Chapter 09.07 Lauren Learns the Nitrogen Cycle

Introduction to Bacon & the Art of Living

The story of bacon is set in the late 1800s and early 1900s when most of the important developments in bacon took place. The plotline takes place in the 2000s with each character referring to a real person and actual events. The theme is a kind of “steampunk” where modern mannerisms, speech, clothes and practices are superimposed on a historical setting. Modern people interact with old historical figures with all the historical and cultural bias that goes with this.

Lauren Learns the Nitrogen Cycle

Copenhagen, August 1891

Dear Lauren,

A father’s relationship with his daughter is very special. It’s magical! This is your turn to get a letter, my precious La. I miss you guys! This week I learned an important lesson, that life is about much more than science, technology, and business.

Jacobus Arnoldus Combrinck, kindsman of the Graaf brothers and founder of the Cape Town butchery that became the Imperial Cold Storage & Supply Company Ltd.

Tribute to Jacobus Combrinck

I got a telegraph on Thursday, 6 August 1891 from David de Villiers Graaff. He told me the devastating news about the death of Uncle Cornelius Combrinck. (1) I was immensely saddened. He was a part of our lives for so long. I practically grew up in his home. He and your grandfather were friends since before I was born. I can almost not imagine going forward without him. The knowledge of his passing left a gap in my heart. When I read David’s message, I took a long walk and cried much.

In my mind, I see him with the two of you on his lap when you were still very small. When we visited at his Woodstock home (2) he would put you on his knee and you would “ride horsie”. I don’t know if you will remember this. You were so small!

You loved going there and he loved having us over. The large apricot trees in his back garden! You and Tristan enjoyed climbing them. He had the biggest garden and tended it with care. I will never forget the last time I saw him just before I left for Denmark. He spoke to me privately and urgently. He told me that he thinks I am finally making a good career choice. He did not like the fact that I rode transport to Johannesburg because he believed that the railroads would soon have put me out of business. The meat industry, he told him, is one of the iconic, almost eternal industries. People would always need food. He told me that the opportunity to become proficient in one aspect of it is something I can build a future on. Now he is gone. Life is short.

Uncle Cornelius never had his own children, but he invested liberally in the lives of others, particularly children. He spared no effort to mentor me, even in times when I made choices that he did not agree with. He took the Graaff brothers into his house and cared for them as if his own.

I understand that he was buried from the Groote Kerk, in Cape Town and laid to rest in the Maitland Cemetary. His life is an example to all of us, little La! He was your age when he started to work in the butchery of Johannes Mechau. His dad passed away and his mother was desperate for extra income. The fact that as a 10-year-old boy he had to earn his living could have been a sign that he was destined for a life of mediocrity and poverty. The opposite was true by his own resolve and willpower! Mechau found that he learned the trade quickly.

He was ambitious and left Mechau’s employment to join the leading pork butcher in town, the Swiss Ithmar Schietlin. When Schietlin returned to Switzerland, Combrinck went into business for himself.

He was very successful. He speculated in the diamond industry in Kimberly. He owned houses in Sea Point, Three Anchor Bay, and Wynberg. He had sheep farms that supplied his own and other butcheries throughout the Colony.

Uncle Jakobus knew the value of a young apprentice from his own experience. He thought it best to select such an apprentice from his own people and in 1870 he visited the farm Wolfhuiskloof in the lovely Franschhoek mountains. Like his own family situation, years earlier, the Graaff family fell on hard times and found it difficult to feed their children. One of the children of Petrus and Anna Graaff impressed Jacobus. The child was lively and intelligent and he suggested that David return to Cape Town with him where he would be taught the butchery trade. The suggestion pleased everybody. This is how it came about that David joined the butchery, Combrinck & Co. (Simons, 2000)

I am sorry that I missed his funeral but I managed to send a telex to the Graaff brothers. It is a comfort to know that you, Tristan, and my parents attended. I wonder how Cecil Rhodes took the news of his passing? (3) (Simons, 2000)

The Best I Can Be

Lauren, I am here to learn the butcher’s trade and the art of curing bacon. One of the best responses possible to honour the memory of Uncle Jacobus is to become the best I can be at these.

As a child on Stillehoogte, I learned that saltpetre is the magical salt that cures meat. A friend of Uncle Jeppe, Dr Eduard Polenski, discovered that nitrites form in bacon brine and suspects that it is the actual compound that changes pork into bacon and not saltpetre (potassium or sodium nitrate). At the factory, I would walk behind Unkle Jeppe on the way to the curing room and he would ask me, “Eben, what changes pork into bacon?” My answer always had to be, “Nitrite!” (4) He would follow this up by asking, “Where does nitrite come from?” upon which I reply, “From the saltpetre, when bacteria change the nitrate into nitrite when it removes the one oxygen atom from the saltpetre molecule.”

To fully comprehend the different nitrogen compounds that play a role in meat curing, there is another compound you must know besides nitrite (NO₂⁻) and nitrate (NO₃-), namely ammonia. In my last letter to you and Tristan, I already introduced you very briefly to it when I told you about ammonium chloride which was another great salt from antiquity that cured meat.

The three cousins of the chemical gas, nitrogen are ammonia, nitrite, and nitrates. These three cousins are key to all life and exist almost everywhere. It occurs naturally in sea salt, in the ground, in salt beds. They are pervasive. Without them, we won’t be able to shoot a gun, fertilize our fields or cure bacon. Some people refer to it as the nitrogen cycle – the fact that nitrogen exists in the atmosphere as a stable gas, that the tight bonds are broken through the action of lightning which then frees the two nitrogen atoms so that one can react with oxygen to form nitric oxide (NO). As it cools down, it reacts further with the oxygen molecules around it to form nitrogen dioxide (NO₂) which is one nitrogen atom and two oxygen atoms. Nitrogen Dioxide (NO₂) reacts with more oxygen and raindrops. Water is H₂O. The two oxygen atoms of nitrogen dioxide combine with the one from water to form 3 oxygen atoms bound together. There is still only one nitrogen atom giving us NO₃– or nitrate. There is now one Hydrogen atom left and it combines with the nitrate to form nitric acid (HNO₃). Nitric acid falls to earth and enters the soil and serves as nutrients for plants.

There is now an interaction where oxygen is added to nitrogen-containing compounds (oxidation) and removed (reduction). Bacteria change decomposing animal and plant matter from ammonia into nitrite and nitrates and eventually back into nitrogen gas which is released into the atmosphere. Certain bacteria change atmospheric nitrogen directly into a form that can be digested by plants. Uncle Jeppe organized a visit for Minette and me to the University of Copenhagen where a professor in biology and chemistry took an entire morning to describe to Minette and I the most recent discoveries in this field.

I wrote to Tristan about nitrate. I told him about saltpetre and nitrite, when I reported on the work of Dr Eduard Polenski and his insight and experiment showing that in bacon cures, nitrate is converted to nitrite. It has recently been shown that there is a conversion of each of these compounds into the other through the action of small organisms, called bacteria in soil and water. It was these discoveries that gave Dr Polenski the insight that it may be bacteria in brine, changing the nitrate ( NO₃-) to nitrite (NO₂⁻). Our visit to the University was breathtaking. I was glad that Minette accompanied me. I needed someone there to help me take notes and to remember every bit of insight shared by the Professors. It is thrilling to share my journey of discovery with all of you!

Discovery of the Microscopic

One of the pillars of understanding nitrogen is its chemical make-up. Another is to understand bacteria and their role in these processes. Some of the reactions in meat curing are driven by chemistry and some by bacteria. Like many of our greatest discoveries, the ancients had a very good idea that the microscopic world must exist.

Bacteria and micro-organisms were discovered between 1665 and roughly 1678. Two of the men responsible for their discovery were Robert Hooke and Antoni van Leeuwenhoek. (Gest, H. 2004) As one can imagine, microorganisms were discovered when the instruments were invented to see them. The first illustrated book on microscopy was Micrographia, published by Robert Hooke in 1665. (Gest, H. 2004)

On 23 April 1663, Hooke reported on two microscopic observations to the Royal Society, one of leaches in vinegar and another of mould on sheepskin. So opened up to humankind the magical world of the minute! The microscopic!

It was the astonishing Antoni van Leeuwenhoek from Holland who introduced us to many micro realities of our world. Here is an interesting list of some of the discoveries of this remarkable man: In 1674, in a single vial of pond scum that he took from the Berkelse Mere, a small lake near Delft, he discovered and described the beautiful alga Spirogyra, and various ciliated and flagellated protozoa. He found in 1674 that yeast consists of individual plant-like organisms. In 1675 he discovered and accurately described and differentiated red blood cells in humans, swine, fish, and birds. In 1677 he was the first to observe sperm cells in humans, dogs, swine, molluscs, amphibians, fish and birds. In 1679 and 1684 he described the needle-shaped microscopic crystals of sodium urate that form in the tissues of gout patients in stone-like deposits called “tophi”. In 1684, he correctly guessed that much of the pain of gout is caused by these sharp crystals poking into adjacent tissues. More than a century would pass before any further advance in the understanding of gout. He found and described in 1680 foraminifera (single-celled protists with shells) in the white cliffs of England’s Gravesend and nematodes in pond water.

Between 1680 and 1701 he carried out many microdissections, mainly on insects, making an enormous number of discoveries: He wrote extensive accounts of the mouthparts and stings of bees. He was the first to realize that “fleas have fleas”. His keen perception enabled him to correctly conclude that each of the hundreds of facets of a fly’s compound eye is, in fact, a separate eye with its own lens. This outlandish (but true) idea was met with derision by visiting scholars. The big breakthrough came in 1683. In his most celebrated attainment, he discovered the bacteria in dental tartar, including a motile bacillus, selenomonads, and amicrococcus.

16 October 1674, Antoni wrote a letter describing his study of the tongue of an ox and his observations of the taste buds. On 24 April 1676 Antoni studied pepper water that has been sitting for three weeks under his microscope. He observed small organisms that he called “little eels” (animalcules). What he was looking at were bacteria. He has discovered a world that we knew very little about!

Antoni was responsible, not just for discovering bacteria, but for discovering important classes of bacteria. He was among others responsible for identifying anaerobic bacteria. (5) (6) In a letter dated 14 June 1680 to the Royal Society, he described his discovery. This would become very important in considering the action of bacteria in meat systems since the environment is often devoid of oxygen.

The important point about bacteria that I want you to focus on is that it plays a pivotal role in the nitrogen cycle as described by Louis Pasteur. It continues the very same interaction with family members of nitrogen in the curing of meat. (Dikeman, M, Devine, C: 436) (6) (7)

Scientists in the late 1800s started to hone in on the particular bacteria responsible for converting nitrate to nitrite. This is becoming very important to us because generally, nitrate exists because of the action of bacteria, but particularly, as Dr Eduard Polenski speculated in 1891, it is the action of bacteria that turns nitrate from saltpetre into nitrite in curing brines and meat that is being cured. The question we have been asking is if this was a fair assumption for him to make and the answer is an overwhelming “yes!”

Since 1868 it has been known that bacteria in soil are responsible for the exact same reduction. It was known for 23 years before Dr Polenski’s 1891 experiments on curing brine and the meat being cured. The reduction of nitrate in soil to nitrite or ammonia was brought about by various forms of microorganisms. The person who demonstrated this in 1868 was the German scientist C F Schonbein. Our French friends, Gayon and Dupetit, confirmed this. (Waksman, SA, 1927 : 181)

Adding carbohydrates, glycerol, and organic acids, in addition to peptone (a soluble protein formed in the early stage of protein breakdown during digestion) to meat through its brine stimulate the reduction of nitrate to nitrite. It was also discovered that an abundance of oxygen hindered it. (Waksman, SA, 1927 : 181) This will prove to be of the greatest importance to meat curing and since we can achieve a brighter colour by adding organic acids, glycerol, carbohydrates and reducing sugars to the brine mix.

One researcher, Maassen, tested 109 different bacteria and found that 85 were capable of reducing nitrate to nitrite, especially Bact. Pyocyaneum. Similar results were found by others who studied this. Not only did they find that many of the bacteria responsible for the reduction were anaerobic (functioning in the absence of oxygen) but that many strict aerobic bacteria were found to act anaerobically in the presence of nitrates. (Waksman, SA, 1927 : 181) This was true of soil and certainly, it should be true in meat and brine systems also!

Ammonium Chloride (Sal Ammoniac)

We have seen that nitrite is formed by removing an oxygen atom from nitrogen. There is another very important way that nitrate is formed namely when ammonia breaks down. The Russian microbiologist Sergei Winogradsky discovered this. Microorganisms, through a process called biological oxidation, change ammonia to nitrite and nitrite to nitrate. Have a look at how oxygen is added at every step. Ammonia is NH₃ and there is no oxygen. Nitrite is formed NO⁻₂which is the nitrogen and two oxygen atoms. From nitrite, through bacterial action, nitrate is formed NO⁻₃. So, from a form with no oxygen, the most oxygenated state is reached namely nitrate with its three oxygen atoms.

We have to understand a bit more about ammonia to see how this works. This will be very important when we look at the decomposition of animal tissue and in animal urine and excrement since it contains copious amounts of ammonia. The building blocks of ammonia is seen in its chemical formulation. Ammonia is a compound of nitrogen and hydrogen with the formula NH_3.

In nature, ammonia exists as NH₃ or its ammonium ion (NH4+). The ammonium ion, in nature, also combines with a metal such as chlorine to form a salt of ammonium. Ammonium is therefore not only important in the nitrogen cycle, but also in meat curing in the form of a salt where a metal such as chloride combined with the ammonium ion to form ammonium chloride (NH₄Cl). It is the NH4 that makes it mildly acidic and the new molecule of sal ammoniac or ammonium chloride is highly reactive with water. Ammonium chloride occurs naturally as a crystal and is formed through the action of bacteria on decomposing organic material. As a salt, it is one of the iconic salts of antiquity.

Natural Sal Ammoniac

Ammonium chloride occurs naturally in the smoking mountains of Turfan and in Samarkand where volcanic fumes are released through vents. The crystals form directly from the gaseous state, skipping the liquid state. The crystal that is formed tends to be short-lived, as they dissolve easily in water. This is the basis for my guess that in Turfan, where ammonium chloride occurs in the mountains and nitrate in the depression these salts were often confused and initially when sal ammonia was the more popular site, nitrate was sold as an imitation of ammonium chloride. Once the crystalline form of ammonium chloride comes into contact with moisture it breaks down to a brownish salt which looks similar to the nitrate salts found on the top layer of soil in the depression between the mountains. I suspect that these nitrate salts were sold as “fake” ammonium chloride because it has overlapping characteristics because of the nitrogen.

Natural Sal Ammoniac occurs in places like the Turpan and Samarkand. An important branch of the silk road runs from Turfan through Samarkand and into Europe. Samarkand is a city in south-eastern Uzbekistan. It is one of the oldest continuously inhabited cities in Central Asia.

In China, ancient names given for Sal Ammoniac are “red gravel” and “essence of the white sea.” There were sal ammoniac mines in Soghd. Mohammadan traders passed it at Khorasan travelling towards China. Kuča still yielded sal ammoniac at the beginning of the 1900s. There are ancient references to white and red varieties of sal ammoniac. The mines in Setrušteh or سمرقند‎ (Samarkand in the Persian language) are described in classic literature as follows. “The mines of sal ammoniac are in the mountains, where there is a certain cavern, from which a vapour issues, appearing by day like smoke, and by night like fire. Over the spot whence the vapour issues, they have erected a house the doors and windows of which are plastered over by clay that none of the vapour can escape. On the upper part of this house the copperas rest. When the doors are to be opened, a swiftly-running man is chosen, who, having his body covered over with clay, opens the door; takes as much as he can from the copperas and runs off; if he should delay he should be burnt. This vapour comes forth in different places, from time to time; when it ceases to issue from one place, they dig in another until it appears, and then they erect that kind of house over it; if they did not erect this house, the vapour would burn, or evaporate away.” (Laufer,1919)

Tibetans received this salt from India as can be seen from an ancient name they gave to it namely “Indian salt.” There are records that it was harvested from certain volcanic springs from Tibet and Se-č’wan. (Laufer,1919) The same vapours are seen in the smokey mountains of Turfan.

Human-Made Ammonium Chloride

Just like saltpetre, sal ammoniac occurs naturally and is also generated through human endeavour. The name, ammonia, came from the ancient Egyptian god, Amun. The Greek form of Amun is Ammon. At the temple dedicated to Ammon and Zeus near the Siva Oasis in Lybia, priests and travellers would burn soil rich in ammonium chloride. The ammonium chloride is formed from the soil, being drenched with nitrogen waste from animal dung and urine. The ammonia salts were called sal ammoniac or “salt of ammonia” by the Romans because the salt deposits were found in the area. During the middle ages, ammonia was made through human endeavour through the distilling of animal dung, hooves, and horns. (Myers, RL. 2007: 27)

The New York Tribune of 31 January 1874 wrote the following. “For centuries sal ammoniac was imported from Egypt where it is sublimed from camels dung.” An article, published in 1786 on Friday, 18 August in the Pennsylvania Packet, described the process of making sal ammoniac in Egypt as follows. “Sal Ammoniac is made from soot arising from the burnet dung of four-footed animals that feed only on vegetables. But the dung of these animals is fit to burn for sal ammoniac only during the four firsts months of the year when they feed on fresh spring grass, which, in Egypt is a kind of trefoil or clover; for when they feed only on dry meat, it will not do. The dung of oxen, buffalos, sheep, goats, horses, and asses, are at the proper time as fit as the dung of camels for this purpose; it is said that even human dung is equal to any other.”

“The soot arising from the burnt dung is put into glass vessels, and these vessels into an oven or kiln which is heated by degrees and at last urged with a very strong fire for three successive nights and days, the smoke first shows itself, and, in a short time after, the salt appears sticking to the glasses, and, by degrees, covers the whole opening. The glasses are then broken, and the salt taken out in the same state and form in which it is sent to Europe.” At this time, Egypt was one of the major suppliers of sal ammoniac to the European continent.

Discovery of gasses

– Joseph Black

At this point in the development of chemical technology, a much bigger development took place in which the discovery of nitrogen and ammonia is only a small part of. In the 1770s scientists started to realise that the atmosphere is made up of various gasses. This was the start of the chemical revolution and the discovery of gasses was, in a way, the major propellant. Up to this time gasses were not regarded as a separate chemical entity and largely ignored in experimental work. The drawback was major and real advances became only possible as this was being resolved. One of its pioneers was Joseph Black (1728–1799). Black is credited with the discovery of carbon dioxide (fixed air).

– Charl Wilhelm Scheele

The Swedish Chemist, Charl Wilhelm Scheele (1742 – 1786) prepared oxygen by heating saltpetre (potassium nitrate, KNO₃) in 1770. Somewhere between 1771 and 1772, he became the first scientist to realise that “air consists of two fluids different from each other, the one that does not manifest in the least the property of attracting phlogiston while the other … is peculiarly disposed to such attraction.” (Smil, 2001: 2) Phlogiston was believed to be the substance present in all material that burns, responsible for combustion. The one substance is obviously oxygen and the other nitrogen.

– Daniel Rutherford

At the same time, Daniel Rutherford (1749–1819), a pupil of Black, obtained his doctorate in Medicine in 1772 from the University of Edinburgh. In his “Dissertatio inauguralis de Aere Fixo Dicto, aut Mephitico” (Rutherford, 1772) he records the following experiment. He placed mice in a closed-in environment. Eventually, the mice will die and Rutherford expected to find was that the only air that is left will not be able to support life and a flame will not burn in it. He removed the fixed or mephitic air (carbon dioxide) with a caustic potash solution (alkali). He found a residual gas still incapable of supporting respiration or fire, similar to carbon dioxide, but unlike carbon dioxide, did not precipitate lime water and was not absorbed by the alkali. He thus discovered a residue of his fixed or mephitic air. He named it “aer malignus” or noxious air.” (Munro and Allison, 1964)

– Joseph Priestley

Priestly, who is credited for the discovery of oxygen (1774 – 1775) presented experimental evidence similar to Rutherford’s before the Royal Society of London. He, however, did not draw conclusions regarding the possible nature of the gas (Priestley, 1772).

– Isolation of Ammonia

The identification of nitrogen was “in the air”, so to speak and as we will see, never far removed from meat curing. Sal Ammoniac (ammonium chloride, NH4Cl) was used since antiquity as curing and preserving agent of meat and was investigated by none other than Joseph Black. In 1756 he became the first to isolate gaseous ammonia by reacting sal ammoniac with calcined magnesia (Magnesium Oxide). (Black, 1893) (Maurice P. Crosland, 2004). Scientists were now widely experimenting with gasses and along with air, gasses like ammonia received a great deal of attention. It would later be discovered that nitrogen is its key constituent in ammonia along with hydrogen.

Following Black, ammonia was, for example, also isolated again by Peter Woulfe in 1767 (Woulfe), by Carl Wilhelm Scheele in 1770 (kb.osu.edu) and by Joseph Priestley in 1773 and was termed by him “alkaline air”. Eleven years later in 1785, Claude Louis Berthollet finally unravelled its composition. (Chisholm, 1911) (Berthollet, 1785)

Priestley, in Part II of his work, Experiments and Observations, described work from between the years 1773 and the beginning of 1774. In this document, he gives a reprint of an earlier publication on effluvia from putrid marshes. Here he identifies ammonia and nitrous oxide. (Schofield, RE. 2004: 98)

His discoveries on ammonia were the result of a consistent application of the English scientist, Stephen Hales’s (1677 – 1761) technique for distilling and fermenting every substance he could get his hands on or capture over mercury rather than over customary water so that the air would “release.” He heated ammonia water and collected a vapour. When it cooled down, it did not condense, proving it was air. He called it alkaline air. (Schofield, RE. 2004: 98, 103, 104)

More experiments showed him that alkaline air was heavier than common inflammable air but lighter than acid air. It dissolved easily in water, producing heat and it was slightly inflammable in the sense that a candle burned in it with an enlarged colour flame before going out. In the end, he not only described ammonia chemically, but also its mode of production, and its characteristics. (Schofield, RE. 2004: 98, 103, 104)

– From Ammonia to Nitrogen

In 1781 the French Chemist, Claude Louis Bertholett became aware that something joined with hydrogen to form ammonia (NH₃₎. Three years later, Claude joined Lavoisier who was responsible for unravelling the composition of saltpetre along with de Morveau and de Fourcroy, in naming the substance azote. (Smil, V. 2001: 61, 62) Lavoisier named it from ancient Greek, ἀ- (without) and zoe (life). He saw it as part of air that can not sustain life. In 1790 Jean Antoine Claude Chaptal, in a French text on chemistry which was translated into English in 1791, gave it the name “nitrogen”. He used the name ‘nitrogène’ and the idea behind the name was “the characteristic and exclusive property of this gas, which forms the radical of the nitric acid,” and thus be chemically more specific than “azote.” (Munro and Allison, 1964) As for ammonia, its modern name was given in 1782 by the Swedish chemist Torbern Bergman. (Myers, RL. 2007: 27) The discovery of hydrogen, the other component in ammonia, is credited to Cavendish in 1766.

A Hint of Nitrogen in Animals

The relation between nitrogen through ammonia and animal bodies was known from early on. In 1785, Claude Berthollet reported to the French Academy of Sciences that he found that the vapour that came from decomposing animal matter was ammonia. When he realised the gas, he found that it was composed of three volumes of hydrogen and one volume of nitrogen, or around 17% hydrogen and 83% nitrogen by weight. He was very accurate in his measurements and the modern values of these are given as 17.75% and 82.25% respectively. (Carpenter, 2003)

Techniques for Testing for Nitrogen

The key to the identification of nitrogen in animal substances was developing the tools to test for it. One of the earliest tests was the oxidation of organic material in the presence of cupric oxide. The gasses resulting from this reaction is then collected and measured. It was extensively developed by none other than Gay-Lussac while he was a professor at the Sorbonne, and later when he was a chemist at the Jardin des Plantes in Paris. (Sahyun, M. (Editor). 1948)

The method of Gay-Lussac was modified by Jean Dumas (1800-1884) and used by Dumas’ contemporary, Liebig. Despite the many alterations of the basic method of micro procedures, the Dumas method would continue to be the preferred one well into the 1900s. In 1841, F. Varrentrapp and H. Will developed a total nitrogen method. This method is based on the liberation of ammonia by heating protein with alkali, followed by gravimetric estimation of the ammonia as its chloroplatinate. (Sahyun, M. (Editor). 1948)

A downside to this method was the fact that it is slow and tedious with fundamental inaccuracies. It had, however, specific technical advantages over that of the Dumas method when applied to metabolic observations and it was used in many early studies. The famous method we are all familiar with today is the Kjeldahl method. It was developed by the Danish chemist, J. Kjeldahl (1849-1900), of Carlsberg, who in 1883 presented a much-improved method for catalyzed digestion of nitrogenous materials in sulfuric acid which allowed for the production of ammonia quantitatively. (Sahyun, M. (Editor). 1948)

Nitrogen in Respiration

Antoine Lavoisier was inspired by Joseph Black, something that Lavoisier was not shy to admit. He wrote Balck a letter, dated 19 November 1790, where he describes experiments on the respiration of human subjects. He showed that oxygen is consumed and carbon dioxide evolved during this process. Interestingly he showed that oxygen consumption increases by some 50% above the basal level after a meal (the modern specific dynamic action of food) and that in severe exercise, oxygen consumption can increase by as much as three-and-a-half times. The measurements were accurate, even by modern standards. Part of the letter states: “Legaz azote ne sert absolument à rien dans l’acte de la res piration et il ressort du poumon en même quantité et qualité qu’il y est entré” which translates to Nitrogen is absolutely useless in the act of respiration, and it appears from the lung in the same quantity and quality that it has entered it.

They had their test subjects exercise in a closed container. They measured for oxygen and carbon dioxide. They also measured the amount of nitrogen ingested during a meal before the experiments started and then, after exercise, the urine and stools were tested to see how much nitrogen was retained in the body or “lost” through the urine and stools.

The experiment was undertaken 18 years after the discovery of nitrogen. It is regarded by many as the first metabolic experiment with nitrogen. The experiments appear (D. McKie, personal communication, 1962) to have been based on studies made by Fourcroy in the late 1780s, using gasometric methods that were published in 1791 by Séguin. They did not find any correlation between nitrogen and respiration. Some researchers of the time still claimed that some nitrogen is lost from the body during respiration. Today, most will simply subscribe to Lavoisier’s view that gaseous nitrogen plays no part in the nitrogen metabolism of the mammalian organism. (Munro and Allison, 1964) They believed that the balance of nitrogen ingested and that which was not recovered in stools or urine was probably lost through what they called “insensible perspiration.” (Carpenter, 2003)

Antoine Lavoisier and Armand Seguin’s experiment of human respiration showed that breathing had no influence on nitrogen levels. It had other positive results. An increase in the output of carbon dioxide (carbonic acid, as they called it) during exercise was demonstrated. They measured this at rest and while lifting weights. This was by itself a step forward. At the time it was believed that the only purpose of respiration was to cool the heart. (Carpenter, 2003)

Lavoisier, in collaboration with a mathematician and one of the greatest scientists of the time, Pierre-Simon Laplace, identified the slow combustion of organic compounds in animal tissue as the major source of body heat. In their experiments, they compared the heat produced by the guinea pig and the production of carbon dioxide with the heat produced by a lighted candle or charcoal. They used an ice calorimeter to measure heat production. The instrument itself is very interesting. It measures the heat generated by relating it to the weight of water released from the melting of the ice surrounding the inner chamber where the animal or burning material is housed. The measurements are crude and not very precise, but results were consistent and it allowed the researchers to draw the conclusion of the origin of body heat. (Carpenter, 2003)

Momentous political movements in France of the time would put an end to one of the most brilliant scientific careers of any person to have lived on earth. Lavoisier returned to further studies on respiration was arrested in 1793 during the Reign of Terror and kept in prison. He pleaded with the judge for a short stay of execution on the day of his trial in 1794, to be allowed one more experiment, but the judge is believed to have replied that the Republic had no need of “savants” (scientists), and he was guillotined the same afternoon. (Carpenter, 2003)

Nitrogen in Animal Matter

Lavoisier introduced order into the study of the new chemistry. One of his great achievements was the vigorous school of chemists he left behind. Some of his students took up the work on organic compounds and applied procedures in which gas was either evolved or removed. Gay-Lussac (a pupil of Lavoisier’s collaborator, Berthollet) and Thénard worked out a system of organic analysis in 1810. Accordingly, the organic material is treated with potassium chlorate and the amount of oxygen and nitrogen liberated is measured (Partington, 1951). The Dumas procedure, which we eluded to above, remained the standard gasometric method of nitrogen analysis. It was developed in 1830. (Partington, 1951). The studies made by Magendie on the importance of nitrogenous components in the diet was one of the matters to be elucidated by the new technique. (Munro and Allison, 1964) Viewed in this way, the persona and influence of Lavoisier continued to directly affect the work he started long after his untimely death.

It was confirmed that animal matter contains nitrogen and it was shown to be absent from sugars, starch, and fats. It was long suspected that wheat flour contained matter with characteristics closely associated with animal matter. This was proved, that gluten (the plant matter) has properties of animal matter, including the development of alkaline vapour when it was allowed to rot. When potatoes were introduced, there was a debate if it could provide an adequate substitution for wheat because it did not have anything resembling gluten. Was it the gluten that made wheat flour good food? (Munro and Allison, 1964)

Bartolomeo Baccari (1682 – 1766) was a professor at the University of Bologna for most of his life. In 1734, one of his papers entitled “de Frumento,” appeared. In this paper, he gives details on how to prepare gluten which was found to be the protein portion of wheat flour. The following is translated from Latin:

“This is a thing of little labour. Flour is taken of the best wheat, ground moderately lest the bran goes through the sieve, for it ought to be purified as far as possible in order that all suspicion of mixture should be removed. Then it is mixed with the purest water and agitated. What remains after this process is set free by washing, for water carries off with itself whatever it is able to dissolve. The rest remains untouched.”

“Afterward that which the water leaves is taken in the hands and pressed together and is gradually converted into a soft mass and beyond what I could have believed tenacious, a remarkable kind of glue and suitable for many purposes, among which it is worth mentioning that it can no longer be mixed with water. Those other parts which the water carries away with itself for some time float and render the water milky. Afterwards, they gradually settle to the bottom but do not adhere together; but like a powder return upward at the slightest agitation. Nothing is more nearly related to this than starch or better, it is indeed starch.”

He classified the starchy material as flour. He described the following characteristics. It ferments to give acid spirits, indicating its “vegetable nature.” On the other hand, it had a characteristic of “animal nature” for “within a few days it gets sour and rots and very stinkingly putrifies like a dead body.”

This was an old way of distinguishing what we call today proteins from carbohydrates. There was a theory at this time that vegetable protein which is consumed by herbivores changes into the flesh and blood of the animal. This was still prevalent during the time of Mulder and Liebig’s. (Sahyun, M. (Editor). 1948) Another question was the source of nitrogen in animal bodies. Since nitrogen is most prevalent in the air around us, some chemists suggested that animals get the nitrogen from the air through a kind of combination that must occur during an animal’s digestion of plant foods “so as to give the ingesta the characteristics that would allow them to be incorporated into the animal’s own tissues either for growth or replacement of worn-out materials.” (Carpenter, 2003) The mechanisms of nutrition were in a developmental process.

François Magendie: Nitrogen as the basis for Nutrition

A major step came from the work of Magendie (1783–1855) who linked the nitrogen of inanimate substances with that of living systems. He was the first to recognise that there is a major difference between the nutritional value of food containing nitrogen and those without it. Magendie grew up in revolutionary Paris and practised as a surgeon before changing to physiology.

In his first work on the subject, reported to the Academy of Sciences in 1816, Magendie addressed the question of whether animals could access atmospheric nitrogen to “animalize” ingested foods of low nitrogen content. (Carpenter, 2003) In his 1816 article, “Sur les propriétés nutritives des substances qui ne contiennent pas d’azote.” (On the nutritional properties of substances that do not contain nitrogen), Magendie famously described experiments on dogs that were only fed carbohydrate (sugar) or fat (olive oil) until they all died in a few week’s time. The conclusion is obvious that a nitrogen source was an essential component of the diet.

As we look back at these early experiments we can see that the results were complicated by vitamin deficiencies, yet they were the first approximations to an ideal—the long-term feeding experiment with purified foodstuffs—which has only been attained in recent years. They can rightfully be seen as forerunners of the classical procedure for establishing whether a nutrient is essential to the body, namely by excluding it from the diet and then looking for symptoms attributable to its deficiency.

In his “Elementary Compendium of Physiology for the Use of Students,” Magendie draws an even clearer distinction between nitrogenous and nonnitrogenous foods. The first edition appeared in 1817 and the third edition was translated into English in 1829. Magendie’s compendium of work is very different from earlier writers like Haller’s “Elementa Physiologiae,” (1757–65). Magendie did not write in Latin and he clearly departed from the primaeval forests of mystery and speculation. His work is done with the illumination of bright sunshine of scientific observation and deductive reasoning.

Again, we have to give credit to the monumental work of Lavoisier. Magendie’s success in the physiology of nutrition directly stems from the influence of Lavoisier’s vigorous school of chemistry, which had grown up in the interval. Megandie followed his 1816 work where he fed dogs only carbohydrates or fat with new experiments. In these, he fed them exclusively on cheese or eggs, both nitrogenous foods. The dogs survived indefinitely, although they were weak. Magendie concluded that “these facts . . . make it very probable that the azote of the organs is produced by the food.”

Magendie’s inquiring mind also extended to views on how the diet was utilized by the tissues of the body. In his textbook (p. 18), he says: … The life of man and that of other organised bodies are founded upon this, that they habitually assimilate to themselves a certain quantity of matter, which we name aliment. The privation of that matter, during even a very limited period, brings with it necessarily the cessation of life. On the other side, daily observation teaches, that the organs of man, as well as those of all living beings, lose, at each instant, a certain quantity of that matter which composes them; nay, it is on the necessity of repairing these habitual losses that the want of aliment is founded. From these two data, and from others which we shall make known afterwards, we justly conclude, that living bodies are by no means always composed of the same matter at every period of their existence. . . . It is extremely probable that all parts of the body of man experience an intestine movement, which has the double effect of expelling the molecules that can or ought no longer to compose the organs, and replacing them by new molecules. This internal, intimate motion, constitutes nutrition. And again (p. 468), … Nutrition is more or less rapid according to the tissues. The glands, the muscles, skin, etc. change their volume, colour, consistency, with great quickness; the tendons, fibrous membranes, the bones, the cartilages, appear to have a much slower nutrition, for their physical properties change but slowly by the effect of age and disease.” (Munro and Allison, 1964) (14)

When one looks back at history, one tries to bridge the linguistic and cultural divide. An important assumption underpinning Magendie’s work is that an animal species could be used as a model for humans; that our bodies are essential of the same general character. A possible reason for this is the interest that existed in France for studies in comparative anatomy. (Carpenter, 2003)

Jean Baptiste Boussingault

Another active investigator in France in the 1830s, with a quite different background from that of Magendie, was also studying the source of an animal’s nitrogen-rich tissues. This was Jean Baptiste Boussingault, the great “farmer of Bechelbrom,” who had learned his chemistry in a school for mining engineers. After a period of adventurous geological exploration in South America, he returned, married a farm owner’s daughter and put his mind to agricultural science. He obtained a position at the Sorbonne in Paris, where he collaborated with J. B. Dumas, one of the leading French chemists, and divided his year between Paris and the farm. (Carpenter, 2003)

It was Boussingault who realised in 1836, over sixty centuries after it was noted and recorded that manure and legumes were beneficial to crop production, that it was the nitrogen content in the soil or fertiliser which is important for plant nutrition. In 1838, he performed a number of experiments where he grew legumes in sand with no nitrogen in it. The legumes continued to grow and the only conclusion he could come to was that they took their nitrogen from the air. How they did it, he still had no idea. (Galloway, J. N, et al., 2013) He was able to show that this was not possible for cereal grain.

His next subjects were cows and horses, whose common feeds were believed to be exceptionally low in nitrogen. First, he wanted to determine the level of feeding that would ensure that his animals are kept at a constant weight, and then for 3 days, he recorded the animal’s feed, what was excreted and, in the case of the cow, its milk. All these were analysed for their nitrogen content. The results for the horse was that he received 8.5kg hay and oats, every 24 hours. The daily nitrogen intake was 139g, and the nitrogen recovered in urine and dung came to only 116g. When the cow was fed on hay and potatoes the figures were as follows. The daily intake of nitrogen was 201g and the recovered output, including 46g from milk, was only 175g. This showed that the animals’ feed provided enough nitrogen to meet their needs. There was no need to speculate about them getting their nitrogen from the atmosphere.

It is important to have some understanding of how these trials were carried out. Many thousands of “balance” trials followed the Boussingault tests that continue to be carried out until today. A drawback was the method he used to test for nitrogen. The system of analysis required the sample to first be dried. There would have been a loss of ammonia when he was drying urine and dung. This probably gives the reason why there seems to have been an apparent “positive” balance in these animals that were assumed to be in a steady state.” (Carpenter, 2003)

Nitrogen and the Nutritional Value of Plants

Boussingault had proposed that the nutritional values of plant food could be extrapolated from their contents of nitrogen. These speculations came from before he did his balance experiments with herbivores. His reasoning was more or less as follows. “Magendie has shown that foods that do not contain nitrogen cannot continue to support life, therefore the nutritional value of a vegetable substance resides principally in the gluten and vegetable albumin that it contains.” Researchers of the time knew that animal bodies contained minerals which they got from the food they ate. Even earlier, two workers had written that: “Beans are so nourishing because they contain starch, an animal matter, phosphate, lime, magnesia, potash, and iron. They yield at once the aliments and the materials proper to form and colour the blood and to nourish the bones”. Perhaps in response to such criticism, Boussingault explained, “I am far from regarding nitrogenous materials alone as sufficient for the nutrition of animals; but it is a fact that where nitrogenous materials are present at high levels in vegetables they are generally accompanied by the other organic and inorganic substances which are also needed for nutrition”. It is clear from the context that the “organic substances” to which he is referring are starches and not any hypothetical trace nutrients. (Carpenter, 2003)

Synthesis by plants

Dumas, a colleague of Boussingault’s concluded in the early 1800s that the plant kingdom alone was capable of synthesizing the kinds of nitrogenous compounds abundant in animal tissues. Then, from the observation that the overall reactions of animals were characterized by oxidation, he made the further generalization that the animal kingdom was only capable of oxidizing the materials that are obtained from its plant food. (Carpenter, 2003)

Ammonia, Nitrite, and Nitrate

Ammonia is changed into nitrites or nitrated through the action of what was called a “microscopic ferment.” The next step would be the discovery of how nitrogen changes into its cousins and enters the earth and living plants and animals.

NollLab_lg — A science class uses microscopes in a lab in 1908. (University Archives Photo)

The afternoons with Jeppe became challenging as I tried to keep up with his lectures. He seemed to remember the names and formulations off by heart and I was not always sure who or what we were talking about. It was nevertheless engaging and I tried to keep up.

– How does nitrogen enter the plant kingdom?

The animal kingdom gets its nitrogen from the plant kingdom. We now return to the matter of how nitrogen enters the plant world. When we looked at the discovery of the microscopic world, we jumped to the discovery of nitrification and the reduction of oxygen in various nitrogen compounds. With the background information on nitrogen and its role in nutrition, let’s look at the progression of thought on ways that nitrogen enters our world.

HB de Saussure (1740 – 1799) discovered that the nitrogen in plants does not come directly from the atmosphere. (Bynum, WF, et al, 1981: 300) He was born in Switzerland and became interested in biology and geography. Most of the discoveries he made while scaling some of the highest mountain peaks and passes in the world. He regarded the Alps as central to understand the geology of the world and spend much time there.

His idea was that nitrogen must be taken up through the roots of plants, through the decomposition of humus (9, 11). (Bynum, WF, et al, 1981: 300) Not everybody agreed with him and a debate developed that raged for almost 50 years. The German chemist, Justice von Liebig (1803 – 1873), was the first to see nitrogen as an essential plant nutrient. This discovery gave him the honour of being regarded as the father of the fertilizer industry. Justice was also an important man in the meat processing industry. He developed the manufacturing process for beef extract and founded a company, Liebig Extract of Meat Company, and later trademarked the Oxo brand beef bouillon cube. (10)

This question of how nitrogen was absorbed by plants remained very controversial (11). Justice believed it is taken directly from ammonia gas in the air. (Craine, JM, 2008: 70) This was the state of affairs until a French chemist, Boussingault (1802 – 1887) demonstrated that plants are incapable of absorbing free nitrogen but were able to flourish even without humus as long as alternative sources of nitrates or ammoniacal salts are supplied. (Bynum, WF, et al, 1981: 300)

Boussingault and his contemporaries saw the uptake of ammonia as purely chemical. (Bynum, WF, et al, 1981: 300) What other way could there be? The great German physiologist, Theodor Schwann, born in 1810, took a step closer to the solution. He discovered that alcoholic fermentation and the fermentation that causes putrefaction was carried out by microbes. (12) (Barnett, JA)

Louis Pasteur, born in 1822 grew up to become very important in the field of science. He was the first one to suggest that microorganisms may be involved in the nitrogen absorption process of plants. (Bynum, WF, et al, 1981: 300) He studied the breakdown and reorganization of material that contained nitrogen by soil bacteria, fungi, and algae. It seemed that nitrogen was not used up but was circulated. Decaying humus gave ammonia, from which microorganisms constructed nitric acid and its compounds. These were then absorbed by plants and turned into proteins and incorporated into living substance. The cycle was completed by the death and natural decay of the plant and the animal. (Bynum, WF, et al, 1981: 300) At the death of the animal, the process of nitrification was reversed and microbes were again responsible for breaking the molecules down until only gaseous nitrogen remained.

The German agricultural chemist, Hermann Hellriegel (1831-1895), discovered that certain plants (leguminous) take atmospheric nitrogen and “replenished the ammonium in the soil through the process now known as nitrogen fixation. He found that the nodules on the roots of legumes are the location where nitrogen fixation takes place.” (Boundless, 2014) Hermann did not discover how this is done. Martinus Willem Beijerinck (March 16, 1851 – January 1, 1931), a Dutch microbiologist and botanist, discovered that the small growth areas on the roots contained bacteria. He called it rhizobia. It is the rhizobia that are responsible for changing the nitrogen to ammonium. Ammonia is NH3 and ammonium is NH4. (Boundless, 2014) Soon more ways were discovered that changed nitrogen in the air into a form that plants can absorb.

Berthelot described in 1885 how lightning was responsible for nitrogen fixation before he too turned his attention to microscopic organisms in the ground that is responsible for nitrogen fixation. (Elmerich, C, Newton, WE. 2007: 3) The energy of a lightning strike disrupts the nitrogen (N₂) and oxygen (O₂) molecules in the air producing highly reactive nitrogen and oxygen atoms that attract other nitrogen (N₂) and oxygen (O₂) molecules that form nitrogen oxides that eventually become nitrates. (Zumbal, 2000: 924) Alternatively, Beijerinck’s rhizobia bacteria fix the atmospheric nitrogen directly (Boundless, 2014) in small growths on plant roots such as beans, peas and alfalfa (Zumbal, 2000: 924), or animal droppings and urea or dead animal or plants provide saprobiotic bacteria, nitrogen or nitrogen-family members that can be changed.

Nitrogen is turned directly into either ammonia (NH₃) or ammonium (NH₄) or into nitrate (NO₃^–). Nitrifying bacteria turns ammonia into nitrite. Nitrite is toxic and nitrifying bacteria change the nitrites into nitrates that either becomes plant food along with nitrate’s that are formed during lightning strikes or are changed back into nitrogen by denitrifying bacteria.

A friend of Jeppe, Dr. Polenski found in 1891, months before I arrived in Denmark, that when he mixed curing brine for bacon with Saltpeter and tested it, that he found nitrate to be present. After a week, when he tested it again, there were only nitrites. The same with the meat that he cured. At the beginning of the week, there was nitrate present in the meat and later he found only nitrites. (13)

The notion that bacteria are responsible for changing the nitrate to nitrite was well established by the time he did the experiment and so, his conclusion that what had happened in the brine was the result of bacteria was reasonable. It would not surprise me if it would be shown that nitrite is responsible for curing and not nitrate. (8)

I realised that saltpetre was a key part of the world we live in. The energy of the acid in the air, harnessed by an entire world of microorganisms that probably occur in every environment on earth and changed into a format that plants and then humans and animals can absorb. An acid, coupled with a salt helps us to preserve meat and change pork meat into bacon, grow plants, feed oceans and drive the processes of the earth. By it we fight wars, we grow crops and we eat and live!

At night after supper, we are reading Foods by Edward Smith. He wrote on bacon and said, “bacon is the poor man’s food, having a value to the masses which is appreciated in proportion to their poverty, and it is a duty to offer every facility for its production in the homes of the poor.” (Smith, Edward, 1876: 65) The reason why it is good for the poor is that it can be cooked in water and the liquid part can be given to the children and the solid part consumed by the parents and “thus both be in a degree pleased, if not satisfied.” (Smith, Edward, 1876: 65)

He continues to say that “it is also the rich man’s food, for the flavour, which is naturally or artificially acquired by drying (and curing), is highly prized, and although it may be taken as a necessary by the rich, it is in universal request as a luxury” (Smith, Edward, 1876: 65)

This is our business plan: to produce the best bacon on earth! Uncle Cornelius passed away after a full life and I can not help to see our current quest as a necessary evolution of time as young and new thoughts replace older methods. Evolution must in the first place be predicated on sound science as well as common sense.

This is then your chance to discover the nitrogen cycle from the perspective of a meat scientist. I miss you, my little girl. There is not a single day that I don’t think of you! It’s late. I am sure that you are fast asleep by this time and that you are holding your bear and dream of the cumming summer.

I learn so much and still, you are my biggest lesson in life. Your love and your spirit have taught me how to live myself!

I count the days till I see you guys again! I miss you all so much and love you!

Your Dad.

Practical Applications for the Modern Bacon Curer

In this section, I highlight some of the points of application in the modern high throughput bacon plant.

A friend of mine from the bacon industry in Castlemaine, Australia recently interacted with me on the matter of total meat content in bacon. Nitrogen is a constituent of the meat protein and important in its nutritional value. This identification and the subsequent determination of a phenomenally stable nitrogen percentage in meat lead to a number of important applications and implications, among others, a way to determine lean meat content and total meat content in meat processing.

A good summary of the thinking early in the late 1800s and early 1900s on the subject exists in the old South African Food, Drugs and Disinfectants Act No. 13 of 1929 (See note 1). It has subsequently been repealed, but the basis of the law is still very much applicable. As an important historical document, it sets out the determination of total meat content. It essentially remained unchanged (apart from minor updates).

The calculations of total meat content are defined in subparagraph 4 (iv) which reads as follows: “In all cases where it is necessary to calculate total meat under regulations 14 (1), (2), (3) and (4), the formula used shall be:—

Percentage Lean Meat = (Percentage Protein Nitrogen × 30 ).
Percentage Total Meat = (Percentage Lean Meat + Percentage Fat).”

The questions of interest are how did they arrive at this and how accurate an indication is it of total meat content? What is the relationship between nitrogen and nutrition? When decay takes place, what happens with the nitrogen in the protein? How does the amount of nitrogen we consume determine the total nitrogen content of our bodies or any animal or plant for that matter? What is the value of nitrogen to the body which makes it essential for nutrition? How does nitrogen move from a plant or an animal into our bodies to provide nutrition? What is the impact of processing on nutrition and the total nitrogen content? Can the standard calculation for fresh meat be applied to processed products? Lastly and equally fascinating, what are other sources of nitrogen that can increase the total nitrogen count and skew the nitrogen count in a product and its relationship and to meat content.

This short series of articles set out to deal with these fascinating issues. In this first article, we will look at the time from the start of the chemical revolution to Boussingault. Sincere thanks to my friend in Castlemaine, Australia for provoking a fascinating line of inquiry!

Notes

(1) After a short service in the Woodstock house, the procession moved to the Groote Kerk where Jacobus has been an elder. The coffin was carried into the church by the Cape premier, Cecil John Rhodes, Sir John Henry de Villiers (subsequent chief justice of the Union), JW Sauer, Onze Jan Hofmeyer, Sir Gordon Sprigg, Colonel F. Schermbrucker, ML Neetling and DC de Waal.

After the service the funeral procession moved to the Cape Town station, where a special train took the mourners to the Maitland Cemetery. The coffin, of Cape teak, was lowered into the ground which Jacobus picked himself.

The grave was filled up and wreaths were laid on top. One from David and Johanna Graaff, a second from John and Rosetta Graaff and a third from Jacobus and Susan Graaff. (Dommisse, E, 2011: 48, 49)

WP_20141019_12_03_36_Pro__highres — Jacobus Combrinck’s grave in the Maitland Cemetery.

WP_20141019_12_04_07_Pro__highres — The funeral procession would have walked along this path from the train tracks at the far top side of the picture to Jacobus’s grave on the right, under the tree, on the right.

The affection from the Graaff brothers who were responsible for erecting the gravestone is evident. At the top, the words, “Ter Dierbare Herinnering aan Jacobus A. Combrinck,” “For affectionate remembrance of Jacobus A. Combrinck.”

Under Jacobus’s birth date and date of passing, the inscription in Dutch reads, “Ik weet op wien te vertrouen,” “I know in whom to trust.”

Underneath is written in Dutch,”Opgericht door zyne dankbare neven de broeders Graaff,” “Erected by your grateful nephews, the brothers Graaff.”

David took over Jacobus’s position in the Legislative Council of the Cape Colony soon after his passing.

The following notice appeared in a colonial newspaper.

The_Colonies_and_India_Sat__Oct_10__1891_ — A notice published on page 11 in The Colonies and Indian under the heading “Colonial, Indian and American News Items” on 10 Oct 1891.

(2) The Woodstock house was previously owned by a highly respected judge, Henry Cloete in the suburb of Papendorp (later to be renamed, Woodstock). He enlarged it greatly. The house was built on an estate where Jacobus planted trees, erected a water mill of his own design, cultivated a splendid flower garden. (Simons, PB, 2000: 14)

(3) Sir Gordon Sprigg, prime minister before Rhodes ousted him, was moved when he heard the news of Combrink’s death. He said, “A good man has gone from among us.” Rhodes apparently only slipped a posy of white and purple violets into his coffin and said nothing. These two powerful men were never the best of friends. (Simons, PB, 2000: 27)

(4) When doing trials at the then Vion Factory in Malton, Ken Pickles was the NPD (New Product Development) manager. A young intern from Brazil would walk behind him and every time we went to the curing tanks, he would ask the young man this question. It’s an image that I will never forget.

(5) An anaerobic organism or anaerobe is any organism that does not require oxygen for growth.

(6) Processed meats many times contain bacteria, many of which are responsible for changing nitrate to nitrite. “This conversion proceeds more rapidly in unpacked bacon than in the vacuum-packed variety, a difference which has been ascribed somewhat surprisingly to the low reducing activity of anaerobic bacteria. (Hill, MJ. 1991: 96)

(7) The nitrate and nitrite in salts are primarily responsible for the curing activity in meat. “The reduction of nitrate (NO3-) salts to nitrite (NO2-) and then to gaseous NO and its subsequent reaction with myoglobin to form the nitrosyl-myoglobin complex forms the basis for cured meat flavour and colour.

It was also later realized that it is bacteria that first converts nitrate into nitrite, which is the mechanism underlying in the preservation of food. Nitrite in meat is responsible for inhibiting the growth in aerobic bacteria (especially the spores of Clostridium botulinum), retard the development of rancidity during storage, develop and preserving the meat flavour and colour, stabilizing the oxidative state of lipids in meat products.” (Dikeman, M, Devine, C, 2014: 436)

(8) The fact that nitrate is not the curing agent, but nitrite was in fact discovered soon asfter 1891. One of the men at the forefront of these discoveries were Prof. D. R. Hoagland, professor of plant nutrition, University of California (www.nature.com). He suggested in 1908 that the “reduction of nitrate to nitrite, nitrous acid and nitric oxide was by either bacterial or enzymatic action or a combination of the two and was essential for NOHb formation. The scientific knowledge led to the direct use of nitrite instead of nitrate, mostly because lower addition levels were needed to achieve the same degree of cure.” (Pegg, RB, Shahidi, F. 2000)

In keeping with our interest in the person and his discovery, the following notice was published at the death of Prof. Hoagland by the University of California.
“1884-1949

Dennis Robert Hoagland, Professor Emeritus of Plant Nutrition, died September 5, 1949. His life had been fruitful in achievement and stimulating in quality.

Professor Hoagland was born in Golden, Colorado, on April 2, 1884. He attended the Denver public schools and in 1903 entered Stanford University, graduating with an A.B. degree in the Chemistry major in 1907. After a fall semester of graduate work, he accepted a position at the University of California in January 1908 as Instructor in Animal Nutrition. From that time until his retirement June 30, 1949, with the exception of the period 1910 to 1913, his academic life was associated with the Berkeley campus.

About 1910 the U. S. Department of Agriculture became concerned with the alleged injurious effects of food preservatives on humans. A consulting board of scientific experts was set up and Professor Hoagland became a member of its staff. This assignment took him to the University of Pennsylvania where in addition to his research he found opportunity to continue his graduate studies in chemistry. It is evident that this early experience introduced him to the intriguing problems of biochemistry and this interest once developed became his major scientific concern the remainder of his career. In 1912 he accepted a graduate scholarship at the University of Wisconsin in the field of Animal Biochemistry, a field there cultivated with distinction by E. V. McCollum and E. B. Hart, and he was awarded the M.A. degree in 1913.

In the fall of 1913 he returned to California as Assistant Professor of Agricultural Chemistry. This area of knowledge, through the stimulating domination of Professor Hilgard, concerned itself with the soil and crop problems confronting California agriculture. Professor Hoagland found no difficulty in adapting himself to this new emphasis. It was probably his diversified early experience that made it possible for him later to develop on this campus a world center for the study of interrelated plant and soil problems. His broad interest did not lead him to scatter his efforts, however. He early demonstrated an ability to clearly outline a segment of the field and vigorously attack it, without restricting his vision of the entire complex problem. It was this quality which enabled him to achieve so significantly.

Professor Hoagland became head of the newly created Division of Plant Nutrition in 1922. Under his guidance and stimulation, this became more than a “Division” in the College of Agriculture: it was in effect what the Germans might have termed an “Institut für Pflanzen und Boden Wissenschaft.” It was a dynamic research center in which both basic and practical problems of plant oil interrelationships were studied with enthusiasm and insight; the laboratory was a magnet which drew students and mature investigators from all parts of the world. His own contributions to the research center’s activities were many and important. It was the early disclosure by himself and associates of the phenomenon of so-called “active absorption” of salts by living cells, both plant and animal, that compelled a complete reappraisal of salt absorption processes. His own research and that of his students led to new discoveries on the need and function of “trace” chemical elements–elements required by living cells in such minute amounts as to escape detection except by the use of the most refined techniques. These and other revelations constituted the leaven which activated investigations in many associated fields. His laboratory was a center with a radiating influence which reached out and touched other great scientific centers, and also the lone worker at an isolated post.

Professor Hoagland entered fully into the academic life of the University. He served as a member, then as chairman, of the Budget Committee and as a member of many other Senate and administrative committees. He was a member of numerous scientific organizations, including the National Academy of Science, and served on important national scientific boards. Many honors came to him. The American Society of Plant Physiologists presented him with the Stephen Hales Award in 1929; the annual $1,000 prize of the American Association for the Advancement of Science was given to him and an associate jointly in 1940. He was selected as Faculty Research Lecturer at Berkeley in 1942 and the same year delivered the John M. Prather Lectures at Harvard. In 1946 he was awarded the Barnes Life Membership in the American Society of Physiologists.

Professor Hoagland was married to Jessie A. Smiley in 1920. She died in 1933 leaving three sons, all of whom are graduates of this University. He did not possess a rugged constitution and the last few years of his life were marred by illness. But almost to the last he kept a faculty for keen appraisal of scientific and social situations and an interest in human events of the most diverse sort. He was a man of judgment, of tolerance, and of discernment, one who abhorred hypocrisy and admired honesty. He was the quality out of which great human structures are built.

W. P. Kelley D. I. Arnon A. R. Davis” (CDLIB)

(9) Humus is decaying organic matter. (Bynum, WF, et al, 1981: 300)

(10) The trademark was granted in 1899 for Oxo.

(11) The German chemist, Justice von Liebig (1803 – 73), continued to believe that plants got their nitrogen from the air (in the form of ammonia). (Wikipedia, Justice_von_Liebig) He has popularised a principle developed in agriculture science by Charl Sprengel (1828) and was called Liebig’s Law of the Minimum, often simply called Liebig’s law or the law of the minimum. It states that growth is controlled not by the total amount of resources available, but by the scarcest resource (limiting factor) (Wikipedia, Law_of_the_Minimum)

(12) He also attributed fermentation to microorganisms.

“Schwann is famous for developing a ‘cell theory’, namely, that living structures come from formation and differentiation of units (the cells), which then constitute the bodies of organisms (Schwann, 1839). His paper on fermentation (Schwann, 1837) was entitled ‘A preliminary communication concerning experiments on fermentation of wine and putrefaction’. Using a microscope, Schwann examined beer yeast and described it as resembling many articulated fungi and ‘without doubt a plant’. His conclusions from his observations and experiments were unequivocal, revolutionary and correct: The connection between wine fermentation and the development of the sugar fungus is not to be underestimated; it is very probable that, by means of the development of the fungus, fermentation is started. Since, however, in addition to sugar, a nitrogenous compound is necessary for fermentation, it seems that such a compound is also necessary for the life of this plant, as probably every fungus contains nitrogen. Wine fermentation must be a decomposition that occurs when the sugar-fungus uses sugar and nitrogenous substances for growth, during which, those elements not so used are preferentially converted to alcohol.

In one of his experiments, Schwann boiled some yeast in a solution of cane sugar in four stoppered flasks. After cooling, he admitted air into the flasks: for two flasks, the air was first passed through a thin red-hot glass tube (analysis showed this air still to contain 19·4 % oxygen); the other two flasks received unheated air. Fermentation occurred only in the latter two flasks. Schwann’s conclusion was important:Thus, in alcoholic fermentation as in putrefaction, it is not the oxygen of the air which causes this to occur, as previously suggested by Gay-Lussac, but something in the air which is destroyed by heat.

In this notable 1837 paper, Schwann anticipated observations made by Pasteur over twenty years later, writing:Alcoholic fermentation must be regarded as the decomposition effected by the sugar fungus, which extracts from the sugar and a nitrogenous substance the materials necessary for its own nutrition and growth; and substances not taken up by the plant form alcohol.

(Barnett, JA. 1998, 2000)

(13) The chemist, Eduard Polenske (1849-1911) (Wikipedia. Pökeln), was born in Ratzebuhr, Neustettin, Pommern, Germany on 27 Aug 1849 to Samuel G Polenski and Rosina Schultz. Eduard Reinhold married Möller. He passed away in 1911 in Berlin, Germany. (Ancestry. Polenske) He was working for the German Imperial Health Office when he made the discovery about nitrite in curing brine. (Wikipedia. Eduard_Polenske)

The Imperial Health Office was established on 16 July 1876 as a focal point for the medical and veterinary in Berlin. First, it was the division of the Reich Chancellery and since 1879 the Ministry of the Interior assumed. 1879, the “Law concerning the marketing of food, luxury foods and commodities” was adopted, including the Imperial Health Office was responsible for its monitoring. Erected in 1900 Reichsgesundheitsrat supported the Imperial Health Office in its tasks. (Original text: “1879 wurde das „Gesetz betreffend den Verkehr mit Lebensmitteln, Genußmitteln und Gebrauchsgegenständen“ verabschiedet, für dessen Überwachung unter anderem das Kaiserliche Gesundheitsamt zuständig war.”) (Wikipedia. Kaiserliches Gesundheitsamt)

The spelling of his surname varies between Polenski and Polenske.

(14) “This prophetic insight into the continual renewal of body constituents, differing in rate in different tissues, succumbed to the theories of Liebig, Voit, Folin and others, and was not regained until more than a century later when Schoenheimer’s publication in 1942 of “The Dynamic State of Body Constituents” demonstrated the instability of tissue components by isotopic means.” (Munro and Allison, 1964)

References

Barnett, JA. 1998, 2000. Extract from lectures. Beginnings of microbiology and biochemistry: the contribution of yeast research. http://mic.sgmjournals.org/content/149/3/557.full

Bynum, WF, Browne, EJ, Porter, R. 1981. Dictionary of the History of Science. Princeton Legacy Library. Macmillan Press.

Craine, JM. 2008. Resource Strategies of Wild Plants. Princeton University Press.

Danchin, A. From Lamarck to Semmelweis, Transformation of chemical biology1800 – 1849: http://www.normalesup.org/~adanchin/history/dates_1800.html

Dommisse, E. 2011. First baronet of De Grendel. Tafelberg.

Associative and Endophytic Nitrogen-fixing Bacteria and Cyanobacterial …

Elmerich, C, Newton, WE. 2007. Associative and Endophytic Nitrogen-fixing Bacteria and Cyanobacterial Associations. Springer.

Laufer, B., 1919, Sino-Iranica, Field Museum of Natural History, Publication 201, Anthropology Series Vol XV, No. 3

Myers, RL. 2007. The 100 most important chemical compounds. Greenwood Press, Westport.

Pennsylvania Packet, Friday, 18 August 1786

Schofield, RE. 2004. The Enlightened Joseph Priestly. The Pennsylvanian State University

Smith, Edward. 1876. Foods. D. Appleton and Company, New York.

Simons, Phillida Brooke. 2000. Ice Cold In Africa. Fernwood Press