Gilbert, early in his work at Rothamsted, noticed that the results of his field experiments were at variance with this "mineral theory," as it was called, of Liebig, and soon found himself involved in a controversy with the great German chemist which was not always free from bitterness. He found that the nitrogen compounds of the atmosphere were sufficient only for a very meagre vegetation. Cereals treated with ammonium salts and other nitrogenous manures showed a far greater increase of produce than when phosphates, potash or other ash constituents only were supplied. "As more nitrogen was assimilated a greater amount of the fixed bases were found in the ash, and he considered that the function of the fixed bases was to act as carriers of nitric acid. These bases--potash, soda, lime and magnesia, were not mutually replaceable, but the predominance of one or the other affected the produce. Luxuriance of growth was associated with the amount of nitrogen available and assimilated, and in the presence of this sufficiency of nitrogen the formation of carbohydrates depended on the amount of potash available." The possibility that the free nitrogen of the air might supply the nitrogenous needs of plants was disproved by growing plants in calcined soil and removing all traces of ammonia from the air before it was admitted into the glass case in which the plants were growing. Determinations were made of the nitrogen in the seed and soil at the beginning of the experiments, and in the plants and soil at their conclusion.
The work on the assimilation of nitrogen by plants extended over three years and was made the subject of a communication to the Royal Society in 1861. The paper, entitled, "The Sources of the Nitrogen of Vegetation; with special reference to the question whether Plants assimilate free or combined Nitrogen," occupies 144 pages of the _Philosophical Transactions_, and is a brilliant example of the scrupulous accuracy and attention to detail which characterised all Gilbert's work. It is divided into two parts--I. "The General History and Statement of the question."--II. "The Experimental Results obtained at Rothamsted during the years 1857, 1858 and 1859." The authors state in the summary of conclusions that "in our experiments with graminaceous plants, grown both with and without a supply of combined nitrogen beyond that contained in the seed sown, in which there was great variation in the amount of combined nitrogen involved and a wide range in the conditions, character and amount of growth, we have in no case found any evidence of an assimilation of free or uncombined nitrogen.