers, through which individual fractions may be separated, which, in turn, may be made visible through a reagent. This qualitative, chromatographic method does not replace a chemical analysis of the fractions, which would be a long, drawn-out and difficult procedure. It allows, however, the interpretation of the chromatographic picture, which shows distinct differences in colors and forms as related to qualitative and biological values. The causative chemical reactions (in most cases we have to do with a mixture of substances that cannot be separated easily) might be determined by other comparative methods insofar as this is possible today. To report this is not the purpose of the present article.

The separation takes place through the capillarity of the filter paper. This must have a homogeneous fiber

structure in all directions. Whatman Filter No. 1, chromatographic quality, is satisfactory. We use circular discs of 6″ diameter.

In order to work out the chemical and biochemical details, one would have to separate sufficient amounts of the mixtures or extracts to be tested, using chromatographic absorption columns with various absorbents; the individual fractions are then collected and enriched, re-dissolved and then made available to further methods of analysis. This is not the subject of our present treatise. Simple circular chromatography as described here suffices for a quick orientation.

The same method has been applied for quality tests of aromatic or other active substances in coffee, tea, tobacco, extracts from drugs, foodstuffs and vitamin preparations, where chemical analyses were too complicated or did not show any

results (as, for instance, in the coffee aroma of various kinds of coffee beans in various roasting processes).

We shall now discuss some of the results obtained.

Evaluation of Chromatograms Preliminary Remarks: Even the best color reproductions do not always give the finest differentiations and hues of the originals. We are also aware that a verbal description is inadequate. Anyone who works with chromatograms himself will understand at once what is meant. In order to learn how to interpret the pictures, one starts with chromatograms of well-defined substances. Gradually a collection of “standards� will accumulate.

The following points have to be considered in reading the chromatogram: (a) Number, width and color of the different zones, as well as their regular or irregular formation and shading. In the

pictures reproduced here in the insert, we distinguish three main zones: an outer and a middle zone, which are mainly due to the organic material to be tested; an inner zone which indicates the presence or lack of mineralization. The width of the zones corresponds to the amounts of characteristic substances. (b) Ring formations between the middle and outer zone and at the edge of the outer zone. (c) Color of the zones: a light to medium brown, evenly distributed, points to a good colloidal humus formation; dark brown enclosures point to acid humus substances; violet radiations point to increasing mineralization and reduced organic substance. In the case of plant extracts, vitamin preparations and foodstuffs, other

colors are observed. These will be discussed later on. (d) Radiation, number, color and shape of spike-like formations.

The violet radiations of the inner zone again indicate the breakingdown tendency toward mineralization. The various phases of fermentation (first, decomposition; second, humus formation; third, mineralization and greatly advanced decomposition) are clearly indicated in the chromatograms of soils and compost.

Interpretation of the Pictures in the Insert For the sake of comparison, we first show some extremes of soil extracts (No. 1-6). Then we show chromatograms of composts from city garbage and manure with and without addition of the B.D. Compost Starter.

Further, we show pictures of composts of the most varied kinds and origins, some of which have been produced with the help of the B.D. Compost Starter (No. 7-12). These reproductions illustrate the manifold possibilities of applying the method.

No. 10,11 and 12 show various stages of the industrial composting of poultry manure: first the raw material, then two stages of transformation. The procedure described here serves as a production control as used by the composting plant in question.

Wherever available, the results of

analysis are reported. All figures of available minerals in soils are reported in lbs./acre. Organic matter is reported in percent.

Illustrations No. 1: An extract of black virgin soil from the Missouri River region in the state of Missouri. This soil possesses a natural, stable humus, an ideal, friable structure, and is of maximum fertility. Attention is drawn to the medium brown edge zone of the chromatogram with darker brown spots. The middle zone extends, spike-like, into the edge zone. The pattern of the radiating forms is harmonious. The inner zone is dark brown; there is no violet coloring.

Analysis of the soil: pH 7.0; available minerals in lbs./acre: nitrate 20, ammonia 5, phosphate 170, potassium 180, exchange calcium 5500, organic matter 5%.

CHROMATOGRAMS OF SOILS

1. VIRGIN SOIL, VERY FERTILE.

2. ADOBE SOIL, DEAD.

3. BOTTOM LAND SOIL, MEDIUM FERTILE. WATERLOGGED.

4. “BLACK DIRT”, VERY FERTILE.

5. FERTILE ORGANIC PASTURE SOIL.

6. WELL AERATED B. D. SOIL. No. 2: As a contrast, we show a black, tough clay adobe soil from California. This soil is caked and crusted when dry, sticky and smeary when wet. It lacks aeration and offers many structural problems. The analysis of available minerals is misleading because this occluded soil was rather infertile. Its microflora

was poorly developed. Plant roots could not utilize the available minerals. Analysis: pH 8.2, nitrate 64, ammonia traces, phosphate 30, potassium 450, exchange calcium 3000, organic matter 2.2%. The absence of valuable humus compounds is shown by the lack of form and faint brown coloration of the edge and middle zone of the chromatogram. The inner zone is comparatively large and contains hardly any humus signs. The capillary radiation is of a violet shade, indicating the mineralization of this soil. Its black color does not reflect the presence of stable humus but is caused by reduced crude organic matter. This soil is rather dead.

No. 3: A heavy, brown clay from a wet, badly drained meadow of a valley in southeastern New York, with a comparatively good stand of grass but many acid grasses and a weak

stand of clover. The raw humus was acid. Observe the absence of the outer zone, which would indicate colloidal stable humus formation. Analysis: pH 5.4, nitrate 72, ammonia 0, phosphate 120, potassium 100, calcium traces, organic matter 2.8%.

No. 4: A peculiar “black dirt� soil from southeastern New York. This is an alluvial soil, probably of a bottom basin formed after the last ice age. It has a high content of organic matter and an unusually high content of nitrogen. It is, however, not a muck or a peat soil. The fertility is high. The soil is well-aerated.

Chiefly vegetables are raised on it. Analysis: pH 6.8, nitrate 128, ammonia 5, phosphate 75, potassium 130, calcium 4000, organic matter 36.5%, total nitrogen (on dry base) 1.56%. A good, stable and colloidal humus condition is present.

No. 5: A medium heavy, good

pasture soil, somewhat waterlogged at times but with a reasonably good humus formation. This soil has been improved over a four-year period from a pH of 5.5 to 6.0, from an organic matter content of 2.8% to 4.5%, nitrates from 30 to 64 lbs./acre.

The edge zone of the chromatogram indicates the degree of humus formation; the middle zone and spikes show the influence of the incomplete drainage to a mild degree. Analysis: pH 6.0, nitrate 64, ammonia 0, phosphate 140, potash 100, calcium low, organic matter 4.5%.

No. 6: A well-aerated, welldrained soil of the same farm in southeastern New York. This field has always given reliable crops. The brown color of the edge and middle zones shows a slightly different but very significant hue, with less gray and more brown and yellow in it.

Analysis: pH 6.2, nitrate 20, ammonia

0, phosphate 170, potash 100, calcium 200, organic matter 4.2%.

These two soils were under biodynamic treatment for many years.

They show the improvement of the organic living condition.

No. 7: Chromatogram from a compost made from a rather poor quality of city garbage, containing little organic wastes but lots of ashes and dust. The garbage was ground up and a pile set up for fermentation without addition of the B.D. Compost Starter. The fermentation period at the time the sample was taken was several months. The lack of a proper outer zone is noticeable on the chromatogram. The middle zone is gray-brown, with rather bulky, unformed spikes. The inner zone is large. Its violet radiation color is an indication of the high amount of mineral matter in this compost. This compost is truly “mineralized�.

No. 8: Exactly the same garbage, set up at the same time as the previously described pile (No. 7), but with the addition of B.D. Compost Starter. The piles were otherwise handled in the same way. The samples were taken in the same hour and the piles were the same age. We notice in the second chromatogram the presence of the outer, light brown edge zone, indicating good humus formation. The spikes of the middle zone are clearly formed and show a lively brown, not gray-brown color.

The inner, mineralized zone is slightly smaller and has a slight brownish hue over the violet radiation, indicating that the mineralization has not advanced quite as far as in No. 7.

The analysis, on dry basis, was:

Although sample No. 8 contained less organic matter, it contained more nitrogen. Its state of humus, its microlife, and its biological value were considerably better, which is also indicated by the higher number of humus-forming actinomycetes in it.

Illustration No. 8 also indicates that this compost had been fermenting too long, because in a properly-handled, B.D. Starter-treated compost, one can stop the fermentation much earlier; in fact, before the third phase of fermentation; that is, here the mineralization phase has started. If the fermentation is interrupted at the right time, the chromatogram should not show a violet inner zone at all.

7. COMPOST FROM POOR CITY GARBAGE WITHOUT B. D. TREATMENT.

8. COMPOST FROM SAME GARBAGE BUT WITH B. D. TREATMENT.

9. COMPOST FROM BARNYARD MANURE AND COFFEE WASTE. B. D. TREATMENT.

10. RAW BROILER POULTRY. MANURE. UNTREATED.

11. COMPOST FROM POULTRY MANURE (SEE #10). B. D. TREATMENT. ADVANCED FERMENTATION.

12. COMPOST FROM POULTRY MANURE (SEE ABOVE). B. D. TREATMENT. It is in such cases as the two reported here that the circular chromatographic method will show distinct qualitative, biological differences, where the chemical analyis will not give the proper answer. We have studied many such

cases of the chemical equality and biological difference of various composts, but space and cost of reproduction do not permit the presenting of more examples. In all these cases, the composts which produced the more colorful, browner, better-formed edge and middle zones and less of the violet radiation in the inner zone were also the ones which, in pot or field tests, produced greater amounts of yield, even if a lesser amount per acre was applied. The composts showing less colorful and less formed chromatograms — no matter what else the chemical analysis may be — produced inferior results in field tests.

No. 9: The chromatogram was made from a properly made and B.D. Starter-treated compost composed of 60% barnyard manure, 30% coffee waste, and 10% soil, including a small amount of garbage. Here we see

an almost ideal humus formation with an intensively brown-colored edge and middle zone, and well-formed small spikes; even the inner zone is still brown and does not as yet show the violet radiation.

The mineralization phase of the compost has not yet started. This compost was produced at Golden Acres Farm, Inc., Newtown, PA. Analysis: pH 7.1, organic matter 26.8%, total nitrogen 2.04%, both on dry base.

No. 10: Chromatogram of fresh broiler-poultry manure as it arrived at the compost plant of M.P.C. Corporation, Gainesville, GA. This poultry manure was collected from many poultry farms and contained the droppings plus a large amount of sawdust or peanut shell litter. The collected, mixed sample, obtained by quartering, was ground up in a laboratory mill and 5 grams were extracted with 50 cc of a 1% sodium

hydroxide solution, of which 5 cc were used for each chromatogram.

The illustration shows that we are dealing with crude organic matter and not compost or humus.

No. 11: Chromatogram from composted broiler-poultry manure, that is, from the material shown under No. 10. The compost was made by mixing said poultry manure (70%) with 20% Hybro-Tite (granite meal of the Potash Rock Company of America, Lithonia, GA), 5% colloidal phosphate(Lonfosco of Loncala Phosphate Co., High Springs, FL) and 5% alfalfa from screenings, all by weight. The B.D. Compost Starter was added while we mixed the compost with a Barber Green Overhead Loader, Model OSM544 with snow buckets. The mixed material was piled in fermentation piles. The actual fermentation was completed in from 16-21 days, or

even less under favorable conditions.

It is important, when using our new hot and fast fermentation, that the piles do not over-ferment but are interrupted at the right moment, when the humus formation is well on its way and the mineralization (phase III) has not yet started. Controlling the composting process with the method described has become an important practice, especially if one wants to preserve the original nitrogen content and to obtain a good, saleable, organic humus fertilizer. This illustration shows a condition where the second fermentation phase has already been overshot and the mineralization has moderately advanced. We see, therefore, the broad edge zone (humus), the wellformed middle zone — both already with a grayish-brown color — and the violet inner zone, still with a brownish hue. A violet mineralized

zone in this case is to be expected because of the addition of natural rock mineral to the original mixture. The compost at this stage smells and looks earthy.

No. 12: Compost from the same material, mixture and procedure as outlined under No. 11, but not quite as far advanced into the mineralization process. The very broad and intensively brown edge-zone color indicates the presence of a very good, stable humus. The middle zone, though small but still with the brownish, not grayish, hue, indicates the transitional state between the second and the third fermentation phase. Actually, this compost pile could have been used for drying and bagging several days prior to the collecting of the sample. The violet inner radiation, still with a slight light brown hue, indicates the beginning of mineralization, as well as the

presence of strictly mineral matter.

Our advice in this case was to stop the fermentation at an even earlier stage. Analysis:

The illustrations so far demonstrate examples of biological values which cannot be taught in a purely chemical analysis.

The circular chromatographic method lends itself to many other problems. In the next chapter a few other applications will be discussed.

II. STUDIES OF VITAMIN PREPARATIONS The question has frequently been asked: Is there a difference between a natural and a synthetic vitamin? This question cannot be answered directly because, if we have a pure, crystalline vitamin as a result of a well-balanced chemical formula, this crystalline, chemically pure product will be the same whether it came fom a natural or a synthetic source. Such is the definition of a chemically pure substance. There will, however, be a certain difference which we may ascribe to the strictly chemical environment of the more or less (usually less) chemically pure and crystalline product. This may be due to so-called impurities or to concomitant association with unseparable by-products. Commercial folic acid preparations contain

numerous impurities which, according to Drs. Warwick Sakami and Robert Knowles of Western Reserve University School of Medicine in Cleveland, 1 have an “intensive effect on certain enzymes” in the body.

In such an entirely different realm as that of metals — for instance silverware, gold or other metal jewelry or steel — one can recognize the “imprint” of the manufacturer by the presence of certain trace mineral impurities which occur in one smelting plant and not in another. The different origin of such silver and gold can then be recognized to a certain extent.

It is also possible for so-called vitamin preparations to be typed according to their origin — natural or synthetic. This means, not that they are identical or not identical, but that the case history of extraction from original source material is reflected

in the final product. Of course, the biological effect of these preparations would be similar.

In order not to be misunderstood, we will therefore use two terms in the following discussion: (1) “vitamin” as a collective term — a combination of various factors (or substances) showing that particular vitamin reaction in a biological assay; (2) “pure vitamin” as defined by the exact chemical name, which may also mean a synthetically pure substance. We will, therefore, speak of a vitamin C preparation, of natural origin whatever this may be, containing the proper potency of C (definition 1) or of pure crystalline ascorbic acid, which may be synthetic or purified to the utmost possible degree (definition 2).

The circular chromatographic method will make this differentiation possible. It is well understood, then,

that the vitamin product (definition 1) will show a pattern including that which remained from the natural environment, while the pure preparation will not show the remnant of a natural chemical environment. In this regard, there may be a difference between the natural and pure vitamin (cf. illustrations No. 12 through 19, 21, 22).

Another question can be asked in this connection: To what extent have the properties of the natural source material been preserved or removed in the manufacturing process? Or, in laymen’s words: To what extent has nature been refined and eliminated in a given product? This problem can be compared with the differences between an unrefined sugarcane juice, molasses, brown sugar and white sugar, or between a refined oil and an unrefined one. Obviously, the superrefined pure substance contains

nothing but itself, while the intermediate steps of extraction or refinement may contain biological values in addition to the one proper only to the pure substance. This same “fading out� of concomitant naturally contained substances can be observed in the decline of nutritional value from whole grain (wheat, for instance) to bran, 90,80,70% extraction flour. This decline can be measured in terms of % protein, amino acid pattern, contents of minerals, trace minerals, vitamins and enzymes.

Again, an example from another realm may illustrate what we mean: Pure corn, olive or other plant oils will all have the same caloric value, namely, 884 calories per 100 grams and 100% fat, but unrefined, red palm oil may contain 100% fat and the same calories, namely 884 calories but, in addition, other intrinsic values,

such as 10,080 I.U. of vitamin A, not even considering the differentiation between saturated and unsaturated fats.

We have to ask our readers to keep the two definitions clear and apart so that they will get an accurate picture of what follows.

The method for the demonstration of differences in vitamin preparations is again the same: circular filter paper chromatography. Two paths are open: to use the vitamin preparation (the eluant) first and the developer (in our case, 0.5% of a nitrate of silver solution) second, or vice versa. In each case, one will have to find out which approach leads to a better differentiation.

For all the illustrations in this report, the developer, nitrate of silver, was used first in order to impregnate or sensitize the filter paper, and the eluant followed. In all cases the

vitamin and enzyme preparations were eluated with a 0.1% sodium hydroxide solution.

No. 13: Chromatogram of a chemically pure ascorbic acid U.S.P. We dissolved 0.1 gram in 10 ml. of 0.1% sodium hydroxide solution.

After 30 minutes, the filter paper absorption was started. We can observe a very small brown edge zone, one small undulated ring zone, two wide belts of the middle zone, all three showing pale shades of grayviolet, and a medium large yellowbrown inner zone.

No. 14: Chromatogram of a commercial product of ascorbic acid U.S.P. quality. Dilution and procedure as under No. 13. The pattern is slightly different; the inner ring of the middle zone is more brownish and the pale ring between the inner and outer ring of the middle zone is sharper; otherwise both patterns are quite

similar.

No. 15: A vitamin C preparation, the vitamin C derived from rose hips.

The manufacturer took specific care to preserve the “natural” quality and did not purify the product to the point where it could be called “chemically pure.” This pattern is quite different from the chemically pure one, showing much more undulation and spike formation, as well as radiation of the inner zone which, in fact, extends all the way out. The middle zone shows a strong brown color; the inner zone contains a yellow-brown which had somewhat faded at the time the chromatogram was used for this reproduction. It is quite obvious that this natural product contains “something” which the pure chemical ascorbic acid did not show. This “something” consists of “impurities,” in a manner of speaking, but they are by no means unessential or

biologically without value. It is known, for example, that natural forms of vitamin E complex lose up to 99% of their potency when separated from their natural synergists.2 In fact, the “impurities” have a biologicaldynamic importance. We know this because we have learned to recognize, by many comparative chromatograms of living substrata, the undulating and radiating forms as well as the intensive colorations as a sign of biological activity, that is, of intrinsic values. At the present stage of our knowledge, it is not possible to identify “chemically” the nature of these concomitant substances, that is, the substances of the chemical or biological environment. We know, however, that the very same undulation and radiation, though with different colors, occur in enzyme studies and with extracts from living seed.

No. 16: Chromatogram of a natural vitamin C preparation by another manufacturer. Again, the manufacturer applied care to preserve the “natural” quality without going into the extremes of chemical refinement. This preparation was made from acerola.

The eluation and procedure with these two preparations (No. 15 and 16) were exactly the same as in No. 13 and 14. All four preparations were diluted so that the same amount of ascorbic acid was present. This fourth preparation shows the characteristic proper to ascorbic-acid but, with regard to the concomitant “impurities” or biologically active accompanying ingredients, it is different from No. 15. Again, we see that intrinsic factors of biological activity are present. It is the presence of these biologically active factors, found in the natural but not the synthetic vitamin, which makes the

chromatogram interesting. As with the soil, a chemical analysis for ascorbic acid (Vit. C) would be the same for No. 15 and 16 as for No. 13 and 14, but the chromatogram shows there is much more to the natural C than just ascorbic acid.

No. 17: Chromatogram of a chemically pure thiamin hydrochloride U.S.P. We dissolved 0.01 gram in 10 cc of the 0.1% sodium hydroxide solution and proceeded as described above. The edge zone is medium brown; the middle is medium-large and gray, with two rings and an undulating separation toward the center; the inner zone is very large with a pink color in its center part, a strongly undulating separation towards the center and a white center ring.

No. 18: Chromatogram of a chemically pure riboflavin U.S.P. We used 0.01 gram and the same

procedure was applied as in all other cases. The outer zone shows a small, well-separated brown ring, a medium dark gray middle zone (two rings) and an intensely yellow-colored, homogeneous small inner zone, separated by a white ring from the middle zone.

No. 19: Chromatogram of a chemically pure nicotinic acid (niacin) U.S.P. Again, 0.01 gram used, procedure the same as above. We see a pale brown, slightly undulated small outer ring. The middle zone is separated from the almost colorless inner zone by a slight radiation (grayviolet ring or halo). The middle zone shows several rings, the outer one dark brown with a reddish tinge, followed by a small halo, then a homogeneous medium brown-violet layer, again followed by a small “halo� ring.

These chromatograms of pure

substances are necessary in order to establish “standard� patterns.

We will now discuss illustrations No. 21,22 and 23, returning later to No. 20.

No. 21: A commercial product of a B-complex, containing refined sources of thiamin hydrochloride (0.3 mg.), riboflavin (0.1 mg.) and nicotinamide (0.66 mg.). The figures in parenthesis represent the amounts used for the chromatograms. This suspension also contained pyridoxine chloride (0.02 mg.) and pantothenic acid (0.55 mg.). The difficulty here is that it is not possible to type such mixtures to exactly the same concentration as is possible with the pure, refined chemicals. In any case, we receive a mixed pattern, divided in the outer, double middle and inner zones. The outer and middle zones show the characteristics of the pure thiamin and niacin pattern, but the

inner zone fails to show the intense coloration of the pure riboflavin, while it still shows the pink of thiamin. Chromatograms of the pure chemicals made with exactly the same concentrations as the mixture have been made but cannot be published here because of lack of space.

CHROMATOGRAMS OF VITAMINS AND ENZYMES.

13. PURE ASCORBIC ACID.

14. A COMMERCIAL VITAMIN C PRODUCT

17. PURE THIAMIN.

18. PURE RIBOFLAVIN.

21. A COMMERCIAL B COMPLEX PRODUCT.

22. AN ALLEGED NATURAL B COMPLEX PRODUCT.

15. A NATURAL VITAMIN C PRODUCT FROM ROSE HIPS.

16. ANOTHER NATURAL VITAMIN C PRODUCT FROM ACEROLA.

19. PURE NIACIN.

20. PURE DIASTASE FROM CLARASE, A STARCH HYDROLYZING ENZYME.

23. A NATURAL VITAMIN B COMPLEX

24. PURE PAPAIN, A PROTEOLYTIC ENZYME No. 22: A vitamin B-complex preparation made from natural sources with preservation of the original natural quality. This mixture is composed somewhat differently from No. 21 and therefore the two cannot be exactly compared with regard to quantitative contents. The purpose of showing this pattern is to

demonstrate again the presence of other biological factors as they become visible by the undulation and spikes. Again, the components of this preparation were not refined to chemical purity but were properly typed as to their contents in specific vitamin potency. The eluation solution contained 0.3 mg. thiamin, 0.6 mg. riboflavin, 0.4 mg. niacin. In this case, the riboflavin (0.6 against 0.1 in No. 21) kicked through with a slightly yellow inner zone.

As we will demonstrate in another paper in the future, the specific “spoke pattern� is significant for the enzymatic and other biological activity too, as one can observe in living tissue and germinating seed tissue extracts.

No. 23: Chromatogram of another vitamin B-complex preparation made by the same manufacturer, who cares especially for the preservation of

natural quality. Again, the vitamins were not refined to chemical purity and the preparation contains other biologically active materials. The formula is the same as the previous one, with the exception of niacin, of which 0.01 mg. were present in the aliquot specimen. The outer ring here is interrupted because of the stronger undulating pattern of the middle zone.

The brown colors are a little darker, the yellow inner zone slightly fainter.

As a whole, the pattern is about the same as in No. 2, pointing again to a strongly active biological quality.

We return now to No. 20.

Illustrations No. 20 and 24 belong to investigations of enzymes, a study which is still going on.

No. 20: Chromatogram of pure diastase (“clarase�), standardized.

Diastase is a starch-digesting, amylolytic enzyme. Its strong reactivity is indicated by the strongly

undulating wavy middle zone. Here even the process of refining the enzyme to standard purity has not destroyed that pattern which we consider to be significant for biological activity in addition to the concomitant chemical nature of the investigated substance. Use was made of 0.1 gram of clarase, diluted in 10 ml. of an 0.1% solution of sodium hydroxide (as eluant). About 1.5 ml. of the solution were taken up by the chromatogram.

No. 24: Chromatogram of pure papain, a proteolytic enzyme obtained from the fruit of papaya. 0.1 gram papain eluated in 10 ml. of 0.1% sodium hydroxide solution, 1.5 ml. being absorbed in the chromatogram.

This is a most peculiar pattern and color, entirely different from all others shown in this paper. At present we have brought in the two enzyme chromatograms only to utilize the

opportunity to have an illustration printed in color. The discussion of the underlying enzyme problem must be reserved for a future publication.

In this communication we have restricted ourselves to presenting mere facts and observed material, omitting details of identification and any discussion of the underlying chemical problems. The purpose of this paper is only to demonstrate the possibilities of application of the method reported. The role of silver nitrate as the most suitable reagent, because of its reducing ability in weak alkaline solution in the presence of naturally occurring biological material, has been established in histochemistry with regard to the staining of tissue in order to detect vitamins, especially C, in situ3.

1 Science (Jan. 30, 1959).

2 Annual Review of Biochemistry (1943), p.

381.

3 Geoffrey H. Bourne and George W. Kidder, Biochemistry and Physiology of Nutrition, II (New York: Academic Press, Inc., 1953), p.

24ff.

CHROMATOGRAMS OF GRAIN AND FLOUR It has been the endeavor of the Biochemical Research Laboratory to build up a series of standards with regard to the quality of agricultural products, seeds, fruits, herbs, etc.

This work has led to many interesting observations. One of our primary interests was to determine the character of a natural product grown under well-known conditions and to follow up the fate of this natural product during processing.

The German philosopher and poet, J. W. von Goethe, coined a phrase 150 years ago which is of the utmost importance with regard to the recognition of natural biological quality: “The whole is more than the

sum total of its parts.” This means that a natural organism or entity contains factors which cannot be recognized or demonstrated if one takes the original organism apart and determines its component parts by way of analysis.

One can, for instance, take a seed, analyze it for protein, carbohydrates, fats, minerals, moisture and vitamins, but all this will not tell how good the seed is or how well it will germinate; it will not tell its genetic background or its biological value.

Biology today recognizes such terms as “directional” or “organizing” factors which cause a living organism to germinate, reproduce, etc. — in other words, to put the right process into the right place at the right time.

Thus, a pattern of growth, of life, of health (normal growth and metabolism) or of disease (abnormal growth, abnormal metabolism) develops. Growth or life forces are at

stake. An older concept spoke of a vital force per se. Most modern scientists are reluctant to accept the existence of such a life force and try instead to demonstrate only the presence of well-defined chemical substances. They are willing to accept such terms as “directional, organizing factors.” They also try to find out whether the seat of life can be discovered in any particular substance or part, for instance, the cell, the plasma, the nucleus, etc. All trials of manifold procedures of investigation have led to great discoveries about the biochemistry of the living cell and/or organism but have not yet given the answer to the question, “What is life?” At best, they have shown how life operates.

The words of Goethe, quoted above, contain a deep philosophical implication which the biologist sees before his observant eyes every day:

the sum total of all the detailed analyses of form and substance does not as yet tell the “why” of life. It only informs about the “how.” The approach to this question leads to a fundamental concept, because in this function we see the outstanding feature of a living organism. Life as a whole is more than the sum of all biochemical reactions or the form pattern of the organism, cell, seed or whatever, which is to be investigated.

Of all the scientists, the physicist, Erwin Schroedinger, whose contributions to the quantum theory of the atom have earned him a Nobel Prize, has probably given one of the most interesting answers. In a series of lectures in Dublin, in February, 1943, he described how an organism maintains itself because of its metabolism, which is in a curious process of exchange of substance.4 Nutrition provides this substance. But,

in addition to the substance, energies are also involved. Usually these energies are described in terms of calories. But it is neither the exchange of substance alone nor the use of caloric energy alone which explains life.

The stroke of art, says Schroedinger, by means of which an organism maintains itself in an orderly state, that is, in a high degree of order (or organization), exists in the fact that in reality this organism continuously absorbs from its environment the principle of order. It is the well-organized state of matter which serves as food, which supplies the more or less complex organic compounds to serve as food. Each higher organism builds itself by absorbing (digesting) material provided by a lower order of life.

There are different degrees of order in our food. There is also the

principle of a dynamic state, an equilibrium or balance involved.

It is not our intention at the moment to enter into a discussion of the philosophical problems involved; in our work we want only to raise the question. In nutrition, however, a concept evolves which indicates that the mere presence of substances does not sustain life or health but that, in food, we receive other qualities which go above and beyond the concepts defining proteins, carbohydrates, fats, minerals, vitamins, enzymes, or calories alone.

The way a plant has grown or an animal has grown and built itself up, whether or not it has received plenty of light, which makes it synthesize its body — these factors have a determining influence upon its quality and true food value.

Our approach was entirely empirical, though we kept an open

mind and had no prejudices in any direction. We wanted to see whether it could be demonstrated that a living seed is different from the very same seed if “life” — that is, the ability to germinate — has been removed. We wanted to see whether that which lives in a living seed can be maintained when one processes this seed for food, for instance by milling it to flour and baking bread.

It is known, of course, that vitamins and enzymes, the powerful biocatalysts in any metabolism, can be destroyed under some circumstances, but we were looking for an easily demonstrable, visible method of making this clear to the observing eye.

Using the circular chromatographic method which has already helped to differentiate a living soil from a dead soil, a biologically better-equipped compost from a poorer one, a natural vitamin with its component

accompanying factors from a pure, synthetic vitamin, we attempted to test the life history of seeds, the pathways of processing.

In this article a few facts and observations derived from the experiment will be described.

Because of limitations of space and available funds, we can give only a few illustrations, but we hope that eventually all the empirical material on hand can be published.

The chromatograms here were made by extracting the wheat seed or flour for several hours with a solution of 0.1% sodium hydroxide. A round paper disc of 7″ diameter had previously been treated or sensitized with a solution of 0.5% nitrate of silver. This was our standard method used for all pictures shown here. The solution to be tested was tested by being sucked up with a wick, then spread by capillarity into the treated

filter paper. Once a certain point was reached, the absorption was interrupted and the paper dried. Then it took several hours to days for the color and pattern to develop.

It is now necessary for the reader to look at the illustrations as we describe them. Take the four pages of illustrations and open to pages 36 and 40 so that you have the whole sheets before you.

Illustrations: No. 1: Extract from a wheat seed.

This particular wheat was grown on a farm in eastern Pennsylvania, where the bio-dynamic method of compost and manure treatment with the B.D. Compost Starter was applied. Variety: Dual wheat, a soft red winter wheat, 1958 harvest. The chromatogram was made on April 7, 1959; therefore the seed was used about 8½ months after harvest. The germination percentage

was 94%, indicating a good quality seed. The following analytical data may be of interest: moisture 12.8%; crude protein as received 10.5%; same on dry basis 12.0%; thousandgrain weight 38 grams. The percentage of essential amino acids in the total crude protein was 21.1%, which is a low average.

The information the chromatogram gives is expressed in terms of form and color. The concentric rings are caused mainly by starch and, to a certain extent, by gluten, as we will see later (illustrations No. 23 and 24).

The spokes more or less protruding from the outer ring area toward the center are caused by proteins and indicate quantity as well as quality of protein. They are also an expression of life, for in “dead� materials they disappear. The pink hue is thiamin, as we found out by comparison with chromatograms of pure thiamin. The

most important features in this chromatogram, however, are the small, round or berry-shaped holes or spots with practically no visible coloring, which appear just outside the darker third ring (counting from the outside). These empty spaces appear only in live material. Between the darker, outermost ring and the lighter second ring (counting from the periphery) is a small ring zone, somewhat lighter-colored, frequently almost white. This halo also is a sign of life and not found in “dead� material.

Under ultra-violet light of a wave length of 2537 and 3660 one can also see fluorescent rings, not visible in daylight, on the outer edge and in the center area, indicating the presence, if any, of niacin and riboflavin. The intensity of color and the form pattern gives a measure, therefore, of certain component parts

of the seed in addition to the “sign of life.” The “sign of life” — that is, the white spots — does not contain any identifiable substance.

No. 2: This wheat was then germinated according to standard laboratory procedures.

The chromatogram made from the swelling seed on the first day of germination is entirely different from the original seed. While the starchy fraction remains rather unchanged, the protein factor has undergone considerable changes, so that the original pattern disappears completely. Enzymatic processes take over which break down the original protein in the germ.

There is still the “ring of life,” the lighter shaded area; in fact, now we see two of these rings. As a whole, we interpret this pattern as being significant of a breakdown process of the seed metabolism; significant too is the contraction of the pattern.

No. 3: On the second day of germination, another entirely different pattern results. The concentric rings, so characteristic of the starches in the seed, are loosened up. This is obvious, because enzymatic processes have now started which begin to mobilize the reserve starch for the new growth and nutrition of the germ.

At this stage, no roots or beginnings of leaves are visible as yet. The white ring of life is very clear.

No. 4: The germ has now properly germinated and started its own life and begins to grow visible; root tips, and eventually the first leaf tip, show up.

We see now in the chromatogram (a) the leftover of the broken-down original seed material, and (b) the influence of the first products of the new growth metabolism (synthesis).

Clearly visible protein spokes show up; they are quite different, however,

from those of the original seed. More of the starch pattern (rings) got lost, the halo or ring of life is a little wider and the earliest beginnings of new, not yet quite empty, spaces show up. We know now that the new life and growth have properly started.

1. WHEAT. WSOLE SEED (G.A. '58) 94 % GERMINATION POWER.

2. SAME WHEAT, FIRST DAY OF GERMINATION.

5. SAME WHEAT. 30' HEAT EXPOSURE. NO GERMINATIO

6. SAME SEED SOAKED ONE DAY.

9. SOIL FROM A PEONY FIELD. POOR SECTION' NOTHING GROWS.

11. CORN N.J. # 9, GROWN BIO. DYNAMICALLY, WHOLE SEED.

10. SOIL FROM SAME

PEONY FIELD WITH GOOD GROWTH.

14. FROM A DEBITTERED BREWER'S YEAST WHICH SHOWED A SATISFACTORY MEDICAL RESPONSE.

3. SAME WHEAT, SECOND DAY OF GERMINATION.

4. SAME WHEAT. THIRD DAY OF GERMINATION.

7. SAME SEED SOAKED 2 DAYS.

8. SAME SEED SOAKED 3 DAYS. NO LIFE.

12. GERM ONLY FROM CORN PICTURED IN # 11.

13. SAME CORN AS IN # 11, FLOUR WITHOUT GERM.

15. ANOTHER YEAST OF LOWER QUALITY; (A PRIMARY FOOD YEAST).

16. A POORER QUALITY

YEAST. The pink hue of this fourth day chromatogram is somewhat more intensive, indicating that more thiamin has been released from the original seed.

There is no space to reproduce all the day-by-day chromatograms which follow — such as those, for instance, which show the chlorophyll and other products of synthesis and assimilation after one or two weeks of growth.

Illustrations No. 5 through No. 8 show examples to the contrary: No. 5: The very same wheat as that used for No. 1 through No. 4 was exposed to 212°F heat in a dry oven for 30 minutes and has lost its ability to germinate. Otherwise, a chemical analysis would show about the same amount of proteins, carbohydrates, fats and minerals. The moisture has been reduced from 12.8% to 10.9%.

This, therefore, is a seed with the same major component parts, even the same amount of heat-stable vitamins, but chemically it has lost some heatsensitive enzymes and, of course, life.

Also the amino acid structure has changed only slightly. In the chromatogram, therefore, we see all that is left and unchanged (let us call it the gross chemical structure), but the empty spaces and the light haloring have disappeared.

This phenomenon was observed many times — that the lifeless substance does not produce these “signs of life.�

No. 6: This seed was then soaked in a germinating tray exactly like the other. It did not germinate. It did get soft, however, and swelled up. We see a pattern entirely different from that of a germinating seed of the same age, that is, on the first day. We still see a halo; we see the influence of

protein; we see thiamin; we see the influence of starch, but now fading.

No. 7: On the second day of soaking, the seed falls apart. All characteristic forms, especially those for proteins, fade out much more. As a whole, it is the typical pattern of a decaying process. While in No. 6 the third concentric ring showed a somewhat irregular and serrated borderline, a phenomenon which we learned is caused by enzymes, this enzyme pattern too fades out. Of course, these enzymes are not the same as those employed in growth.

But even a decaying process needs enzymes.

No. 8: On the third day of soaking, the process of disintegration has continued and the fading out has progressed. It is interesting that even here the pink color indicating thiamin is still present.

The comparison of No. 1 through

No. 4 versus No. 5 through No. 8 teaches us several lessons: (1) The difference between growth and decay, which at the same time is the difference between life and death; (2) Progressive changes and their different paths; (3) The difference of a sprout from day to day; this is very important from a nutritional point of view, since it indicates that the intrinsic nutritional value might be different. Judging from the pattern of the second day, we might draw the conclusion that the seed is in between old and new life, while on the third and fourth day — that is, when the first roots begin to show — the sprout begins to unfold its own life.

For those who like to eat sprouts because they feel that the living sprout might transmit more than just the mere substane of the seed, this knowledge may be important. Our personal opinion, based on our present

knoweldge, is that one should eat the seed which produces the pattern of No. 4 rather than that of No. 2. We purposely do not say second, third or fourth day because there might be slower or faster germination due to the age and viability of the seed or temperature and moisture conditions.

Also, different seeds — wheat, rye, oats, alfalfa — may behave quite differently and require different time intervals. Therefore, the state of development, not the measure of time alone, should decide.

In any case, a dead seed, or a seed with low viability, does not provide what the sprout-eater is looking for.

Now we ask you to turn the sheet of illustrations around for a while and unfold the color sheet opposite page 40. We begin with No. 17.

No. 17: Shows the chromatogram of a different wheat, also grown in 1958, at a bio-dynamic farm in New

Jersey. This wheat analyzed as follows: moisture 11.7%; crude protein as received 10.4%; same on dry base 11.8%. The germination percentage was 92%; the thousandgrain weight 39.0 grams, which is quite good. The percentage of amino acids of the total crude protein was 29.2%. The percentage of the 15 most important amino acids which we analyzed was rather high, namely, 89.6%. This already points to a high quality protein; for instance, lysine, which is usually low in grain (average for wheat in literature 2.7%) was in this case 3.8%. Accordingly, in the pattern of the chromatogram we see the “spots of life� larger and more pronounced. The protein pattern also is different from other wheat origins.

While the general principles in a chromatogram apply at all times, seeds or any produce from different places of origin show variations of

these principles.

No. 18: The same wheat as in No. 17 was now exposed to the rough heat treatment-exposure in a dry oven for 30 minutes at a temperature of 212°F. The germination of this wheat after exposure was the same as before, namely, 92%. We, therefore, have in this case heat-resistant enzymes and proteins. This is a characteristic of many bio-dynamically grown wheats, as well as of some other types of organically grown wheats. The chromatogram of the heat-exposed seed, therefore, shows the signs of life again, inasmuch as this seed still germinated at the 92% level. This, therefore, is a seed with a high degree of vitality.

Now we ask the reader to return to No. 1, the whole living seed, and to follow the next investigation group, pictured in No. 25 and No. 26, then No. 19 through No. 28 and No. 29.

The purpose of our investigation was to take the seed apart, grind it to flour and separate the component parts, and finally to put the component parts together.

No. 25: Demonstrates the chromatogram of the germ alone and No. 26 the chromatogram of the entire wheat berry but without the germ.

Neither one, separated, shows the whole pattern of the seed; neither one is able to germinate; neither one reflects a living entity in its completeness, nor would the germ without the kernel be able to survive.

The chromatographic pattern of the germ (No. 25) has all the markings of protein and enzyme influences prevalent. The starch pattern is weak but still the last traces of the signs of life are recognizable.

The kernel without the germ (No. 26) shows a strong starch and a lesser protein pattern. If one would project

both pictures, one on top of the other, the original pattern of No. 1 would result, but without the empty space labeled as “sign of life.” By separating the germs from the kernel, a deep incision into the structure of “life” has been made, even though, bio-chemically, the germ still contains many valuable ingredients. We observe a somewhat different situation if the whole seed is gently ground to a whole-wheat flour, without heating up.

No. 19: Shows the chromatogram of the protective layer, the bran and the aleurone layer, that is, the protein — and enzyme-carrying layers of the kernel. Here everything is still present - the protein, the cellulose, some enzymes, some starch and even the “empty space of life,” but the pattern looks somehow squeezed, irregular and slightly damaged.

No. 20: Here the coarse bran

(about 3%-5% of the whole wheat flour) has been separated and chromatographed. Some proteins and enzymes adhere to the bran, hence a pattern which allows recognition of its origin, but much squeezed and damaged. This is truly a tortured pattern. This, by the way, is the stuff that livestock frequently get in bran concentrates with some life attached as in the leftovers from flour milling.

No. 21 shows another situation: bran and aleurone layer together. The characteristics of bran are minimal but the aleurone layer comes through clearly.

No. 22: This is the chromatogram of the white flour of an 80% extraction from which the bran and most of the aleurone layers have been screened off. There remains some protein, a few enzymes and almost all starch, and that is what the pattern indicates; also thiamin kicks through

clearly. The gluten has now been separated by washing away the watersoluble starch.

No. 23 is the chromatogram of the gluten, that portion which is so significant for wheat. We still recognize the last remnants of the protein spokes.

No. 24 is the pattern of the pure starch without the gluten. This is the typical starch pattern: concentric rings of different coloration. The water left over after the elimination of gluten and starch was concentrated and now contains minerals, all other watersoluble substances which have remained and, above all, enzymes.

No. 27 shows the water-soluble fraction after removal of gluten and starch. The edge pattern is typical for the presence of certain enzymes which can be identified by comparison with suitable enzyme patterns.

No. 28 shows the chromatogram of

the water-soluble extraction from the aleurone layer. This chromatogram has a very significant enzymatic pattern and tells exactly where the most powerful enzymes are located in the berry. Enzymes, too, are proteins of a very complex structure. The protein spokes of the enzymes are quite different from the mass or crude protein; hence we have a radiating pattern all the way through.

No. 29: All the parts which we separated out of the original whole grain — the protective layer, the bran, the aleurone layer, the gluten, the starch, the water-soluble residues — have now been mixed together again, so that the substance of the original grain has been collected and reunited.

The chromatogram of this composite mixture of all the component parts of the grain in No. 1 shows all the markings of starch, of protein, of thiamin and of cellulose, but it does

not show the enzymatic pattern which had been destroyed in the process of taking apart, even though we could catch it in the residue. But, reunited, these enzymes were not effective any more. As we can see, however, the sum total of the parts does not give us back the original manifoldness, color and form of the living seed. It is now the sum total of chemicals and of substance but no longer of life. The original life has vanished, as have the empty spaces and the halo, which were so significant in the living originals.

The truth of Goethe’s words, “The whole is more than the sum total of its parts,� has been demonstrated in an experiment which in every step follows a rational schedule of the separation and identification of the parts.

Whoever studies these chromatograms carefully and studies

the chemical background which leads to them, moves in a world of observable reality. A new vista opens before his eyes. He need not speculate, philosophically, about what life is; he can see with his own eyes to what extent life exists, how and when, and under what conditions it is lost. If he has an unprejudiced mind and proceeds empirically and not speculatively, basing his conclusions on phenomena and nothing else, he can conclude: Life is an expression of the whole, untampered with, not yet separated, and left in the state in which nature has produced it. The implications of this statement are many; the consequences are great.

This writer and his co-workers became fascinated by the problem and its many possibilities of application.

17. WHEAT BIODYNAMICALLY GROWN. 92 % GERMINATION (AS '58).

18. SAME WHEAT AS IN

#17.30' HEAT EXPOSURE. 92% GERMINATION.

21. BRAN AND ALEURONE LAYER ONLY OF SAME WHEAT AS IN #1.

22. WHITE FLOUR MADE FROM SAME WHEAT AS #1.

80 % EXTRACTION.

25. GERM ONLY OF SAME WHEAT AS #1.

26. BERRY WITHOUT GERM OF SAME WHEAT AS IN #1.

29. MIXTURE OF FLOUR' BRAN, STARCH AND GLUTEN OF SAME WHEAT

AS IN #1. THE ORIGINAL PLATTERN CANNOT BE RESTORED.

30. FLOUR FROM SPRING WHEAT

PROTECTIVE LAYER, BRAN AND PART OF ALEURONE LAYER OF WHEAT AS IN #1

20. BRAN ONLY OF SAME WHEAT AS IN #1.

23. GLUTEN EXTRACTED F OM SAME WHEAT FLOW AS IN % 22.

24. PURE STARCR -

TRACTED FROM SAME WHEAT FLOUR AS IN # 22.

27. WATER SOLUBLE RESIDUE AFTER GLUTEN AND STARCH EXTRACTION (ENZYME PATTERN).

28. WATER SOLUBLE RESIDUE FROM EXTRACTION OF ALEURONE LAYER (ENZYME PATTERN).

31. DOUGH MADE FROM

SAME FLOUR.

32. BREAD MADE FROM DOUGH AS IN # 31 The reader is now asked to look at illustrations No. 9 and No. 10, showing the same soil chromatographed from the same field of a farm in Indiana. The farmer grows peonies. On the field is a section which is barren, while the rest of the field grows splendid peonies.

Chemically, there was no significant difference in the analyses of both

sections, the poor and the good one.

The matter was referred to this laboratory. Chromatogram No. 9 tells us that this section lacks air and circulation (drainage problems), so that structural problems exist, causing the roots to die out, while No. 10 from the good section does not show this damage. The poor section contained 50% very fine silt particles, the good spot only 35.3%. The poor spot was occluded.

We return once more to the problem: whole seed and separation into germ alone and kernel alone, without seed, this time demonstrated with corn. We picked out a corn, N.J. No. 9, which grew very well at Golden Acres Farm, Newtown, PA; in fact. of all the various varieties grown there in 1959, it performed best and stood up best.

No. 11 shows the chromatogram of the whole seed from the corn which

germinated 96%.

Corn chromatograms are, of course, different from wheat; the protein enzyme spokes are different; the empty spaces do not show up as holes, but the light ring of life can be seen (third color change, counting from the outside, before the protein spokes). Thiamin and enzymes show up in the radiating pattern and in the pinkish hue.

No. 12 is the chromatogram of the germ of this corn. We see the squeezed pattern; we even see some of the empty spaces (= holes), the sign of life, but we see a damaged pattern because the germ was separated from the whole.

No. 13: Again the chromatogram of the rest of the seed without the germ shows all that there is — starch, protein, zein, thiamin influences — but not the whole.

By the way, pattern No. 11 points

to a better quality of corn.

Chromatograms of poorer quality corn are quite different. This method could be used for grading.

We became further interested in the case history of the pathway from grain to dough to bread which is illustrated

by No. 30 through No. 32.

No. 30: Shows the chromatogram of the flour (whole wheat) from a hard spring wheat organically grown in Kansas. The wheat had a good chemical composition of 14% crude protein, as received at 7% moisture, or 16.0% dry base. Even for a hard spring wheat, this is considered to be a good quality. (Due to lack of space we cannot show the chromatogram of the seed.) The germination was 94%, but the wheat did not have the vitality to germinate after exposure to heat.

The flour, therefore, even though it was whole-wheat flour, had lost some of the qualities of the seed. Maybe the

miller contributed his share to the final product, too.

No. 31 is the chromatogram of the dough made from this flour. The dough contained the flour ground just before baking; that is, the flour had not steamed out or settled. In a settled flour, the pink of thiamin should kick through more strongly, as was the case in No. 30.3 lbs. of flour were mixed with a solution of 3½ teaspoons salt, 1½ oz. honey, 1½ oz. sugar, 2 ozs. of dry yeast, in ½ cup of tepid water, plus 3½ cups of scalded milk. The flour was mixed by hand with all the ingredients but the yeast; then the yeast was added, mixed again for 3 minutes, 3 oz. of shortening added and the dough kneaded for 15 minutes.

The dough was put in a cold oven to rise for 1 hour and 15 minutes; then it was punched down, folded four ways and turned over. It was then cut into three loaves, shaped into round balls,

settled for 10 minutes, shaped into loaves and allowed to rise in the pan for another 45 minutes. The bread was baked for 15 minutes at 400°F; then for 20 minutes at 375°F. We mention these details because later experiments showed that the mixture, rising time, frequency of rising, and baking temperature and time have a tremendous influence on the quality of bread demonstrated by the chromatograms.

Since this publication is limited to 56 illustrations, it is intended to discuss all further observations on dough and bread problems, on baking time and heat, in one of the next issues of Bio-Dynamics.5 Here we can present only a glimpse of the problem for the time being.

The pattern of the dough is entirely different from that of the grain and flour. The pinkish thiamin hue comes very much to the fore. The proteins

are visible as small spokes protruding from the outer ring inward. The starches are completely changed. The pattern, however, is most significant for the action of the yeast; as other experiments have taught us, it shows how well and to what extent the yeast can revitalize the flour mixture. A new life principle enters. Even if a flour were really “dead,� the yeast could introduce a new life principle.

Much depends now on the quality and vitality of the yeast and on the procedure and time of rising.

No. 32: The chromatogram of the bread actually tells how good the bread is, that is, how much of the original seed, flour, dough and yeast are left. As far as we can judge from our present experience, the bread pattern in most cases is an arrested dough and fermentation pattern. Few markings of the original grain and seed have been retained. The reader

is advised to compare No. 32 (the bread) with No. 1 or No. 17 to realize how much of the origin has survived, and also to compare it with No. 25 (the germ pattern) and No. 26 (the berry pattern), and finally with No. 30 (the whole-wheat flour pattern). He will discover indications of all these patterns, but much reduced and changed.

We have worked with many different breads and will devote a special publication to the subject.

While this particular bread of the No. 32 pattern was one of the better breads we have tasted, a true wholewheat bread, we were disappointed that the original life and seed pattern did not penetrate more strongly. We would like to see a loosened-up outer ring pattern, for instance. The yeast has definitely reinforced the thiamin hue and even helped to maintain it in the bread, but many intrinsic factors

of protein, starch and enzymes are no longer present.

Our laboratory then began to study yeast, doughs and bread. So far, of the many breads we have tested, only three have shown a reasonably satisfying pattern. It is now necessary to check into all the problems of bread-making. The question has been raised, but it will take much time and patience to collect all the answers.

The first step was the investigation of yeasts. The opportunity arose because we got many different yeasts for study. I have always had a great interest in yeast; in fact, many years before the war, I produced a patent on a procedure for yeast-testing— the evaluation of yeast performance, quality, and vitality — and cooperated with one of the largest companies producing yeast in Europe.

The reader should now turn to the color sheet opposite page 36 again —

No. 14, No. 15, and No. 16.

No. 14 shows a debittered Brewer’s yeast with marked protein and enzyme signs. This yeast was used medically and apparently performed satisfactorily, according to our informant.

No. 15 shows another yeast, a primary food yeast, also with a satisfactory protein and enzyme pattern but not as diversified as No. 14.

No. 16 shows a yeast which was given to us as an excellent food yeast.

We cannot agree. This pattern is not one of great enzymatic activity. It lacks all the detail the other two yeasts show.

Our yeast studies still continue. It is quite evident that the process of growing yeast, its nutrition, its aerobic or anaerobic growth conditions, have a decisive influence upon its quality. Since there is no

more space for illustrations, we must let the case rest at this time.

The scientific reader must forgive us for getting carried away at times in our report with philosophical problems and with our enthusiasm. It is customary to report only data, but this produces a rather dry, objective style. However, the problems and the vistas which have opened before our eyes are so paramount, and lead to such an entirely new concept of investigation, that we felt entitled to bring up basic and fundamental questions related to the preservation of life, especially as related to our study of seed to bread. We believe that much more work still needs to be done, but we have now also added a new approach to find out about the expression of life itself through the use of chromatography.

The phenomena observed cannot be disputed, no matter what the

explanation or theory may be.

4 In What Is Life?, published in New York by Macmillan in 1946 and based on the Dublin series, Schroedinger says (pages 74-75 and 77): “How would we express in terms of statistical theory the marvelous faculty of a living organism, by which it delays the decay into thermodynamical equilibrium (death)? We said: ‘It feeds upon negative entropy,’ attracting, as it were, a stream of negative entropy upon itself, to compensate the entropy increase it produces by living and thus to contain itself on a stationary and fairly low entropy level . . . Hence the awkward expression ‘negative entropy’ can be replaced by a better one: entropy, taken with the negative sign, is itself a measure of order.

Thus the device by which the organism maintains itself stationary at a fairly high level of orderliness (fairly low level of entropy) really consists in continually sucking orderliness from its environment. This conclusion is less paradoxical than it appears at first sight. Rather could it be blamed for triviality. Indeed, in the case of higher animals we know the kind of orderliness they feed on well enough, viz. the extremely well-ordered state of matter in more or less complicated organic compounds, which serve them as

foodstuffs. After utilizing it they return it in a very much degraded form — not entirely degraded, however, for plants can still make use of it. (These, of course, have their most powerful supply of ‘negative entropy’ in the sunlight.) An organism’s astonishing gifts of concentrating a ‘stream of order’ on itself and thus escaping decay into atomic chaos — of drinking orderliness from a suitable environment — seem to be connected with the presence of the ‘aperiodic solids,’ the chromosome molecules, which doubtless represent the highest degree of well-ordered atomic association we know of — much higher than the ordinary periodic crystal — in virtue of the individual role every atom and every radical is playing here.”

5 Although this material was never published in a formal way, Dr. Pfeiffer did discuss some of the issues mentioned above in an article entitled “Bread-baking, Cookery & Related Questions,” Bio-Dynamics, 65 (Winter, 1963), pp. 14-23.