Bacterial pigments and their applications

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Process Biochemistry 48 (2013) 1065–1079

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Process Biochemistry

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Review

Bacterial pigments and their applications

Chidambaram Kulandaisamy Venil a, Zainul Akmar Zakariab, Wan Azlina Ahmada,∗

a Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysiab Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia

a r t i c l e i n f o

Article history:Received 11 March 2013Received in revised form 9 May 2013Accepted 1 June 2013Available online 10 June 2013

Keywords:BacteriaPigmentsEconomicsChallengesNovel strategiesApplications

a b s t r a c t

Natural pigments sourced from ores, insects, plants and animals were the colorants used since prehistoricperiod. Synthetic dyes which took the place of natural pigments in the middle of 19th century still rulethe field to the maximum extent in spite of its hazardous effect to humans, animals and environment. Asan alternative to synthetic pigments, bacterial pigments due to their better biodegradability and highercompatibility with the environment, offer promising avenues for various applications. The industry isnow able to produce some bacterial pigments for applications in food, pharmaceuticals, cosmetics andtextiles. Extraction of bacterial pigments in relatively pure and concentrated forms is the main techno-logical challenge. Optimization of fermentation process and the medium components are reported as keystrategies for economic recovery of pigments. Research work needs to be carried out to formulate thefermentation media for each bacterial pigment on large scale by using economical and easily availablesources for commercial process. Recent advances in synthetic biology, metabolic engineering efforts ofbacteria will greatly expand the pigments that could be produced economically in sufficient amounts forindustrial application. This review summarizes the current technology status and challenges, economics,novel strategies for production of bacterial pigments and metabolic engineering of bacteria with a focuson applications of bacterial pigments in food industry, pharmaceutical industry, dyeing as well as onother applications.

© 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10661.1. Background/history of pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10661.2. Synthetic dyes vs natural pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10661.3. Current technology and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067

2. Bacterial pigment production technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10682.1. Strain development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10682.2. Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10682.3. Recovery and separation of bacterial pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10692.4. Novel strategies for production of bacterial pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10692.5. Metabolic engineering of microbes for natural product biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070

3. Economics for pigment production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10704. Applications of bacterial pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071

4.1. Applications as food colorant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10724.2. Applications in pharmaceutical industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10724.3. Applications in textile industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10744.4. Applications in other aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075

5. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1075Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077

∗ Corresponding author. Tel.: +607 5534546.E-mail addresses: [email protected], [email protected] (W.A. Ahmad).

1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.procbio.2013.06.006


1066 C.K. Venil et al. / Process Biochemistry 48 (2013) 1065–1079

1. Introduction

The new found awareness in human safety and environmen-tal conservation has kindled fresh enthusiasm for natural sourcesof colors. Natural colorants or dyes derived from flora and faunaare believed to be safe because of non-toxic, non-carcinogenicand biodegradable in nature [1]. Traditional sources of colorantsinclude natural products such as flavonoids and anthraquinonesproduced by plants and animals. For example, carminic acid, a deepred anthraquinone produced by scale insects is now used as a pig-ment in paints, crimson ink, cosmetics and food colors [2]. As thepresent trend throughout the world is shifting towards the use ofeco friendly and biodegradable commodities, the demand for natu-ral colorants is increasing day by day. Natural pigments are sourcedfrom ores, insects, plants and microbes. Among microbes, bacteriahave immense potential to produce diverse bioproducts and onesuch bioproduct is pigments. The production and application ofbacterial pigments as natural colorants has been investigated byvarious researchers [3–5].

Bacterial pigment production is now one of the emerging fieldsof research to demonstrate its potential for various industrial appli-cations [4]. Most of the bacterial pigment production is still atthe R&D stage. Hence, work on the bacterial pigments should beintensified especially in finding cheap and suitable growth mediumwhich can reduce the cost and increase its applicability for indus-trial production [5]. There are many studies in the literature onbacterial pigments which focus production and application of spec-ified pigment in each case. A comprehensive material of suchstudies on bacterial pigments is rare in literature. So, based on accu-mulated data in the literature and with an intention to encourageresearchers and stake holders alike to explore and exploit this over-flowing source of bacterial pigments, this review paper presents anoverview of varied studies on bacterial pigments and their wide-ranging applications. This endeavor has the potential to lead to amore sustainable and environment friendly way to color the worldwith bacterial pigments.

1.1. Background/history of pigments

Dyeing was known as early as in the Indus Valley period(2600–1900 BC); this knowledge has been substantiated by find-ings of colored garments of cloth and traces of madder dye in theruins of the Indus Valley Civilization at Mohenjodaro and Harappa.So, natural dyes, dyestuff and dyeing are as old as textiles them-selves. Man has always been interested in colors; the art of dyeinghas a long past and many of the dyes go back into prehistory. Itwas practiced during the Bronze Age in Europe. The earliest writ-ten record of the use of natural dyes was found in China dated 2600BC [6]. Primitive dyeing techniques included sticking plants to fab-ric or rubbing crushed pigments into cloth. The methods becamemore sophisticated with time and techniques using natural dyesfrom crushed fruits, berries and other plants, which were boiled

into the fabric and which gave light and water fastness (resistance),were developed. The cochineal dye was used by the people of Aztecand Maya culture period of Central and North America. By the 4thcentury AD, dyes such as woad, madder, weld, Brazil wood, indigoand a dark reddish-purple were known [7]. Henna was used evenbefore 2500 BC, while saffron is mentioned in the Bible [8]. Useof natural pigments in food is known from Japan in the shosointext of the Nara period (8th century) which contains references tocolored soybean and adzuki-bean cakes, so it appears that coloredprocessed foods had been taken at least by people of some sec-tions. Thus, studies on natural pigments are greatly impulsed bytheir multiple functions [9]. The art of coloring spread widely withthe advancement of civilization [10].

Before the advent of synthetic pigments, natural pigments werethe only source of color available and were widely used and traded,providing a major source of wealth creation around the globe. It hasbeen used for many purposes such as the coloring of natural fibers(wool, cotton, silk), fur and leather. They were also used to colorcosmetic products and to produce inks, watercolors and artist’spaints [1]. Since the introduction of synthetic dyes by Perkin in 1856(Table 1), many convenient and cheap synthetic pigments haveappeared, and the use of natural dyes has decreased due to the rela-tively cheaper synthetic pigments [11]. Over the course of the 20thcentury, naturally occurring organic pigments have been almostcompletely displaced by synthetic molecules such as phthalocya-nines that range from blue to green, arylides that are yellow togreenish or reddish-yellow and quinacridones ranging from orangeto violet [12]. Advances in organic chemistry enabled mass pro-duction of these compounds relatively cheaper thereby allowingthem to displace natural product pigments, whose procurement isoften more challenging. Current applications of synthetic pigmentsare in the textile industry, leather tanning industry, paper produc-tion, food technology, agricultural research, light-harvesting arrays,photo electrochemical cells and in hair colorings.

1.2. Synthetic dyes vs natural pigments

Synthetic additives are severely criticized and consumers showinhibition towards these products [13]. In the 1960s in US, theenvironmental activists demonstrated against the use of suchfood additives and this attitude was spread out widely. Activistscampaigned for natural colorants highlighting their nutritionalcharacteristics as a sales tool. Thus, a worldwide tendency to usenatural colorants was generated. Currently, people interpret thecontent of synthetic products as contaminants and the tendencyhas been reinforced [10]. Because of their pharmacological prop-erties, the number of advantages of using natural pigments, oversynthetic colorants, has further boosted.

Natural pigments and synthetic dyes are extensively used invarious fields of everyday life such as food production, textile indus-tries, paper production, and agricultural practices [14]. Accordingto green technology, less toxic products and more natural starting

Table 1Historical developments in color [3].

Year Development

1856 Perkin’s mauve pigment was discovered and coaltar dyes were synthesized1884 Monascus sp. was traditionally cultivated and utilized in the orient for making red rice wine, red shaohsing wine and red

Chinese rice1954 The first carotenoid pigment from Cryptococcus was marketed1963 Production of carotenoid pigments from Rhodotorula sp. startedEarly 1970s Astaxanthin was isolated from Phaffia rhodozyma (in honor of Prof. Herman Jan Phaff) grown on exudates of deciduous trees in

Japan and AlaskaLate 1970s and early 1980s Production of beta carotene from Dunaliella salina took place1985 Betatene Limited Corporation was established for cultivation of D. Salina on large scale for producing natural beta carotene

products


C.K. Venil et al. / Process Biochemistry 48 (2013) 1065–1079 1067

Table 2Biologically active pigmented compound from bacteria and its market potential [15].

Pigment and structure Bacteria Functions

Bacillus subtilis Used in foods, vitamin enriched milkproducts and energy drinks

Flavobacterium, Agrobacterium aurantiacum Food supplement for humans and asfood additives for animals and fish

Serratia marcescens, Pseudomonas,Pseudoalteromonas, Alteromonas denitrificans,Hahella, Vibrio

Antibacterial, antifungal, antimalarial,antibiotic, immunosuppressive,anticancer

Cyanobacteria Dietary supplement rich in proteins

Chromobacterium violaceum, Alteromonasluteoviolacea

Antiviral, antibacterial,antiulcerogenic, antileishmanial, andanticancer properties, potent cytotoxiceffects against U937 and HL60Leukemia cell lines

material is favorable for today’s production lines. It is well knownthat some synthesized dye manufacturing is prohibited due to thecarcinogenicity of the precursor or product and also the effect of dis-posal of their industrial wastes in the ecosystem. Natural pigmentsnot only have the capacity to increase the marketability of products,they also display advantageous biological activities as antioxidantsand anticancer agents. It is therefore, essential to explore variousnatural sources of colorants and their applications. The industry isnow able to produce some bacterial pigments for applications infood, cosmetics or textiles [15]. Products are of good market valueif they are colored with natural compounds (Table 2).

Since time immemorial, natural products such as isoprenoids,alkaloids and flavonoids, have been used by humans as colorants,flavors and fragrances. Due to the scarce availability and high priceof natural products, synthetic compounds have gained more andmore importance in the food industry in the last decades. However,nowadays customers demand natural products as a consequenceof proven toxicological effects of some synthetic compounds [16].Natural products were also of great importance as pharmaceuticaldrugs. However, due to the limited chemical diversity and struc-tural complexity of such synthetic libraries, as well as great successof natural pigments on the market in the last years, screening ofuntapped novel bacteria for new pigments is expected to be con-tinued in the future [17].

1.3. Current technology and challenges

The pigmented bacteria can be sourced from various environ-mental sources which can be cultured and purified. Various growthmedia can be used to isolate different types of bacteria producingpigments. However, due to the high cost of using synthetic medium,there is a need to develop new low cost process for the productionof pigments. The use of agro-industrial residues would provide aprofitable means of reducing substrate cost. Pigment produced bybacteria can be separated using solvent extraction and further char-acterized using various instrumental based analytical techniquessuch as TLC, UV–vis, FTIR, ESI-MS, NMR, HPLC and Gel PermeationChromatography.

Extraction of bacterial pigments in relatively pure and concen-trated forms is the main technological challenge. Bacteria producetwo types of pigments: those that predominantly remain boundto the bacterial mycelia and those that are secreted into the fer-mentation broth. Whereas pigments from the former class canbe conveniently recovered by disrupting the filtered mycelia withacetone, secreted natural products are typically recovered byextracting the aqueous broth with large quantities of organic sol-vents such as ethyl acetate. To mitigate environmental and healthconcerns associated with solvent use, alternative separation tech-nologies such as spray-drying (widespread in the food and feed



industry) and solid-phase extraction (commonplace in the finechemical industry) may be appropriate [18].

The utility of a pigment for industrial applications is dictated notonly by its inherent properties but also by the ability to produceit in sufficient quantities. Although there are several challengesassociated with scaling up of pigment production, much of thetechnology to overcome these challenges is already in place, whichprovides a potential route for reintroducing bacterial pigments toa cost-sensitive world. For example, the pragmatic constraint ofculturing a large number of semi-solid media plates, which wouldrequire the use of many petri-dishes as well as large incubators,can be overcome using fermentation tanks (much like those in abrewery). In fact, such fermentation technology is already used toproduce natural products from bacteria for pharmaceutical, animalhealth, and agricultural applications. A more significant challengelies in the need to increase the production of the pigments frombacteria to make its manufacture economically viable. Here, recentdevelopments in molecular biology could be of use. The genesresponsible for the biosynthesis of numerous pigments have beencloned, and recombinant DNA technology has been harnessed tooverproduce these pigments [19,20]. For example, scientists atAmgen, Inc. were able to engineer a widely used non-hazardousstrain of Escherichia coli to overproduce indigo (which at one pointwas exclusively derived from the woad plant) in fermentation tanks[21].

Biosynthetic pathways can also be manipulated to engineera pigment’s molecular structure and consequently its color. Forexample, Streptomyces coelicolor, which produces the blue pig-ment actinorhodin can be genetically modified to produce a relatedpolyketide called kalafungin, which is bright yellow. Alterna-tively, actinorhodin biosynthesis can also be engineered to produceorange or yellow-red anthraquinones [22,23].

Finally the bacterial pigments must have acceptable stabilitywhen exposed to environmental stresses, especially UV light. UVlight initiates undesirable free-radical reactions in industry thatultimately lead to their degradation. A variety of UV absorbers (suchas benzotriazole- and triazine-based molecules) and free-radicalscavengers (such as hindered amines) are already used in the indus-try and are commercially available [18], whereas their effectivenessin conjunction with bacterial pigments remains to be studied. Thecurrent technology will have the potential to enhance the utility ofbacterial pigments besides its challenges.

2. Bacterial pigment production technologies

2.1. Strain development

Traditionally, strain improvement was achieved mainly by mul-tiple rounds of random mutagenesis and selection, which are stillvery useful nowadays [24,25]. In the latest decade, the developmentof gene deletion approaches [26–28] enabled efficient genome DNAinactivation and greatly improved metabolic engineering of bacte-ria. A more systematic and integrated approach for biotechnologicalprocess development for strain development became prevalent.The motivation for industrial strain development is economic, sincepigment concentrations produced by wild strains are too low foreconomic process. It is very important to isolate strains which pro-duce pigments with shorter fermentation times.

Improvement of microbial strains for the overproduction ofindustrial products has been the hallmark of all commercialfermentation processes. The strain improvement by commonmutagens such as ultraviolet (UV), ethyl methane sulfonate (EMS)and 1-methyl-3-nitro-1-nitrosoguanidine (NTG) is convenient andcan yield a several-fold enhancement of pigments in the process,as proved in several cases [29,30].

2.2. Fermentation

A lot of attention is now paid to the biotechnological syn-thesis of the colors through the bacteria. Microbial fermentationand gene manipulation have been investigated with respect tothe production of pigment. The biotechnological production ofthe natural colors has two fundamental approaches; first is tofind new sources of colors and then enhance their color pro-duction capacity. The other approach is to obtain enhanced andconsistent yields from the already recognized good sources of thecolorants either through the strain improvement or through opti-mizing the process parameters to maximize the yield. There isalso the obstacle of research and development investment andmanufacturing facilities. The appropriate use of the fermentationphysiology together with the metabolic engineering [31] couldallow the efficient mass production of the colorants from thebacteria. With the advances in the gene technology, attemptshave been made to create cell factories for the production ofpigments through the heterologous expression of biosyntheticpathways from either already known or novel pigment producers[32].

Optimization of fermentation processes is an important‘strategy’ needed to achieve high-level production of valuablefermentative products [33]. Medium optimization is one of theimportant processes for getting maximum pigment yield and itinvolves several factors such as medium components, operatingconditions, pH, temperature, aeration and agitation, etc. Classicalway of one factor at a time optimization is accepted well but it hasmany disadvantages like more number of experimental runs, labo-rious, time consuming, etc. Response Surface Methodology (RSM)approach of screening and optimization has gained importance formedium and process optimization of different pigment produc-tion. This technique solves multivariate data which is found fromappropriately designed experiments to solve multivariate equa-tion simultaneously [34]. Moreover, the main advantage of RSMis to reduce number of experimental trials needed to evaluatemultiple variables and their interactions. Therefore, RSM is lesslaborious and brief than the classical methods required for opti-mizing a process [35,36]. Plackett–Burman design was employedby several researchers for screening of significant factors involvedin a particular process [37]. RSM also successfully applied for opti-mization of medium components and fermentation conditions withless number of experiments [38–40]. Also, it is used to investigatethe interaction and quadratic effects among the variables in theprocess.

Khodaiyan et al. [41] optimized the canthaxanthin productionby D. natronolimnaea HS-1 from cheese whey (low cost substrate)by statistical experimental methods. Using sequential optimizationstrategy (fractional factorial design followed by central compositedesign coupled with response surface analysis), pH, concentra-tion of cheese whey and yeast extract were shown to increasethe production of canthaxanthin (2871 ± 76 �g/L) by this bac-terium. Su et al. [42] identified the optimal composition of thecultivated medium for prodigiosin production by S. marcescens.Prodigiosin production was significantly high upon cultivating ina medium containing sucrose and glycine as the carbohydrate andenergy source. When sucrose and glycine were added to the cul-tivate medium, the prodigiosin yield by S. marcescens increased2.12-fold and 2.15-fold, respectively. Furthermore, the inorganicsupplement, KH2PO4, greatly accelerated the cell growth and sub-sequently accelerated the prodigiosin production. However, otherinorganic components did not provide any outstanding results,even a negative effect. Using RSM analysis, an optimal concen-tration for peptone and KH2PO4 was identified. This predictedprodigiosin production was also confirmed experimentally withonly an 8.66% deviation. Under this optimal composition, the



prodigiosin production was enhanced by nearly 8.42-fold (from273.3 to 2423.39 mg/L).

Wang et al. [43] employed Plackett–Burman and Box–Behnkendesigns to examine the effects of cultivation conditions on violaceinproduction by Duganella sp. B2. The results of their study indicatedthat the concentrations of potassium nitrate, l-tryptophan and beefextract, the volume in the flasks, and the pH had significant effectson violacein production. The yield of violacein by Duganella sp. B2reached 1.62 g/L under the optimized conditions and was increasedapproximately 4.8-folds. It was 3.8-folds of the optimized produc-tion by C. violaceum so far reported [44].

Substrates for the bioproduct production were highly influencedby the cost of the bioprocess. So, there is a need to select cheapand efficient substrates for producing the bioproducts economi-cally. Various agricultural products and byproducts such as corncob [45], sugarcane bagasse [46], grape waste [47], jackfruit seed[48], corn steep liquor [49], wheat substrates [50] and cassava [51]were successfully utilized for the production of pigments.

From an industrial point of view, canthaxanthin production viacarotenogenic strains can be more efficient and more economi-cal if the cost of microbial fermentation is minimized by use oflow-cost substrates [52]. In recent years, agro-industrial byprod-ucts such as cheese whey [53], sugarcane molasses [54], glucosesyrup [55], peat hydrolysate [56], cellobiose [57] and beet molasses[58] have all been used as carbohydrate and nutrient sources forcarotenoid production. Beet molasses is a by-product of beet-sugarfactories and contains approximately 50% (w/w) of total sugars(mainly sucrose). Beet molasses constitutes a valuable nutrientsource for microorganisms because it contains growth substancessuch as trace elements, biotin, pantothenic acid, betaine, inositoland glutamic acid [59]. This substance is the most valuable byprod-uct of the beet-sugar industries and may be an inexpensive mediumfor carotenoid production via microbial fermentation [60].

The production of the intensive blue pigment is one of the moststriking features of the Rheinheimera baltica group, but yet nothingis known about its chemistry, production dynamics, and ecolog-ical role. There are many recent indications that production ofpigments greatly depends on environmental conditions includinginteractions with other bacteria in the surrounding environment[61]. Angell et al. [62] could demonstrate that production of ablue pigment with antibiotic activity (pyocyanin) by Pseudomonasaeruginosa was induced when kept in a coculture with an Entero-bacter species (Pup14B). Some dual microbial systems have beencharacterized on the molecular level, and several small signalingmolecules are known. Therefore, pigment production depends onenvironmental parameters such as growth medium and interspe-cific interactions.

Janthinobacterium lividum S9601 strain (deposited as FERM P-15894), isolated from refuse cocoons or stained silk yarns alsoproduces a derivative of violacein, which is used as a dye with excel-lent color tone, fabric hand and fastness of dyeing (JP 10113169). Aprotease and violacein are simultaneously produced using a liquidculture of Chromobacterium and Janthinobacterium sp. containingdecomposed matter of animal hair and dipping protein fiber prod-uct of animal hair or silk effectively improves a feeling of fibersand carries out dying at the same time (JP 6341069) [63]. Researchwork needs to be carried out to formulate the fermentation mediafor each bacterial pigment on large scale by using economical andeasily available sources for commercial process.

To develop a process for the maximum pigment produc-tion, standardization of medium and fermentation condition iscrucial. The application of statistical experimental design tech-niques in the fermentation process development can result inimproved and closer conformance of the output response, reducedprocess variability, and reduced development time and overallcosts.

2.3. Recovery and separation of bacterial pigments

Necessities both in quantity and quality for bacterial pigmentshave increased as new research areas on their biological andpharmacological properties were developed. The separation andpurification processes, however, still have many bottle-necks thatconstrain large-scale preparation of prodigiosin. The conventionalmethod for the separation and purification of prodigiosin was basedon extraction using organic solvents [64]. A complicated and time-consuming process was required, since prodigiosin produced byS. marcescens was mostly bound to bacterial envelopes and onlya small part was released to the broth [65]. In addition, a largeamount of solvent was exhausted and the yield was very low toget the product with high purity.

Non-ionic adsorption resins have been applied in the isolationand purification of many organic macromolecules, including sepa-ration of organic acids, peptides, proteins, nucleic acids and othercompounds [66,67]. Their high loading capacity made it possible toseparate compounds in large amounts. The target product could beadsorbed on to the selected resin directly from the culture broth.Cell separation and prodigiosin extraction steps, therefore, can beeliminated with the novel method. This technology can lower thecost of operation because of its low consumption of extraction sol-vents and reusable adsorbents.

On the basis of non-ionic adsorbent, Wang et al. [65] haveprovided an effective adsorption method for the separation andpurification of prodigiosin directly from culture broth with highquantitative recovery. This technology not only eliminated the cellseparation step, but also yielded a concentrated and partially puri-fied product ready for subsequent purification. The total recoveryof this process (83%) was much higher compared with the conven-tional extraction and silica-gel chromatography process (50%). Inaddition, the adsorption resin (X-5) used in this process had a highloading capacity and could easily be regenerated. These advantagesmade it possible to separate and purify bacterial pigments on a largescale.

To meet the demand for violacein, it is extracted economi-cally using simple technical processes from other species also. Amarine sediment bacterium Pseudoalteromonas sp. strain BlackBeauty (originally deposited under file number DSM 13623)gave 13-fold yield and the crude dye was extracted from thecell mass by slurrying with hot methanol as disclosed in PatentNos. AT000000369438, DE000010063712, EP000001341925,US020040053375, WO002002050299 [63]. Carotenoids may beextracted directly with sunflower oil instead of organic solvents,thus eliminating possible toxic reactions due to trace concen-trations of acetone or hexane and hence facilitating pigmentassimilation by animals (Dufosse, 2006). Several technologicaladvances are still necessary to improve for recovery and separationof bacterial pigments from culture broth to reduce the energy andcost intensive process.

2.4. Novel strategies for production of bacterial pigments

Biotechnology industries are in continual quest for the discoveryof novel bacterial pigments and the enhancement of productivity ofvalue products to retain their global competitiveness. Traditionalmethods employed to achieve these goals include microbial strainselection, culture improvement, media development, and, bioreac-tor and process design. These methods, however, suffer from severedrawbacks such as the long time required for successful outcomes,high expenses and, in many cases, low success rate.

Over the last five years the two novel strategies for over-production of industrially desirable bacterial products (pigments,enzymes) was introduced. These strategies are based on bacterialresponse to the bacteria in their vicinity (quorum sensing) and, to



Table 3Economics for pigment production.

Color Synthetic pigment Natural pigments

Insect/plant pigment Bacterial pigment

Name Price/kg (USD) Name Price/kg (USD) Name Price/kg (USD)

Violet FD&C Blue No. 1 (Brilliant Blue FCF, E133) 42–60 NA NA Violaceina 5000 × 105

Red FD&C Red No. 40(Allura Red AC, E129)

24–42 Cochineal (Insect) 50–80 Prodigiosina 5000 × 105

Annato extract (Plant) 80Orange/yellow FD&C Yellow No. 5 (Tartrazine, E 102) 500 Saffron (Plant) 1400 Carotenoids 1000

a The price mentioned for violacein and prodigioisn are for standards.

their surrounding environment (elicitation). While research intothe quorum sensing process has been continuing with an impres-sive pace, the activity is limited to research at bench scale mainlyin biomedical areas. However, as the range of quorum sensing-affected physiological activities show, there is great potential forthe use of this communication process for bacterial pigments forindustrial exploitation [68].

The production of bacterial pigments, including prodigiosin andviolacein, is known to be controlled by quorum sensing systems[69,70]. In quorum sensing, a bacterial cell can sense the cell densityby the accumulation of signaling molecules. Pantoea agglomeransproducing deep blue pigment production was observed with highcell density when grown on G agar plate. These results suggestedthat the production of the blue pigment may be regulated by quo-rum sensing [71].

Bacterial pigment production by quorum sensing carry poten-tial promise for use in pharmaceutical and biotechnology industrywhere bacterial communication may be used for the overpro-duction of commercially desirable bioproducts. The minimalrequirements for the cultures when biotic elicitors and quorumsensing molecules are used and the high increases in the productiv-ity of the desired products make these molecules suitable potentialsources to be exploited as alternative methods for industrial-scaleoverproduction [68].

2.5. Metabolic engineering of microbes for natural productbiosynthesis

Metabolic engineering has recently been embraced as an effec-tive tool for developing whole-cell biocatalysts for natural productsynthesis. Microbial catalysts now provide a practical means toderive many valuable products in sufficient quantities to supportresearch and applications. Recent advances in the study of natu-ral products from bacteria have laid the foundation for engineeringthese molecules and for developing cost-effective ways to man-ufacture them. Bacterial fermentations and bioconversions play acentral role in the production of natural products [72].

Many naturally occurring bacteria possess highly efficientmechanisms for natural product synthesis. These mechanismscould potentially be engineered into bacteria for natural productsynthesis with enhanced efficiency [73]. Fermentation microbio-logists search for a rare overproducing strain in nature, and thenfurther deregulate the bacteria so that it overproduces huge quan-tities of a desired commercially important product such as ametabolite (pigment) or an enzyme. Deregulation is brought aboutby nutritional as well as classical and molecular genetic manipu-lations to bypass and/or remove negative regulatory mechanismsand to enhance positive regulatory mechanisms [74].

Microbial production of natural products is a promising alterna-tive with several advantages. Since many decades microorganismshave been increasingly used to produce a broad range of value-added compounds with numerous applications in the food,chemical and pharmaceutical industries. Important examples ofthese compounds include many (proteinogenic) amino acids,

organic acids, vitamins, fine-chemicals, biofuels as well as a largenumber of pharmaceutical drugs [75–77]. Microbial productionhas the advantage of being much more environmentally friendlycompared to chemical synthesis, since it avoids the use of organicsolvents, heavy metals and strong acids or bases. Furthermore,in contrast to natural product synthesis, microbial productionbased on renewable feed stocks is inexpensive and the rapidgrowth of microorganisms allows short production times. Unliketraditional synthetic chemistry-based routes, microbial fermenta-tions are readily scalable from the lab bench to industrial-sizedfermenters of several hundred cubic meters. In addition, thegenetic accessibility of industrially relevant microorganisms suchas Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis,Pseudomonas putida allows construction of tailor-made recombi-nant strains by metabolic engineering to modify metabolic profilesaccording to individual production purposes. Since recombinantmicroorganisms are usually void of competing pathways to theheterologously expressed pathway desired natural products aretypically made in the cell as chemically distinct substances [78].This in turn simplifies downstream processing in comparison toproduct purification from other sources.

With the application and integration of the latest tools andstrategies in systems biology, protein engineering, process engi-neering, high-throughput technologies for cultivation, screeningand analytics, metabolic engineering of bacteria for natural productsynthesis is expected to become increasingly powerful. Togetherwith the advances in the field of synthetic biology, future metabolicengineering efforts of bacteria will greatly expand the number ofnatural products that can be produced economically in sufficientamounts [79].

3. Economics for pigment production

The market for the bacterial pigments produced by bioprocessesis hard to estimate, due either for the lack of the statistics of theregional, low-technology products such as annatto extracts, or thefact that the production is pulverized over many small companiesworldwide [80–82]. At one side, there is a growing preference forthe bacterial pigments in food industry, textile dyeing, pharma-ceuticals and cosmetics; at the other side, in some cases, naturalpigments may be several times more expensive than syntheticanalogs. A unique example is the �-carotene produced by bacteriawhich has an approximate cost of US$1000/kg against US$500/kgby synthetic means (Table 3); although more costly, �-caroteneproduced by the bacterial means competes in a market segmentswhere it is important that all the pigments be “natural” [83].

Increasing globalization, restructuring, and internationalizationhas been a key trend shaping the pigment industry over the pastseveral years. Global demand for organic pigments and dyes isexpected to reach almost 10 million tons by 2017 according toGlobal Industry Analysts. The global dye manufacturing industryoriginally dominated by suppliers from UK, Switzerland, Germanyand later shifted to Asia over the past 20 years. Textile industry willremain the largest consumer of organic pigments and dyes, while



Table 4Price comparisons for the production of 50 L of prodigiosin from Serratia marcescens UTM1 using commercial and agricultural-based growth medium.

Prodigiosin production Medium/raw materials Quantity used (kg) Cost (USD) Total cost (USD)

Synthetic medium Nutrient broth 0.4 58.61 58.61Agricultural basedsubstrate

Agriculture Based substrate 0.2 0.10 5.91Nutrient Broth 0.044 5.81

faster growth is expected to occur in other industrial sector suchas printing inks, paints, coatings and plastics. There is an increas-ing thrust towards the use of natural dyes due to the forbidden useof synthetic compounds (banning of azo dyes in Europe). Marketvalue will benefit from consumer preferences for environmentallyfriendly products.

Development of bacterial strains that can utilize cheap andrenewable substrates will make the price of pigments competitivewith synthetic pigments. Therefore discovering cheap substratesfor pigment production is believed to reduce the production cost(Table 4). Although the price of bacterial pigment will be relativelyhigher compared to the synthetic dyes, the production cost can bereduced via:

• Use of agricultural wastes such as pineapple wastes, sugarcanebagasse and molasses as growth medium for cultivation of bacte-ria.

• Use of locally isolated wild type bacterial strains – eliminates thecost for any genetic alterations etc.

• Use of simple extraction techniques.

The bacterial pigments will offer good opportunities due to theirenhanced environmental acceptability and superior performancecharacteristics, classical or conventional grades are expected tocontinue to dominate the organic market.

4. Applications of bacterial pigments

Pigments produced by bacteria are of traditional use in orientalcountries and have been a subject of intense research in the presentdecades because of its potential for applications. Bacterial pigmentsoffer the following benefits and advantages [84–86]:

• Increasingly attractive to science because of broad ranging activ-ities.

• Easy propagation and wide strain selection.• High versatile and productive over other sources.• Fermentation is inherently faster and more productive produc-

tion compared to any other chemical process.• Easy to manipulate genes.• Simple and fast culturing techniques allowing continuous biore-

actor operation.• Structural complexity suits for industrial needs.• Bacterial pigments extracted using simple liquid–liquid extrac-

tion technique minimizing operation cost.• Cheap substrates used for bulk production.

Among the molecules produced by bacteria are carotenoids,melanins, flavins, phenazines, quinones, bacteriochlorophylls, andmore specifically monascins, violacein, prodigiosin or indigo [15].The success of any pigment product manufactured by fermenta-tion depends upon its acceptability in market place, regulatoryapproval and the size of capital investment required to bring theproduct to market. A few years ago, there were doubts aboutthe successful commercialization of fermentation-derived foodgrade or cosmetic grade pigments because of the high capitalinvestment requirements for fermentation facilities and the exten-sive and lengthy toxicity studies required by regulatory agencies.

Nowadays fermentative food grade pigments are in the market likeriboflavin, �-carotene and phycocyanin. Pigments like indigoids,anthraquinones and naphthoquinones will hold potential appli-cations in food industry in near future [87]. Public perception ofbiotechnology-derived products also had to be taken into account[88].

There are several groups of bacterial pigments which are in gen-eral noncovalently bound to proteins [89].

(a) Pigment–protein complexes are organized as photosyntheticunits and consist either as photosynthetic reaction centers oras light-harvesting complexes. Pigmentation of purple bacteriahas been studied extensively [90].

(b) Phenanzine pigments [91,92] are known from several bacte-rial genera in more than 50 varieties, each of which containsa substituted phenazine ring system, and together they repre-sent every color of the visible spectrum. Phenazines are derivedfrom the shikimic acid pathway via phenazine-1,6-dicarboxylicacid and seem to be precursors for further metabolism or areused as redox systems.

(c) Other bacterial pigments such as carotenoids protect theorganism from ionizing radiation. Ionizing radiation produceselectrons, hydroxyl radicals, and hydride radicals which arecapable of altering biopolymers, for example, proteins and DNA.Higher pigmentation of bacteria due to increased UV radiationhas been reported for bacteria in surface waters [93].

(d) In addition, violacein, a pigment with putative antibiotic and/orantiviral activity, has been shown to even influence protozoangrazing [94].

Among the natural pigments produced by bacteria reported sofar (Table 5), most researches have focused on yellow and red pig-ment production, such as monascue produced by Monascus sp. [95],carotenoid from Phaffia rhodozyma [96], Micrococcus roseus [97],Brevibacterium linens [98] and Bradyrhizobium sp. [99] and xan-thomonadin from Xanthomonas campestrispv [100]. However, studyof blue bacterial pigments is limited, probably because not manybacteria are capable of producing blue pigment. The research con-cerning violacein has mainly focused on its medical application.In addition to its application in dyeing fabrics [101], violacein hasalso exhibited cytotoxic activity in human colon cancer cells [102],antileishmanial [103], antiulcerogenic [104], antiviral, antibiotic,antitumoral and anti-Trypanosomacruzi activities [105]. Recently,prodigiosin has been considered effective as a biological con-trol agent against harmful algae in natural marine environmentsbesides its role in textile dyeing and medicinal uses.

Currently pigments of various kinds and forms have beenused as additives or supplements in the food industry, cosmetics,pharmaceuticals, livestock feed and other applications [106–108].However, because of the problems of the synthetic pigments thatcause toxicity and carcinogenicity in the human body, their use isgradually decreased. Therefore interest in natural pigments, thatcan replace synthetic dyes is increasing [109,110]. Recently inresponse to this trend, the tendency to use natural pigments asadding natural materials in the natural dyeing, healthy functionalfoods, cosmetic products for human health and safety have beengradually expanded [111–113].



Table 5Natural pigments produced by bacteria [115].

Bacteria Pigments/Molecule Color Applications

Agrobacterium aurantiacum, Paracoccus carotinifaciens,Xanthophyllomyces dendrorhous

Astaxanthin Pink-red Feed supplement

Rhodococcus maris Beta-carotene Bluish-red Used to treat various disorders such as erythropoieticprotoporphyria, reduces the risk of breast cancer

Bradyrhizobium sp., Haloferax alexandrines Canthaxanthin Dark-red Colorant in food, beverage and pharmaceuticalpreparations

Corynebacterium insidiosum Indigoidine Blue Protection from oxidative stressRugamonas rubra, Streptoverticillium rubrireticuli, Vibrio

gaogenes, Alteromonas rubra, Serratia marcescens,Serratia rubidaea

Prodigiosin Red Anticancer, immunosuppressant, antifungal, algicidal;dyeing (textile, candles, paper, ink)

Pseudomonas aeruginosa Pyocyanin Blue-green Oxidative metabolism, reducing local inflammationChromobacterium violaceum, Janthinobacterium lividum Violacein Purple Pharmaceutical (antioxidant, immunomodulatory,

antitumoral, antiparasitic activities); dyeing (textiles),cosmetics (lotion)

Flavobacterium sp., Paracoccus zeaxanthinifaciens,Staphylococcus aureus

Zeaxanthin Yellow Used to treat different disorders, mainly with affecting theeyes

Xanthomonas oryzae Xanthomonadin Yellow Chemotaxonomic and diagnostic markers

4.1. Applications as food colorant

The development of foods with an attractive appearance is animportant goal in the food industry. Increasingly, food produc-ers are turning to natural food colors, since certain artificial coloradditives have demonstrated negative health issues following theirconsumption. Due to the lack of availability of natural food col-orants, its demand is much sought especially in the food industry.This demand can be fueled by research to offer a more naturalhealthy way of coloring foods and provide a clean label declaration[114]. It is therefore, essential to explore various natural sourcesof food grade colorants and their potentials. Though many natu-ral colors are available, microbial colorants play a significant roleas food coloring agent, because of its production and easy downstreaming process. Industrial production of natural food colorantsby microbial fermentation has several advantages such as cheaperproduction, easier extraction, higher yields through strain improve-ment, no lack of raw materials and no seasonal variations [115].Microorganisms could be made to produce colorants in high yieldby inserting genes coding for the colorant, even colorants not nat-urally produced by microorganisms (e.g., turmeric) could be madein this way. These pigments are looked upon for their safe use asa natural food colorants and will not only benefit human healthbut also preserve the biodiversity, as harmful chemicals releasedinto the environment while producing synthetic colorants couldbe stopped [116].

Scientists have isolated food grade pigments from bacteria(Fig. 1; Table 6) and blue pigment from cultured soil bacteria thatcould offer a natural color with an excellent stability and toxico-logy profile for food. The researchers from the East China Universityof Science and Technology reported that the blue pigment tapsinto the trend for edible natural pigments [117]. Food makers haveincreasingly been looking for alternatives to artificial food colorssuch as sunset yellow, Tartrazine and quinoline yellow. While theEuropean coloring market faces an annual growth rate of just 1%between 2001 and 2008, the coloring food stuffs market is rippingahead on growth of 10–15%. Thus bacterial colorants in addition tobeing environment friendly, can also serve the dual need for visu-ally appealing colors and probiotic health benefits in food products[118].

Microbial colors are in use in the fish industry already, for exam-ple to enhance the pink color of farmed salmon. Further, somenatural food colorants have commercial potential for use as antiox-idants [15]. Nowadays some fermentative food grade pigmentsare in the market: Monascus pigments, astaxanthin from Xantho-phyllomyces dendrorhous, Arpink Red from Penicillium oxalicum,riboflavin from Ashbyagossypii, and carotene from Blakeslea trispora

[119] which are considered safe and approved by FDA. The suc-cessful marketing of pigments derived from microbes, both as afood color and a nutritional supplement, reflects the presence andimportance of niche markets in which consumers are willing to paya premium for ‘all natural ingredients’.

The number of approved colorants for food industry is limited.Some approved food colorants are known by their chemical name(canthaxanthin) while others are known by source (fruit juice orvegetable juice). The biocolorants identified by their chemical namecan be synthesized easily by cheaper biotechnological sources.Biotechnology may play a crucial role for large fermentation ofnatural biocolorants. Technological limitations are the major bot-tleneck for the commercial exploitation of the source materials. Thesuccess of any pigment produced by fermentation depends uponits acceptability in the market, regulatory approval, and the size ofthe capital investment required in bringing the product to market[15].

4.2. Applications in pharmaceutical industry

Most studies investigating microorganisms have shown the effi-cacy and the potential clinical applications of pigmented secondarymetabolites in treating several diseases and they also have cer-tain properties like antibiotic, anticancer, and immunosuppressivecompounds. Significant progress has been achieved in this field, andinvestigations of bioactive compounds produced by these microbesare rapidly increasing. As such, the number of compounds iso-lated from bacteria is increasing faster when compared with othersources [130].

Anthocyanins are involved in a wide range of biological activities[131] that affect positively the health properties and decrease therisk of cancer [112,113,132,133], reduce inflammatory insult [134]and modulate immune response [110].

The genus, Streptomyces or Serratia can produce a red sub-stance of pyrrolylpyromethene skeleton, which is one of followingsubstances: prodigiosin, metacycloprodigiosin, desmethoxy prodi-giosin, and prodigiosin 25-C. These substances have been knownto have an antibiotic and antimalarial effect, especially prodigiosin25-C that shows immunosuppressing activity [135]. Immunosup-pressive activity of prodigiosin was first described by Nakamuraand co-workers in 1989. These researches showed the presenceof prodigiosin and metacycloprodigiosin in culture broth of Ser-ratia and observed selective inhibition of polyclonal proliferationof T-cells as compared to that of B-cells. Besides that, the cyto-toxic potency of prodigiosin has also been investigated in thestandard 60 cell line panels of human tumor cells derived fromlung, colon, renal, ovarian, brain cancers, melanoma and leukemia.



Fig. 1. Structure of microbial food grade pigments [94].

Inhibition of cell proliferation as well as induction of cell deathhas been observed in these cell lines. In vitro anticancer activityhas also been reported for different prodigiosin analogs and syn-theticindole derivative of prodigiosin [136]. The antiproliferative

and cytotoxic effects of prodigiosin have been observed not onlyin cultured tumor cell lines but also in human primary cancer cellsfrom B-cell chronic lymphocytic leukemia patients [137]. The useof prodigiosin for treating diabetes mellitus has also been reported

Table 6Food grade pigments from bacteria.

Bacteria Pigment Color Applications Statusa

Flavobacterium sp., Paracoccus xanthinifaciens Zeaxanthin [120–122] Yellow Additive in poultry feed, strengthen yellowcolor of skin of animals, accentuate the color ofyolk of egg

DS

Streptomyces sp. Carotenoids [123,124] Yellow Food colorants, feed additive, coloration ofornamental fishes

DS

Photosynthetic bacterium, Bradyrhizobium sp.,Halobacterium sp.

Canthaxanthin [125,126] Dark red Food colorants RP

Agrobacterium aurantiacum, Paracoccuscarotinifaciens, Halobacterium salinarium

Astaxanthin [127–129] Pink-red Natural nutritional component, foodsupplement

RP

a RP, research project; DS, development stage.



Fig. 2. Benefits of bioengineered natural, ‘green dyes’.

by Hwanmook et al. [138] where prodigiosin was found to be anactive component for preventing and treating diabetes mellitus.

Violacein is produced by several bacterial species, includingthe Gram-negative species Chromobacterium violaceum, Janthi-nobacterium lividum, Pseudoalteromonas luteoviolacea, Ps. sp. 520P1and Ps. sp. 710P1 [139–141]. Violacein was reported to haveantiprotozoan [142], anticancer [143,144], antiviral [145], antibac-terial [146,147] and antioxidant activities [148]. Mojib et al. [149]described the antimycobacterial activity of two pigments, violacein,a purple violet pigment from Janthinobacterium sp. Ant5-2 (J-PVP),and flexirubin, a yellow-orange pigment from Flavobacterium sp.Ant342 (F-YOP) might be valuable compounds for chemotherapyof tuberculosis. These characteristics provide the possible applica-tions of violacein for therapeutic purposes [150]. Thus pigmentsfrom bacteria offer the wide range of biologically active propertiesand continue to provide promising avenues for applied biomedicalresearch.

4.3. Applications in textile industry

The textile industry produces and uses approximately 1.3 mil-lion tons of dyes, pigments and dye precursors, valued at aroundU$23 billion, almost all of which is manufactured synthetically.However, synthetic dyes have some limitations, primarily, (i) theirproduction process requires hazardous chemicals, creating workersafety concerns, (ii) they may generate hazardous wastes, and (iii)these dyes are not environment friendly.

Until the second half of the 19th century, all dyes used in textileswere naturally derived. However, with the synthesis of mauveineby Perkin in 1856, the synthetic dye industry has grown at a vigor-ous rate and all but totally eradicated the use of natural dyes. Thelarge number of synthetic dyes in use today bears witness to the cre-ativity and innovation of textile chemists in successfully satisfyingthe dyer’s demands for simple, reproducible application processes,and the consumer’s demand for quality products at a reasonableprice.

Biosynthesis of colorants (natural dyes) for textile applica-tions has attracted increased interests in recent years (Fig. 2)[151]. The currently used colorants are almost exclusively madefrom nonrenewable resources such as fossil oil. The productionof the synthetic colorants is economically efficient and technicallyadvanced with colors covering the whole color spectrum. However,synthetic colorants are facing the following challenges: depend-ence on non-renewable oil resources and sustainability of currentoperation, environmental toxicity, and human health concerns of

some synthetic dyes. Thus, biosynthesis of pigments through fer-mentation processes can serve as major chromophores for furtherchemical modifications, which could lead to colorants with a broadspectrum of colors [152].

Practically, fermentation of microorganisms such as fungi andbacteria could be a valuable source of manufacturing colorants.Microorganisms produce a large variety of stable pigments such ascarotenoids, flavonoids, quinones, and rubramines, and the fermen-tation has higher yields in pigments and lower residues comparedto the use of plants and animals [152]. Besides, some natural col-orants, especially anthraquinone type compounds, have shownremarkable antibacterial activity in addition to providing brightcolors [153], which could serve as functional dyes in producingcolored antimicrobial textiles.

Alihosseini et al. [154] characterized the bright red pigmentprodigiosin from Vibrio spp. and suggested that it could be usedto dye many fibers including wool, nylon, acrylics and silk (Fig. 3).Yusof [155] reported the capability of using pigment from Serratiamarcescens to color five types of fabric namely acrylic, polyestermicrofiber, polyester, silk and cotton using tamarind as mordant.However, the dyeing performances are different, depending onthe types of fiber. From the colorfastness testing, the dyed fabricsalso have the ability to maintain its color under several externalconditions such as perspiration, washing, and rubbing/crocking.Similar textile-dyeing ability was also reported for Janthinobac-terium lividum [101] and gave good color tone when applied onsilk, cotton and wool (bluish-purple, all natural fibers), and nylonand vinylon (dark blue, both synthetic fibers). Dyeing was per-formed by a simple procedure consisting of either dipping in thepigment extract or boiling with the bacterial cells. Color variationwas achieved by changing the dipping time and the temperature ofthe dye bath.

Ahmad et al. [5] characterized the red pigment prodigiosin(Serratia marcescens) and violet pigment violacein (Chromobac-terium violaceum) and tested its dyeing efficiency in differentfabrics i.e., pure cotton, pure silk, pure rayon, jacquard rayon,acrylic, cotton, silk satin and polyester. Their results suggested thatprodigiosin could be used to dye acrylic and for violacein intensecolorations was oberved in pure rayon, jacquard rayon and silksatin.

Ahmad et al. [5] observed the applications of prodigiosin andviolacein in batik making. “Batik” is a popular gown like dress wornmostly by woman in South East Asian region. The most popularmotifs include leaves, flowers and geometrical design. The chosenpattern was first drafted onto the fabric by a “Batik-Tulis” maker i.e.,



Fig. 3. Colored multifibers fabric with red pigments from Vibrio spp. strain KSJ45 [154].

the painter, using pencil. Then, melted wax (mixture of beeswaxand paraffin wax) was applied over the drafted motifs using a tech-nique called “canting”. The beeswax holds the fabric while paraffinwax will allow cracking, which is a typical characteristic of batik.Wherever the wax has seeped through the fabric, the dye will notpenetrate. After waxing process, the fabrics were dyed using theextracted bacterial pigments using the brushing technique. Thecolor tone was adjusted by adding either ethyl acetate (for red andpurple pigment) or acetone (yellow pigment). This was followedby immersing the fabrics into boiling water containing fixer (alum,iron sulfate, copper sulfate) to remove excessive wax as well as fix-ing the bacterial pigments onto the fabrics. The “batik” was then letto dry under mild sunlight (Fig. 4).

4.4. Applications in other aspects

Ahmad et al. [5] evaluated the potential of prodigiosin in col-oring candles, paper, soap and pencil case pouch and also testedthe potential of prodigiosin and violacein as ink in ball point penand high lighter pen (Table 7). Commercial candles (fluted andtranslucent) were placed in a beaker and heated until completelymelted before the addition of the bacterial culture broth. The mix-tures were homogenized and poured into the mold. The wicks wereimmediately placed into the center of the mold and the candleswere left to cool at room temperature for 1 h. Translucent candleshowed a more intense coloration compared to the fluted candle.To evaluate the potential application of prodigiosin in paper mak-ing, bacterial culture broth (3 mL) and one teaspoon of cornstarchwere homogeneously blended in one half of the thick pulp, whereasthe other half was not added with pigment (control). The pulp wasthen evenly spread onto a net to drain the excess water followedby 24 h drying [156]. The prodigiosin – dyed paper was exposed tosunlight and fluorescent light for 4 h while paper unexposed to light

Fig. 4. Applications of bacterial pigments – finished product of Batik making [5].

acted as control. Initial intense red coloration on the paper was sub-stantially reduced to light red upon exposure to both sunlight andfluorescent light, with sunlight exerting the higher fading effect.This effect can be attributed to wider range of light wavelengthsfor sunlight compared to the fluorescent light [157].

The bacterial pigments were evaluated for its potential role asink in ballpoint pen and highlighter pen. There are two types ofballpoint-pen ink namely oil-based ballpoint-pen ink and water-based ballpoint-pen ink. The basic components in ballpoint-peninks are coloring agent, solvent and resin. Dyes and pigments whichare soluble or dispersible in aqueous media can be used as coloringagents in inks. Besides the basic components, several other com-pounds were also added to the inks as additives that include aminederivatives as the pH-controlling agent or mildew-proofing agent,fluorine-containing surfactant that is responsible to increase sol-vent penetrability as well as defoaming agent, rust proofing agentand lubricant. The addition of shear-viscosity reducing agent suchas cross-linked acrylic resin and fatty acid metal salt can preventthe leakage of ink due to the gap between the ball and the tip whenthe pen is not used. Typical ink for the highlighter pen would consistof liquid vehicle, colorant and acidic buffer solution.

Liquid vehicle is the major component in the ink and is usedto carry the other highlighter ink component to the substrate.Liquid substrate can be of any liquid type including surfactant,solvent, co-solvent, buffer, biocide, viscosity modifier, stabilizingagent, complexing agent and water. To evaluate the role of bacterialpigments as ink in ballpoint pen, three types of tests were carriedout namely the Ink-Rubbing test, Ink-Follow test and Stability of Inktoward Light test. Similar tests were carried out for the highlighterpen except the Ink-Follow test was replaced by the Ink-Drop test.During the ballpoint-pen test, different ink compositions were pre-pared and the results clearly showed that color intensity increaseswith amount of pigment added. The violacein Ink 4 gave dark pur-ple while the prodigiosin Ink 4 resulted in intense red coloration.The use of excess solvents would result in smearing of the ink whichmay not be suitable for ballpoint application. The potential of viola-cein as ink for highlighter pen was evaluated by preparing differentcompositions of the ink in the presence of citric acid and glyceroland the ink was then used to highlight printed words. Based on thesmearing effect, glycerol performed better as solvent compared toethyl acetate. Although solvent can include any liquid capable totransfer the colorant and acid buffer to the substrate, a good solventis a liquid which can evaporate in short time and having low degreeof smear [5]. In fact, research work on pigments from bacteria needsto be intensified to suit its various industrial needs.

5. Future perspectives

The preference for natural colorants over synthetics started withthe green movement of the 1960s and shows no sign of decreasing.This may result from a perceived uneasiness with the safety of thecolorants on the part of the consumer, but another factor, perhaps



Table 7Other applications of bacterial pigments [5].

Applications Pigment Picture

Coloring candle Prodigiosin

Coloring paper Prodigiosin

Coloring soap Prodigiosin

Coloring pencil case and pouch Prodigiosin

Ink

Ballpoint Pen INK Prodigiosin

Violacein

Highlighter Pen INK Prodigiosin

Violacein

more important, is that most governments allow more flexibilityand leniency in the use of natural colorants. Production of colorsby fermentation has a number of advantages: cheaper production,possibly easier extraction, higher yields, no lack of raw materials,and no seasonal variations. There is an increasing interest involvingbacteria as a possible alternate source of colorants used in foods,textile, pharma etc. In this direction, biotechnology may play acrucial role for large fermentation of biocolorants.

Acknowledgements

The authors are thankful to the Ministry of Agriculture, Malaysiafor the Technofund grant (TF0310F080) and Universiti TeknologiMalaysia for the Post Doctoral Fellowship to Dr. C.K. Venil. Alsowe would like to thank Universiti Teknologi Malaysia’s ColorBacResearch team Nur Nazrina Ahmad Sabri, Nur Zulaikha Yusof, AliReza Khasim and Nordiana Nordin.



Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.procbio.2013.06.006.

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Bacterial pigments and their applications

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