A HISTORY OF HUMIC ACID RESEARCH

MORE THAN TWO CENTURIES OF HUMIC ACID RESEARCH

—WHY SO LONG?

 

BY MICHAEL SUSIC

RESEARCH SCIENTIST

2 FEBRUARY 2001

(Revised 10 June 2008)

 

ABSTRACT

Even though humus was named in 1761 and the extraction of humic acids was reported in 1786, humic acid structure and origin has only recently been determined. Such a time span from the discovery of a substance to the elucidation of its origin and structure is indeed rare, perhaps unique. Yet humic acids are extremely common, and also are precursors to such important natural resources as coal and petroleum. The pressure to improve agriculture in the 18th and 19th centuries was the impetus behind the effort to understand humic acid chemistry, and the growing importance of coal and petroleum in the 20th century further increased this pressure. There has been no shortage of research effort, with about 10,000 reports in the scientific literature. Despite this, the work has been conflicting and confused. There are two principal reasons why progress has been so slow. First, humic acid chemistry was difficult to unravel with the technology that was available before WWII. Second, as a consequence of this, researchers became stubbornly polarized in their interpretation of data and refused to acknowledge valid results that were different to their own. When adequate technology for the study of humic acids became available after WWII, the previous unhealthy attitudes were continued and major errors went unchallenged for long periods of time. It is hoped that with the latest humic acid discoveries a more sensible approach will prevail, and that future progress will be much more rapid.

 

Introduction

Within the past 150 years or so, the structures of naturally-occurring organic compounds have generally been elucidated within decades of their discovery. A classic example is the structure elucidation of DNA. Even if the exact sequence of amino acids and sugars in for example, a glycoprotein, has not been determined, the number and types of amino acids and sugars is well-known. In contrast, the situation for humic acids has been very different. Humic acids* are brown-black polymeric acids of plant origin that are ubiquitous at the Earth’s surface. They contribute to soil fertility, a fact that has been recognized since ancient times. As the study of humic acids progressed from 1786 when they were first extracted from peat bogs in Germany, their importance has become clearer. Not only are they important for soil fertility, but they are also precursors of kerogens, peat, asphalt, bitumen, petroleum, and coal. But they also have deleterious effects, and in more recent times it has been discovered that humic acids affect the environment by complexing with metals and organics, which can modify the toxicity of heavy metals, pesticides, and herbicides. They can also interfere with the efficiency of industrial processes such as alumina extraction from bauxite, adversely affect water purification, and form carcinogens during raw water disinfection.

Along with a good climate, fertile soils are a necessity for human existence, survival, and enjoyment of life. The cultures of many ancient people were based on an agricultural existence. To them fertile soils were the responsibility of the gods, whom they frequently implored for good crops. As knowledge of Man’s environment slowly grew, especially in the Middle Ages, humans turned their minds to more concrete reasons for good crop yields, rather than the mere whims of mythical gods. Differences in plant litter were a fairly obvious candidate for study, apart from the non-descript entities such as wind, earth, and fire which were in vogue at the time. Hence, the linking of soil fertility with plant litter was no surprise. But the complex nature of this could not have been foreseen. After all, it is now known that plants are composed of a plethora of chemical compounds, and even more are produced in the soil due to microbial alteration of them. Knowledge about this in an era of wind, earth, and fire thinking had to come slowly by observation, experiment, and recording of results. Man’s obvious need for and concern about food, or the scarcity of it, brought humus, and later, humic acids, into sharp focus.

The pressure to improve agriculture in the 18th and 19th centuries was the impetus behind the effort to understand humic acid chemistry. Then, the growing importance of coal and petroleum in the 20th century further increased the pressure. However, from the late 1700s to the mid 1800s researchers were only just discovering such simple things as oxygen, hydrogen, carbon dioxide, and the noble gases, yet the question was being asked:

What are humic acids?

The question came far too early, and with the inadequate knowledge and technology of the time, incorrect viewpoints became stubbornly entrenched. This attitude then persisted even when adequate technology became available after WWII.

* The term humus originally meant all organic compounds of plant origin in the soil, and is used as such in this report. The term humic acids is used for the brown-black, polymeric, alkali-soluble acids found in soils, plants, seagrasses, fungi, sediments, and terrestrial and marine waters.

Plants, Humus, and Humic Acids

Humus is the Latin name for soil, and plant matter in soils was first named as such by Wallerius (1) in 1761. de Saussure (2) introduced it specifically for the dark-colored compounds that later came to be called humic acids. During the period 1630-1750 a search was made for the principle of vegetation, namely, what makes plants grow. Before that time it was known that not all soils had the same fertility, but no-one knew or attempted to find out why this was so. The botanist Linnaeus (1707-1778) classified soils in a manner similar to his classification of plants (refer to Waksman [3]). So when Wallerius defined humus in terms of decomposed plant matter, it was quickly seized upon as the substance that makes plants grow. It was claimed that plants grew by absorbing organic carbon from humus. Surprisingly, this idea persisted until the late 1800s (4,5) despite the fact that de Saussure (2) in 1804 demonstrated that plants derive at least some of their carbon from atmospheric carbon dioxide, and that Leibig (6) in 1841 demonstrated that plants can grow without humus. From 1865 onwards (refer to Waksman [3]) attention was drawn to the role of microorganisms in humus and soil, first by Kette and then by others. Only at this time did the carbon cycle come to be understood. A basic tenet of the carbon cycle is that organic carbon from plants is decomposed by microorganisms to carbon dioxide and returned to the atmosphere, to be reabsorbed by plants. The idea that humic acids per se stimulate plant growth has persisted with the use of commercial humic acid fertilizers and soil conditioners. However, it is now generally believed* that plant hormones, impurities in these preparations, are responsible for increased plant growth. I have shown (unpublished data) that the growth of wheat and tomato seedlings in tap water is not accelerated by the addition of highly purified humic acids. In contrast, I have shown that traces of the steroids pregnanediol and pregnanetriol rapidly accelerate the growth of barley seedlings in tap water. This confirms that trace impurities in humic acid preparations could be responsible for the accelerated plant growth. Although it was originally believed that humic acids directly influenced plant growth by providing a source of carbon, it is now known that the effect, apart from such impurities as growth hormones, is indirect. Humic acids alter soil properties by making them more friable, by buffering pH, by allowing soils to swell with moisture, by complexing with trace metals and making them more readily available to plants, and by releasing bound nutrients such as phosphates from clays. Because of the refractory nature of humic acids, they also control the release rate of carbon to the atmosphere. Soils are a huge buffer of humic acids (7), and since microorganisms decompose them only very slowly (8), soils put a brake on what would otherwise be a rapid return of carbon to the atmosphere. Most other organics such as proteins, lignins, and polysaccharides are recycled far more rapidly.

The earliest studies of humus failed to realize that plants are composed of an enormous number of different organic compounds, and that microorganisms can feed on them and elaborate even more compounds. To elucidate the composition of such a huge mixture would have been a daunting task with the limited technology available in the early 1800s. Fortuitously, humic acids are so refractive (to both chemical and microbial decomposition) that they tend to dominate soil organic matter, which meant that the early workers did not have to be very concerned about separating out other compounds. Initially organic chemistry was poorly understood, but once a basic understanding was established, an explosion in knowledge occurred. This explosion in knowledge about the composition of humus began around 1871 with the discovery of proteins in soils, and continued to around 1920, especially with the work of Schreiner and Shorey (9a, b), who helped to discover such organic components as sterols, hydrocarbons, fatty acids glycerides, resin esters, chitin, cellulose, xylan, sugar alcohols, lecithin, pyridines, amides, amino acids, purine bases, vanillin, numerous aliphatic and aromatic acids, and elemental carbon in humus. Nucleic acids and lignin lagged behind somewhat, and plant hormones a lot more, but by the late 1930s the composition of humus was well known. From when the term humus was coined until its composition was established, about 170 years elapsed, a very long time for a given field of research. But this was only part of the story, because humic acids, the major humus components, defied structural elucidation for much longer. This was not through a lack of effort, though. A review of soil humic acid research was made by Bremner (10) in 1954, and he stated: “The literature concerning the humic fraction of soil organic matter is so extensive that no attempt can be made here to review work carried out on the subject before 1940.” This effort did not abate, and as evidence for this, a search of Biological Abstracts between 1969 and March 1989 (inclusive), gave 3001 reports for the keywords humic substances, humic acids, and fulvic acids. By modelling the data, I estimated that up to 1996 about 7600 reports in European languages, several hundred in Russian, and several in Chinese and Japanese had been published in the scientific literature. Therefore, until now, about 10,000 reports have appeared in the scientific literature. This compares favorably with other very important organic compounds, such as glucose with about 60,000 reports.

* There are some researchers who believe humic acids induce membrane permeability, which allows a more efficient uptake of nutrients and accelerates plant growth. However, for this scenario, humic acids also are not a direct source of carbon.

History of Research

Humic acids were first extracted from peat bogs in Germany by Achard (11) in 1786. They were extracted from plant matter by Vauquelin (12) in 1797, and later from soils by many investigators. de Saussure (2) and Döbereiner (13) began crude studies of humic acids in 1804 and 1822, respectively. The first comprehensive studies were published by Sprengel (14) in 1826. Sprengel extracted humic acids from soil with alkali, the same method that Achard used for peat, and this has been the preferred method ever since for extracting humic acids. As early as 1819 Braconnot (15) added acids to starch and sucrose and formed a dark precipitate that looked like humic acids extracted from soils. This began a major effort to prepare as what was then termed artificial ulmin. Glucose was soon found to give the same type of substances, and by 1835 an explanation for the transformation of carbohydrates to synthetic humic acids was given by Malguti (16). When cellulose was transformed to synthetic humic acids by Mulder (17) in 1839, the genesis of humic acids from polysaccharides was thought to have been confirmed. Only time would tell what a mistake this was because the debate was still raging in 1985, 146 years later! Even though there was agreement about the origin of humic acids at this time, there was much debate about the classification of humic acids. Because humic acids are ubiquitous and can be extracted in different proportions by many solvents, they were extracted from many sources by varying methods. Naturally, the resulting extracts had different solubilities, colors, and textures, the principal properties used to differentiate compounds at the time. This led to the invention of a plethora of new names, confusing their identity, and wasting time on classifications instead of investigating their properties. The multiplicity of names was gradually abandoned, probably because a consensus could not be reached. By the mid 1800s humic acids were commonly characterized by chemical formulas (18,19).

Around the 1870s two discoveries had a major effect on views about humic acids. First, it was demonstrated that other organic compounds with structures as simple as carbon tetrachloride could give dark-colored substances that looked like natural humic acids. Both sugars and these chemicals obviously could not be precursors of humic acids, so the polysaccharide origin of humic acids began to lose favor. Second, the chemical formulas and compositions of humic acids became more and more diverse and confusing, incorporating not only carbon, hydrogen, and oxygen, but also nitrogen, anhydrides, ethers, ketones, hydroxyls, alkyl groups, aromatics, and furans. This complexity, together with the loss of favor of the polysaccharide origin, led to the idea that humic acids were elaborated by microorganisms. Microbiology was a newly discovered and popular field at the time, and was rapidly applied to the humic acid problem, although without any proof. Still, those who favored the microbial transformation of organics argued successfully that such a plethora of elements and groups as had been reported to exist in humic acids could only come from a sort of organic soup, something that could be elaborated by microorganisms. The idea that humic acids come from polysaccharides was strongly revived around 1914 by researchers such as Gortner (20) and Marcusson (21) who were working on coal research. The impetus for this was the discovery that the furan structure was prominent in coal and humic acids. Fischer and Schrader (22), also coal researchers, in 1921 demonstrated that microorganisms rapidly consume polysaccharides, and therefore were adamant that polysaccharides could not be precursors of humic acids. They also claimed that lignin degradation, which was much slower, correlated with the production of humic acids (Note: the correlation was poor). Now that microorganisms were given a raw material, the theory was readily accepted by many researchers, but not all. For example, researchers like Marcusson (23) and Hilpert and Littman (24) vehemently opposed the lignin origin theory. But the lignin origin theory came to predominate, and coal texts such that of Hendricks (25) in 1945 simply repeated the popular concept. Concurrently a protein-lignin origin theory evolved because many researchers found small amounts of nitrogen in humic acids. However, this theory began to wane in the 1950s as more and more evidence accumulated to indicate that the nitrogen was due to proteins loosely complexed to humic acids. In 1938 Waksman (3) wrote a popular book called Humus in which he strongly supported the microbial alteration of lignins as the way in which humic acids were formed. Waksman stated in his book: “No other phase of chemisty has been so much confused as that of humus…” and considered his book to be the first study of humic acids from many different aspects, although he did admit that Wollny did it in 1897 but without the inclusion of microorganisms. Waksman emphasized that the role of bacterial and fungal alteration of plant matter was all-important in humus formation, no doubt because he was a microbiologist. But Bremner (10) in 1954 cautioned against Waksman’s claims, by stating: “Much useful information concerning soil organic matter has been obtained by non-isolative methods of investigation such as Waksman’s system of proximate analysis. It is generally realized, however, that such methods are of very limited and uncertain value, and that to achieve any real progress in the elucidation of the chemical nature of soil organic matter we must return to the isolative method of investigation used by Schreiner and Shorey at the beginning of the century.” (Note: Schreiner and Shorey discovered many components of humus.) This caution went largely unheeded and in the 1950s the transformation of lignins by microorganisms became the dominant theory, and the polysaccharide origin theory was forgotten. Humic acids then came to be seen as some mysterious products derived in an aura of mystery so complex that it could probably never be completely understood. No doubt this led to the fanciful, hypothetical structural drawings of humic acids that began appearing at this time.

The lignin origin theory of necessity gave birth to the concept that humic acids are aromatic rather than aliphatic. This feature created a debate of its own, namely, whether humic acids are aromatic or aliphatic. Many researchers then began looking for just aromatic compounds in humic acids. By the time Waksman wrote his book, the multiple names according to hypothetical classifications had been discarded, as well as the chemical formulas, although Waksman lamented that many chemists still held on to ideas that gave rise to those procedures. The only names that have survived are humus, humic acid, humin, and the term fulvic acid, coined by Oden (26) in 1919. It was not until 1957 that Shapiro (27) again made an attempt to define humic acids by a chemical formula. The disfavor of chemical formulas became so great that I have not seen a single report from the past 60 years, apart from the one by Shapiro, that gave a non-hypothetical chemical formula. The standard procedure has been to quote the carbon, hydrogen, oxygen, and nitrogen (if any) composition of humic acids. The chemical formulas were disdained because the data was so variable and researchers felt that the alteration of lignins by microorganisms was far too complex to be desribed by simple chemical formulas. Shapiro’s work was outstanding because it introduced chromatography, solution-phase IR, and organic solvent extractions into humic acid analysis. However, it was totally ignored, probably because it showed that humic acids, or at least the ethyl acetate soluble fraction, are aliphatic, not aromatic.

In the late 1950s it became popular to use GC and GC-MS as a tool to investigate organic compounds, and humic acids were no exception. Because humic acids are polymers the technique was useless for unaltered humic acids, so they were oxidized according to the much earlier work of Bone et al. (28). Even though GC was popular, the oxidized products were also identified by more conventional methods (29). The products contained mainly aromatic compounds, especially benzene polycarboxylic acids, similar to the mellitic acid that had been obtained by Fischer and Schrader (22) in 1921. Results were interpreted to confirm that humic acids are aromatic compounds, and therefore have a lignin origin. However, the oxidation studies had major flaws which were ignored. Reuter et al. (30) in 1983 demonstrated one of these flaws, namely that the amount of aromatics produced was an artifact of oxidation severity (Note: aromatics can be readily formed from aliphatics such as plant polyketides). The oxidation work had become so entrenched that even in 1989 another report on this oxidation work was published (31). In 1966 a popular book on humic acids was published by Kononova (32), which like Waksman’s book before it, strongly supported the lignin origin theory. In the 1970s KBr IR and solid-state NMR studies became popular, but they demonstrated that humic acids are primarily aliphatics. By the early 1980s many researchers had come to realize that the situation regarding humic acid research had become intolerable. But the problems were viewed differently. One group, mainly supporting the lignin origin theory, helped to form the International Humic Substances Society (IHSS) in 1982. The IHSS was formed to better coordinate humic acid research, especially by collecting a bank of standard humic acid samples so that researchers could work on documented samples in the hope of reducing the huge variability in data. Another group wanted to abandon the lignin origin theory in favor of a theory based on the aliphatic structure of humic acids. For example, in 1985 Farmer and Pisaniello (33) wrote:

“Soil organic matter is by far the most abundant form of organic matter at the Earth’s surface…Yet the nature of its distinctive constituents, the humic substances, remain unknown.”

Waksman had made a similar statement 47 years earlier, so this statement highlighted the lack of progress in humic acid research. Anderson and Russell (34) in 1976 had shown that by polymerizing maleic anhydride, synthetic, aliphatic humic acids were obtained. Farmer and Pisaniello made it quite clear that NMR studies proved that humic acids were aliphatics, not aromatics. However, this evidence did not sway Schnitzer (35), a foremost worker in the humic acid field at the time. He blamed the problem on an incomplete understanding of NMR data. Another popular book on humic acids, Humus Chemistry, had been published by Stevenson (36a) in 1982, again strongly supporting the lignin origin theory of humic acids. Surprisingly, its second edition (36b) of 1994 was not much different. However, more and more researchers in the 1980s and 1990s like Ikan et al. (37) began to report that humic acids contain primarily an aliphatic structure. Also, humic acids of marine origin had been discovered in 1972 by Nissenbaum and Kaplan (38), and were always recognized to be purely aliphatic. Harvey et al. (39) in 1984 even proposed a fatty acid origin for these humic acids. In my studies (40) hydrophobic humic acid derivatives eluted from silica gel gave IR and NMR spectra similar to those of fatty acids, although they were a pale yellow color and gave an intense fluorescence, which distinguished them from fatty acids.

In contrast to the studies of oxidized humic acids, studies of reduced humic acids were few. Interestingly, one study (41) from 1966 found that most of the products were low molecular weight aliphatic compounds, and another (42) from 1987 found that the most prominent structures were due to either 1,4-butanediols or aliphatic ether polymers. With the ever-increasing evidence during the 1980s and 1990s about the aliphatic nature of humic acids, most researchers came to accept that humic acids have at least some aliphatic structure. But the idea of genesis by microorganisms strongly persisted. In stark contrast to this idea, we reported (43) in 1991 that humic acids actually occur in plants, well before plant matter reaches the soil. This was in agreement with a report (44) from 1987 in which humic and fulvic acids were isolated from plant leachates. I also found that humic acids occur in very high concentrations in senescent plant matter that has had no contact with the soil, and in fungi, seagrasses and rotten fruits that do not even contain lignin (40). These results have since been replicated (45,46), and indicate that the microbial origin idea of humic acids is incorrect. In fact, brown leaves, yellow hay, yellow wheat field stubble and burnt toast are all everyday testimony of the in situ chemical conversion of polysaccharides to humic acids. This can be readily demonstrated by extracting the humic acids with alkali, measuring the fluorescence emission and excitation spectra, and comparing them to the spectra of humic acids obtained from soil extracts.

Due to the utter confusion in humic acid research, in a 12-year study using the modern techniques of FT-IR, NMR, GC-MS, HPLC, FT-MS, and fluorescence, I reexamed much of the humic acid research of the past 150 years or so. I discovered that humic acids can be readily synthesized from polysaccharides and sugars, albeit with some reactions that have only become known since the late 1940s. By chromatographing derivatives of synthetic and naturally occurring humic acids on silica gel, I have been able to fractionate them into groups with similar properties. These synthetic and naturally-occuring humic acids have almost identical UV-visible, fluorescence, IR and NMR spectra, together with other similar chemical properties. Many solution IR spectra have very sharp bands, and many NMR spectra have sharp peaks, like one finds in normal IR and NMR organic chemistry, permitting the unequivocal assignment of moieties and structures. This is a summary of the data (40):

1. Microbial alteration of plant organics does not form humic acids. The processes are entirely chemical.

2. Humic acids are entirely aliphatic copolymers of several principal monomeric units originating from polysaccharides. Because there are so many possible monomeric units in the molecule, the variability in composition is enormous. However, the conversion process consists of a small number of well-known reactions to form copolymers of molecular weight near 1000 u.

3. The aliphatic structures readily change to aromatic structures, which however, are not related to lignin-derived compounds such as vanillic and syringic acids.

4. They possess a unique conjugated chelate (keto-enol) moiety that changes the structure of humic acids depending on their environment. This gives them almost infinite variety, no doubt contributing to the mystery that has surrounded them.

5. They stick to almost any substance, including large proteins, minerals, and soil particles. Because of this they can as an example solubilize 10x their own weight of clay particles. If they are not rigorously separated from these particles, they can appear to be extremely large molecules.

6. They degrade readily by well-known chemical pathways (e.g. decarboxylation) to form kerogen, bitumen, asphalt, shale oil, petroleum, and coal tar, the exact conversion probably depending on time and external conditions in the natural environment.

7. Their natural conversions can be readily mimicked and altered in the laboratory, but with greater control. Therefore new compounds unknown in nature but related to components of kerogen, bitumen, asphalt, shale oil, and petroleum can be synthesized in the laboratory.

Lack of Technology

In the late 1800s when it was discovered that compounds other than sugars such as carbon tetrachloride give dark-colored products, the nature of the products could not be distinguished due to a lack of technology. At the time solubilities, colors, textures and degradation of molecules were the principal analytical tools, quite inadequate for distinguishing any differences in the dark-colored products. When I repeated the reactions with polysaccharides, sugars, acetone, and acetic anhydride, and analyzed the products by FT-IR, NMR, and GC-MS, differences in the products were obvious. Some sugars did indeed form humic acids, others did not, and the chemicals such as acetone and acetic anhydride formed very different polymers. Therefore, with adequate technology, there would have been no valid reason to dismiss the polysaccharide origin of humic acids as occurred in the late 1800s. Also, in the late 1800s humic acids were found to contain a plethora of moieties, which indicated to researchers at the time that humic acids must have a far more complex origin than simply polysaccharides. However, humic acids are just one component of humus which contains many organic compounds. Without adequate separation methods in the late 1800s, the humic acid preparations would have been impure and contained many other organic compounds. Humic acids per se may not have contained the many elements and moieties that were found. Therefore, the speculation that microorganisms must produce humic acids because of their complexity would have been unfounded with the help of appropriate technology. Only after WWII were such tools as chromatography, IR, NMR, GC, and MS widely introduced in analytical chemistry. For humic acid studies these tools were indispensable. Prior to this time structural elucidations were painstakingly achieved by disassembling molecules piece by piece using chemical reactions. Then the molecule was synthesized and its properties compared to the natural compound. This approach was useless for humic acids because the only way that the molecule could be disassembled was by oxidation, which destroys the entire molecule and the products are mainly carbon dioxide with some oxalic, succinic, and maleic acids, various keto acids and minor amounts of vanillic and syringic acids if the sample has a source containing lignins. The situation is further complicated because benzene polycarboxylic acids are formed as artifacts of the reaction. So all of this information was useless for structural elucidation.

There can be little doubt that before WWII the technology for the structural elucidation of humic acids simply did not exist. But with the huge amount of work that had been done, the foundation should have been set to resolve the matter quickly after WWII with the advent of adequate technology. However, this was not the case because in 1985 researchers were still arguing about such basic matters as whether humic acids were aromatic or aliphatic! The next section investigates the reasons for this.

Unchallenged Errors

Fischer and Schrader (22) claimed that humic acid formation correlated with lignin degradation, and this gave many researchers confidence that their speculation about microorganisms mediating humic acid formation was correct. However, even if a correlation is high, and Fischer and Schrader’s certainly was not, it does not prove a process. In this case it would still have been necessary to actually feed lignins to microorganisms and measure the humic acids, if any, produced. This experiment was NEVER DONE, or at least never reported. Yet without it the microbial alteration of lignins is simply speculation. Then to make matters worse, Waksman (3) claimed that microbiology alone could determine humic acid composition. No doubt this ridiculous overstatement was simply his biased opinion as a microbiologist. But the unfortunate part is that his errors were not properly challenged. Bremner (10) did it 16 years later, albeit not very strongly, but contemporaries were silent. This meant that by the 1950s the belief in the microbial conversion of plant lignins had an almost religious awe to it. Marcusson (23) in 1925 had extracted humic acids from Sphagnum peat, a source that he correctly identified as having very little lignin (47). To have any credence the lignin origin theory must explain this discrepency, but it has simply been ignored. Further, Grosskopf (48) in 1929 had found humus-like substances forming at cellulose (Note: not lignin) decomposition sites on the cell walls of conifer needles. Again, the evidence was ignored.

Another mandatory experiment was also NOT DONE. The microbial alteration of organics to form humic acids would be meaningless if the humic acids already existed before the plant matter reached the soil. Vauquelin (12) in 1797 had extracted humic acids from plant matter, so why was this not checked? We have shown (43) that there is an abundance of humic acids in plant matter, and that the humic acids are leached into the soil from the senescent plant matter by rainfall. One can see this phenomenon every day with leaves lying in puddles of water after heavy rains, and the water becoming reddish-brown in color. Frimmel and Bauer (44) in 1987 inadvertently did this experiment in the laboratory, and it has been done frequently since (45,46), proving that the role of soil microorganisms in humic acid formation has been a red-herring. Yet, these simple experiment were not done until the 1990s!

Shapiro (27) in 1957 made major advances in humic acid research by introducing chromatography, solution IR, and organic solvents. He clearly showed that humic acids, or at least the ethyl acetate-soluble humic acids, are aliphatic. Why were his methods not adopted, and why was his work totally ignored? When I reexamined his work, I found that solution IR was far superior to any other analytical technique, yet no researcher had adopted this technique! However, his contemporaries spent much time trying to find aromatics by the oxidation method, so one can only conclude that the researchers were solely trying to prove a preconceived idea, no more. If the oxidation method had been useful, one could be a little less harsh in criticizing this work, but it had three major flaws. First, most of the molecule was destroyed by the oxidation process, so attention was directed to the 2-5% (in rare cases more) of material that remained, which was mainly aromatic. The best scenario that could be deduced from such a wholesale destruction of the molecule is that aromatics are less susceptible to oxidation than aliphatics. Second, aliphatics, especially polyketide-type structures, can form aromatics readily, and Reuter et al. (30) quite clearly showed that the aromatics in these experiments were artifacts of the process. Third, the aromatics that were obtained in the greatest amount were benzene polycarboxylic acids, compounds that are not lignin degradation products. Lignin degradation products such as vanillic and syringic acids were obtained in trace amounts, but these compounds are ubiquitous and could have been contaminants. These serious errors were challenged in a small way by Reuter et al. (30), but many years after this method was at its peak of popularity. Why was it not challenged earlier, and strongly, because of its serious flaws?

Solid-state NMR studies in the 1970s and 1980s, although lacking sharp peaks, clearly showed humic acids to be mainly aliphatic. The highest aromaticity obtained was 20-40% (37). When it was made known by Farmer and Pisaniello (33) in 1985 that humic acids are aliphatic, and that aromatic moieties are probably contaminants, Schnitzer (34) continued to defend the aromatic structure and lignin origin theory. NMR had been the technique of choice for structure elucidation of organic compounds, both big and small, for around three decades, yet amazingly Schnitzer blamed the discrepency between NMR data and the supposed aromaticity of humic acids on a poor understanding of NMR data! Surely the obvious conclusion should have been that humic acids simply are not aromatic compounds, or at least that they are principally aliphatic. A report by Schulten and Schnitzer (49) in 1993 certainly showed far more aliphatic content than was previously accepted, but the hypothetical humic acid structure was based only on evidence obtained by Schnitzer and colleagues. Farmer and Pisaniello had earlier made the criticism that aromatic structural evidence seemed to come only from laboratories that Schnitzer was associated with. Why was this ignored and once again a highly aromatic structure presented?

Popular books about humic acids were written by Waksman (3) in 1938, Kononova (32) in 1966, and Stevenson (36a) in 1982. Each one strongly supported the aromaticity and lignin origin theory of humic acids. This had a strong influence on humic acid research, and because the authors refused to address the evidence in favor of the polysaccharide origin of humic acids, probably many researchers were discouraged in the past 70 years from pursuing this line of research. The 2nd edition of Stevenson’s book (36b) in 1994, though acknowledging that there is a polysaccharide pathway (because marine humic acids are clearly aliphatic), still promoted the aromatic character and lignin origin theory of humic acids. However, now it was even more complicated in the guise of the polyphenol theory. Surely by now the time had come to give equal space to the evidence that indicated the entire theory was wrong! As the Humic Acids Research Group states on its web site (50), “Without structural knowledge of humic substances, we will continue to rattle around in the black box of ignorance when asked to predict and forecast the impacts of chemical and biological actions on our environment.” One of the main reasons that we have been “rattling around in the black box” for so long is that major errors went unchallenged far too long. In 2003 a book written by KH Tan (51) made a significant step forward by discussing the carbohydrate origin theory of humic acids in detail. Tan correctly pointed out that humic acids are formed in environments that lack lignin, so this theory obviously needs attention. However, the carbohydrate origin is proposed to occur via Maillard condensation, and Tan believed that sugars could escape rapid microbial assimilation by “hiding” in soil interstices. In my personal discussions with him, he was unaware of the xylan (hemicellulose) origin of humic acids (via rapid biochemical conversion of xylose to furfural followed by oxidation to 4-oxobutenoic acid that then polymerized to humic acids) at the time that he wrote his book, but was favorable to the idea after becoming aware of it.

Why so Long?

The search for humic acid structure and origin has taken so long for two major reasons. Because of the pressure to provide adequate food, the question: What are humic acids? was asked far too early. Further pressure came from the discovery that humic acids are precursors of such important natural resources as coal and petroleum. Before WWII the technology simply was not available to answer this question satisfactorily. However, after WWII, with the advent of chromatography, IR, GC, NMR, and MS, it should not have taken long to answer the question. After all, a huge amount of work had been done to lay the foundation of humic acid research. The other reason for the tardiness of humic acid research has been the failure of the scientific method to curb speculation. Major errors went unchallenged completely or at least partially for long periods of time. Researchers refused to address alternative evidence that contradicted their own views, debates became acrimonious, and opinions polarized. Due to the polarization of opinions, strong evidence for the polysaccharide origin of humic acids was not even mentioned in textbooks, yet major flaws in the research promoting the lignin origin theory were ignored.

In conclusion one must strongly speculate that researchers in the field of humic acids have been overly obsessed with the need to consolidate careers, defeat competitors, and save face with colleagues. After all, why care about the exact structure of humic acids if you lose your colleagues and/or job in the process?  

References

1. Wallerius, J.G. De Humo (Diss. Upsala, Sweden) Agriculturae fundamenta chemica spez. (1761).

2. de Saussure, T. Recherches chimiques sur la vegetation (Paris, France) (1804).

3. Waksman, S.A. Humus, 2nd ed. (Bailliere, Tindall &Cox, London, UK, 1938).

4. Grandeau, L. Recherches sur le role des matieres organiques du sol dans les phenomenes del nutrition des vegetaux (Nancy, France) (1872).

5. Johnson, S.W. How crops feed. A treatise on the atmosphere and the soil (Orange Judd, New York, USA) (1883).

6. Leibig, J. v. Organic chemistry in its applications to agriculture and physiology, Trans. Webster, J.W. and Owen, J. (Cambridge, UK) (1841).

7. Susic, M. and Isdale, P. Hydraulic and Environmental Modelling of Coastal, Estuarine and River Waters (eds. Falconer, R.A., Goodwin, P. and Matthews, R.G.S.) 588-597 (Aldershot, UK, 1989).

8. Moran, M.A. and Hodson, R.E. Limnol. Oceanogr. 35, 1744 (1990).

9. Schreiner, O. and Shorey, E.C. (a) J. Am. Chem. Soc. 30, 1599-1607 (1908), (b) J. Biol. Chem. 8, 385-393 (1910).

10. Bremner, J.M. J. Soil Sci. 5, 214-232 (1954).

11. Achard, F.K. Crell’s Chem. Ann. 2, 391-403 (1786).

12. Vauquelin, C. Ann. Chim. 21, 39-47 (1797).

13. Döbereiner, Phytochemie 1822, 64 (1822).

14. Sprengel, C. Kastener’s Arch. Ges. Naturelehre 8, 145 (1826).

15. Braconnot, H. Ann. Chim. Phys. 12, 172-195 (1819).

16. Malguti, M. ibid., 59, 407-423 (1835).

17. Mulder, G.J. J. prakt. Chem. 16, 495-497 (1839).

18. Berzelius, J.J. Kgl. Svenska Vet. Akad. (Stockholm,Sweden) (1883).

19. Hermann, R. J. prakt. Chem. 27, 165-172 (1842).

20. Gortner, R.A. J. Biol. Chem. 26, 177-204 (1916).

21. Marcusson, J. Chem. Ztg. 44, 43 (1920).

22. Fischer, F. and Schrader, H. Brennstoff-Chem. 2, 37-45 (1921).

23. Marcusson, J. Z. angew. Chem. 38, 339-341 (1925).

24. Hilpert, R.S. and Littman, E. Ber. deut. chem. Ges. 67B, 1551-1556 (1934).

25. Hendricks, T.A. in Chemistry of Coal Utilization (ed. Lowry, H.H.) 1-24 (Wiley, New York, USA, 1945).

26. Odén, S. Kolloidchem. Beih. 11, 75 (1919).

27. Shapiro, J. Limnol. Oceanogr. 2, 161-179 (1957).

28. Bone, W.A., Parsons, L.G.B., Sapiro, R.H. and Groocock, C.M. Proc. Roy. Soc. A, 148, 492-522 (1934).

29. Wright, J.R. and Schnitzer, M. Can. J. Soil Sci. 39, 44-53 (1958).

30. Reuter, J.H., Ghosal, M., Chian, E.S.K. and Giabbai, M. in Aquatic and Terrestrial Humic Materials (eds. Christman, R.F. and Gjessing, E.T.) 107-125 (Ann Arbor Science, Ann Arbor, USA, 1983).

31. Griffith, S.M. and Schnitzer, M. in Humic Substances II (eds. Hayes, M.H.B., MacCarthy, P., Malcolm, R.L. and Swift, R.S.) 69-98 (Wiley-Interscience, Chichester, USA, 1989).

32. Kononova, M.M. Soil Organic Matter, 2nd Eng. ed., Trans. Nowakowski, T.Z. and Newman, A.C.D. (Pergamon, Oxford, UK, 1966).

33. Farmer, V.C. and Pisaniello, D.L. Nature 313, 474-475 (1985).

34. Anderson, H.A. and Russell, J.D. ibid., 260, 597 (1976).

35. Schnitzer, M. ibid., 316, 658 (1985).

36. Stevenson, F.J. Humus Chemistry (a) (John Wiley , New York, USA, 1982), (b) 2nd ed. (John Wiley, New York, USA, 1994).

37. Ikan, R., Ioselis, P., Rubinsztain, Y., Aizenshtat, Z., Pugmire, R., Anderson, L.L. and Ishiwatari, R. Naturwiss. 73, 150-151 (1986).

38. Nissenbaum, A. and Kaplan, I.R. Limnol. Oceanogr. 17, 570-582 (1972).

39. Harvey, G.R., Boran, D.A., Piotrowicz, S.R. and Weisel, C.P. Nature 309, 244-246 (1984).

40. Susic, M. Structure and origin of humic acids and their relationship to kerogen, bitumen, petroleum and coal. At http:/humicacid.wordpress.com

41. Mendez, J. and Stevenson, F.J. Soil Sci. 102, 85-93 (1966).

42. Martin, F., González-Vila, F.J. and Almendros, G. The Science of the Total Environment 62, 121-128 (1987).

43. Susic, M., Boto, K. and Isdale, P. Mar. Chem. 33, 91-104 (1991).

44. Frimmel, F.H. and Bauer, H. The Science of the Total Environment 62, 139-148 (1987).

45. Davies, G., Fataftah, A., Ghabbour, E.A., Jansen, S.A., Radwan, A. and Raffauf, R.F. Sci. Total Environ. 201, 79-87 (1997).

46. Davies, G., Fataftah, A., Radwin, A., Willey, R.J., Ghabbour, E.A. and Jansen, S.A. in The Role of Humic Substances in the Ecosystems and in Environmental Protection, (eds. Drozd, J., Gonet, S.S., Senesi, N. and Weber, J.), 567-572 (Polish Society of Humic Sustances, Wroclaw, Poland, 1997/8). 

47. Hänninen, K.I. The Science of the Total Environment 62, 193-200 (1987), and references therein.

48. Grosskopf, W. Brennstoff-Chemie 10, 293 (1929).

49. Schulten, H.-R. and Schnitzer, M. Naturwiss. 80, 29-30 (1993).

50. At http://www.hagroup.neu.edu

51. Tan, K. H. Humic Matter in Soil and the Environment Principles and Controversies (CRC Press, Boca Raton, USA, 2003).

THE CRITICAL EXPERIMENT THAT WAS NEVER DONE

 

18 February 2011

The structure of humic acids has always been in dispute. However, the origin and genesis of humic acids has had much less controversy. The reason for this is that a critical experiment was never done or at least never reported until we did this in 1989 and 1991 (Susic, M. & Boto, K.G. J. Chromatogr. 482, 175-187 (1989); Susic, M., Boto, K. and Isdale, P. Mar. Chem. 33, 91-104 (1991)), and by that time the theory of microbial conversion of plant matter in the soil developed in the 1920s and 1930s had been almost universally accepted. In the above studies we demonstrated that humic acids per se were already present in plant matter before it even reached the soil. Later research showed that humic acids were also present in fungi and sea grasses. The 1991 research report stated: “Humic acid persists throughout the lifespan of a leaf but is lost to the soil when the leaf decomposes.”

Why this critical experiment was never done or reported is an enigma, and has resulted in total ignorance regarding the origin and genesis of humic acids. Therefore, I recently revisited this matter in detail and have shown conclusively that senescent plant matter contains enormous amounts of humic acids that are readily extracted and have fluorescence emission spectra consistent with humic acids extracted from soils and Leonardite coal deposits.

Commercially available sugar cane mulch was extracted, filtered and purified as shown in the following photos:

Extracting dry, senescent sugar cane mulch with 0.1 M KOH	The extract was filtered, centrifuged and then acidified



The acidified humic acid was filtered and collected	The collected humic acid was dried
The humic acid was purified several times by redissolving in KOH, centrifuging, acidifying, filtering, drying, and saving in this vial. A single extraction gave 5.12 g pure humic acid from 310 g of sugar cane mulch, which was 1.65% of the senescent plant matter.

Note that not all of the humic acid can be extracted with alkali because much seems to bind to proteins or other organic matter. Therefore, an estimate of 3% humic acids in senescent plant matter is not unrealistic.

Fluorescence spectroscopy is a powerful tool to compare humic acids, and this technique was used to compare the purified humic acids extracted from sugar cane mulch with humic acids from other sources. The scans are reproduced here:

These series of fluorescence scans demonstrate the similarity of alkali soluble humic acid fractions derived from soil, Leonardite coal deposits and kelp.

From these scans a small difference between the alkali soluble humic acids (soil) and ethanol soluble humic acids (Leonardite/soil) can be observed. The alkali soluble sugar cane mulch humic acids fluorescence exactly matches the ethanol soluble (Leonardite/soil)  humic acids.

The fact that humic acids are generated in senescent plant matter is always before our very eyes, yet this was missed for about 200 years! Anywhere in nature where there is a black, brown or yellow color, often it is from humic acids. From the yellow fields of ripe wheat in Australia to the yellow fields of ripe corn in the USA to the dark Negro River flowing into the lighter Amazon river in Brazil, these colors are due to humic acids. The photos below show the ubiquitous presence of humic acids in the environment. The plant matter can be readily extracted with alkali and purified, and fluorescence spectra are similar or identical to those obtained from humic acids extracted from soils.

11 July 2010
In reply to Dr. Kim H. Tan.

Hello Kim

I am very happy to hear from you again and I hope that all is well with you. I would like to give you some background information regarding my humic acid research. I have not been involved in this now for about 10 years or so, but at present I have the opportunity to do a small amount of work. I am extracting quite large amounts of humic acids from dry plant material and hope to make this information known later this year or next year under the title: “The Critical Experiment That Was Never Done.” I want to elaborate on our previous work where we showed that humic acids are formed in senescent plant matter well before this gets into the soil or near microorganisms.

When I first began humic acid research in 1986 as an organic analytical chemist I was quite excited as it was part of a large project looking at fluorescent banding in corals which is now routinely used for rainfall reconstruction and climate work. However, my excitement soon turned to disappointment as I researched the literature. The limited literature that I consulted was very inconsistent and confusing, and then I came across the then recent 1985 paper by Farmer and Pisaniello, which made me realise that I would have to start from the beginning to make any headway. I had a great librarian (Ms Inara Bush) and I must give her much of the credit for going back in history and finding all sorts of obsure manuscripts and reports. One must remember that this was in the late 1980s and early 1990s when the Internet was only beginning and it was much more difficult than today to locate information. For example, she found the first report of humic acid extraction (by Achard) in the Natural History Museum, London, but they were reluctant to try to photocopy the article because of the age of the document. She somehow managed to convince them to do it! The University of Queensland and the State Library of Victoria also provided many articles, and my ability to read some German, even some of the old style writing, was extremely useful. Another set of articles about fumaric, maleic and 4-oxo-2-butenoic acids, which were key papers describing the conversion of furfural into these acids, were in Spanish. Since I can also read some Spanish (but no more languages) I was very lucky.

After receiving hundreds of manuscripts and reports dating back to 1786 I decided that I could achieve something useful by deciding for myself what was more correct and what was less correct. The papers that inspired me were the one by Shapiro from 1957 and the one mentioned above by Farmer and Pisaniello. Especially the paper by Shapiro made me realise that I should work with organic solvents, so my main tool became solution FTIR because by default almost nothing else worked. Of course, I now also had a treasure of knowledge about the history of humic acid research and felt compelled to share it so that past errors did not have to be repeated. However, another disappointment has been the reluctance of long-time workers in the humic acid field from accepting the discoveries that I made. I have contacted most of the office-bearers worldwide from the IHSS, but with little response. Some of this lack of acceptance is understandable since few soil scientists would understand the complex organic chemistry involved, but on the whole, we need a new generation of researchers to forget the old dogmas and take a new approach. I am happy that in your recent book you have taken a much more enlightened view of the humic acid saga, and I know first hand that it has enlightened researchers in the field.

Many regards

Michael