Microbial Production of Indigo

While synthetic indigo has enjoyed a virtual monopoly for nearly a century, an environmentally friendly microbial production of indigo is under development. The microbial production of indigo has been known since the 1920s. Indigo production with hydrocarbon degrading bacteria expressing mono-oxygenases or dioxygenases has also been investigated in search of a possible alternative for the chemical synthesis of indigo. In 2002 Berry et al. and his companions developed a fermentation process where indigo was produced from glucose with recombinant Escherichia coli that had been modified with Pseudomonas putida genes. However, the method produced also indirubin, which gave an undesirable red hue to the dyeing.

Several bacteria, most notably Pseudomonas species, can use a variety of organic compounds such as naphthalene, toluene, xylene and phenol as their sole carbon source. In many instances, the genes encoding the enzymes for the degradation of these organic compounds are located on large, naturally occurring plasmids. For example, pseudomonads that contain NAH7 plasmid could grow on naphthalene as a sole carbon source. The clone bank was then introduced into E. coli cells. During the characterization of one of the transformants that could convert naphthalene to salicylic acid, it was observed that when the growth medium contained tryptophan, it turned blue. A thorough analysis of the blue color revealed that the transformed E. coli cells were synthesizing the dye indigo. This synthesis is achieved in four steps:

• Conversion of tryptophan in the growth medium to indole by the enzyme tryptophanase, which is produced by the E. coli host cell.
• Oxidation of indole to cis-indole-2,3-dihydrodiol by naphthalene dioxygenase, which is encoded by the DNA that was cloned from NAH7 plasmid.
• Spontaneous elimination of water.
• Air oxidation to form indigo.

In addition, introduction of the gene for enzyme xylene oxidase, which is encoded in the TOL plasmid, can convert tryptophan to indoxyl, which then spontaneously oxidizes to indigo. In pathway A, the naphthalene dioxygenase is derived from the NAH plasmid. In pathway B, the xylene oxidase is from the TOL plasmid. E. coli transformants that synthesize indigo contain either pathway A or B. The conditions for large scale growth of an E. coli strain capable of synthesizing indigo, including temperature, pH and the amount of tryptophan that must be added to the medium to give maximum yields, are being tested.

Although this system has not yet been commercialized, a microbial process for the synthesis of indigo might include a bioreactor in which the recombinant E. coli is chemically immobilized to a solid matrix (e.g. cellulose or silica gel). The unit could be run continuously by adding tryptophan to one end and removing indigo at the other. Genencor International, of Rochester, New York, is experimenting on a process to produce indigo using biotechnology. However, at this stage the technology is expensive and production costs might be prohibitive.

The research and development efforts made in the field of microbial synthesis of indigo from 1927 onwards has been critically reviewed. The highlights of this critical review indicated that biosynthesis of indigo could be divided into three periods: biosynthesis by wild microbes, whole cell catalysis by engineering bacteria and biotransformation regulated by metabolic engineering. Most aromatic degrading microbes and their relevant enzymes possess the ability to convert indole to indigo.

New technologies such as directed evolution, metagenome and two-phase reaction systems could facilitate in-depth investigations of the enzyme resources, and they will play a crucial role in indigo biosynthesis research. Meanwhile, hydroxyl-indoles and indigo derivatives produced in the process are promising pharmaceutical and chemical precursors with great research interest. However, the transformation interactions between intermediates and by-products are still unclear. Besides, low indigo yield and efficiency with high cost have hampered practical production. Therefore, it is essential to combine the molecular biology and metabolic engineering technologies to investigate the mechanisms and industrial application of indigo biosynthesis in the future.

Production of Synthetic Indigo

Demand for natural indigo dramatically increased during the industrial revolution, in part due to the popularity of Levi Strauss’s blue denim jeans. The natural extraction process was expensive and could not produce the mass quantities required for the growing garment industry. So, chemists began searching for synthetic methods of producing the dye. Indigo has been prepared by many methods.

In 1865, the German chemist Adolf von Baeyer began working on the synthesis of indigo. He described his first synthesis of indigo in 1878 from isatin, second synthesis from cinnamic acid and third synthesis from 2-nitrobenzaldehyde. But these synthesis routes were not economically feasible for large scale production. Therefore, the search for alternative starting materials continued. The synthesis of N-(2-carboxyphenyl) glycine from aniline provided a new and economically attractive route. This led the development of a commercially feasible manufacturing process by BASF in 1897. The development of different methods of indigo synthesis and the chemical reactions involved are shown in Figure below.

The third indigo synthesis, from 2-nitrobenzaldehyde (1882), was simple and gave a good yield of indigo, but again was economically impractical due to the high cost of the starting material, 2-nitrobenzaldehyde. This route to indigo is shown in Figure below, now commonly called the Baeyer–Drewson process Adolf von Baeyer was awarded the Nobel Prize for chemistry in 1905 in recognition of his works on indigo, among his many other chemical accomplishments. However, economically practical syntheses of indigo were later developed by a Swiss-German chemistry professor, Karl Heumann (1850–1894), and by a German industrial chemist, Johannes Pfleger (1867–1957).

Heumann’s first synthesis, in 1890, used the industrial chemical aniline as a starting material. It was converted into N-phenylglycine, which was internally condensed into indoxyl in molten alkali at ∼300°C. The indoxyl was quickly oxidized by atmospheric oxygen, dimerizing into indigo. Unfortunately, the yield of product was too low by this route to make it commercially attractive.

His second synthesis at the same time used the more expensive fine organic chemical anthranilic acid as the starting material. In the same sort of reactions utilized by his first route, Heumann obtained a high yield of indigo in this alternate procedure. The process was scaled up to an industrial level (several thousands of tons per annum) by BASF and Hoechst in 1897. Thus, commercial production of indigo began in 1897. By 1900, it equaled the yield of farming 250,000 acres of indigo containing plants.

By 1914 BASF was producing 80% of the world’s synthetic indigo, as a result of which Indian exports of natural indigo fell from 187,000 tons in 1895 to 11,000 tons in 1913 (Freeman, 1997). Thus by 1913 natural indigo had been almost entirely replaced by synthetic indigo. In 1901, Pfleger, working for Hoechst, modified Heumann’s first method by adding sodamide (NaNH2) to the alkaline flux. Sodamide is a very powerful dehydrating agent, and it drove the ring closure reaction, to form indoxyl, to completion.

Sodamide reacts with excess water, thus lowering the overall reaction temperature from almost 300 to 200°C. This results in a much more efficient reaction process. Use of the relatively cheap aniline as the starting material and of sodamide as the condensation agent were the two key factors in the economic success of the BASF–Hoechst industrial indigo synthesis. These synthesis routes are shown in Figure’s below.

Improved synthesis of N-phenylglycine

In 1925 BASF researchers devised an improved synthesis of N-phenylglycine from the N-methylolation of aniline with formaldehyde and hydrogen cyanide, followed by saponification of the resulting nitrile intermediate. This modification provided an additional economy in the overall indigo production method. BASF’s indigo capacity could not meet the huge global indigo demand during the 1960s and 1970s. The increasing prices encouraged quite a few competitors to invest in indigo production, particularly in China.

Production of Indigo through Natural Resources i.e. Plants

Natural indigo was the only source of the blue color until about 1900. The raw materials used in the natural production of indigo are leaves from a specific plant species containing only a small amount of the dye (about 2–4%). Therefore, a large amount of plant material is required to produce a significant quantity of dye. To ensure a controlled supply, indigo was planted in many parts of the world. There are three types of plants with about 300 species that make indigo.

Leguminosae (pea family)

The most famous indigo bearing plant is I. tinctoria, commonly known as indigo. It is a shrub originally grown in the tropics, particularly in India, Southeast Asia and the Middle East.

Cruciferae (cabbage family)

This plant is Is. tinctoria, commonly known as woad. The plant was grown in the Mediterranean and Western Asia. Woad is also grown in North America and in Europe.

Polygonaccae (dock family)

Dock is the name applied to a group of broad-leaved wayside woads. Rhubarb comes from the same family. This species is commonly called Japanese or Chinese indigo. The most important indigo species is I. tinctoria. The species is also known as Indigofera sumatrana. A common alternative used in the relatively colder subtropical locations such as Japan’s Ryukyu Islands and Taiwan is Strobilanthes cusia. In Central and South America, the two species I. suffruticosa (Añil) and Indigofera arrecta (natal indigo) were the most important. In temperate climates, indigo can also be obtained from woad (Is. tinctoria) and dyer’s knotweed (Polygonum tinctorum)

Indigo extraction

The traditional methods of extraction of indigo from the plants I. tinctoria and woad (Is. tinctoria) differ from each other.

Extraction from I. tinctoria

Indian method: Cultivation of indigo for extraction of dye is an age-old practice in India. Although there are several variations, traditionally the cut plant is tied into bundles, packed into the fermenting vat and covered with clear water. The vats, which are usually made of brick lined with cement, have an area of about 400 square feet and are 3 feet deep, arranged in two rows over each other. The top vat is known as the fermenting vat and the bottom as the beating vat. The indigo plant can steep up to 10–15h, during which natural fermentation sets in. The liquor, which varies from a pale straw color to a golden yellow, is then run into the beating vat, where it is agitated either manually or mechanically. The color of the liquid becomes green, then blue, and finally indigo separates out as flakes and is precipitated to the bottom of the beating vat. The indigo can thoroughly settle, when the supernatant liquid is drawn off. The pulpy mass of indigo is then boiled with water for a few hours to remove impurities, filtered through thick woolen or coarse canvas bags, then pressed to remove as much of the moisture as possible, after which it is cut into cubes and finally air dried.

Japanese method: Historically, the Japanese have used another method, which involves extracting indigo from the Polygonum plant. In this process the plant is mixed with wheat husk powder, limestone powder and lye ash. The mixture can ferment for about one week to form the dye pigment, which is called Sukumo.

Extraction from Is. tinctoria

In the traditional method of producing indigo dye (also called woad) from woad, the leaves were crushed to a pulp, which was kneaded into balls, which were then allowed to dry for several weeks. These dried balls could then be stored. The balls needed to be couched. The couching meant crushing the balls into powder and wetting it and allowing the material to ferment for several weeks. After couching, the woad was dark clay like material that was dried and packed tightly before use.

The dye from woad was very impure and it gave only light colors, whereas the indigo from the tropics was of better quality and could be used to produce darker blues. This was the reason why the exotic indigo from the Indigofera species could overtake woad so completely.

The modern extraction method of indigo from woad uses the water solubility of the indigo precursors in steeping the leaves in hot water. The precursors are broken down to indoxyl and sugar moieties by enzymes in the plant. Subsequent aeration produces indigo by oxidation of indoxyl.

Coloring component in plant species

Indigo as such does not exist in the leaves of indigo producing plants. Instead, there are its precursors, indican in Indigofera and P. tinctorium species, and isatan B in addition to indican in Is. tinctoria. Indican in fresh green leaves is stable, as it is attached to glucose, forming a stable indicant glucoside. However, when the leaves are fermented, indican is hydrolyzed (cleavage of sugar residue) by an enzyme glucosidase present in the leaves to yield indoxyl, which transforms rapidly into indigo by oxidative dimerisation.

Surprisingly, indican is also biosynthesized in the human body from the amino acid tryptophan. Part of the indican is degraded by intestinal bacteria to the smelly indole. Some of the colorless and water soluble indican is also eliminated via the kidneys. In rare cases certain people are unable to metabolize the indican properly, and they excrete traces of the intensely blue indigo in their urine. This medical condition is also known as PUB, ‘purple urine bag syndrome’.

Natural indigo purity

The purity of plant-derived indigo even with the modern extraction method is somewhat low when compared to synthetic indigo. Natural indigo contains impurities such as indirubin, indigo-brown, indigo gluten and mineral matter. The indigo purity for woad has been reported to be 20–40%, and for Indigofera indigo from 50% up to 77%. There is also the question of the efficiency of the extraction; the theoretical yield of indigo formation from indoxyl molecules has been discovered to be approximately 60%. So, 40% of the indoxyl is lost during the process to impurities such as isatin and indirubin and other by-products of the reaction.

Revival of natural indigo

Since the commercialization of synthetic indigo, the use of natural indigo has almost become extinct. In recent years, the demand for natural dyes has been increasing in many countries, because of health and pollution effects and a revival of interest in the relationship between dyes and culture. In the present time, indigo is still cultivated for dyeing on a small scale in India and in some parts of Africa and Central America. It is frequently grown as a secondary crop.