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5.1 Introduction

Phenazines cover nitrogen containing colored redox active heterocyclomers of biological and chemical origin. More than 6,000 phenazine derivatives have been described with one or other bioactivity (Mavrodi et al. 2006; Pierson and Pierson 2010). However, nearly 100 natural phenazines were reported exclusively from bacteria of diverse vicinity. Based on the types and position of functional groups present on structure, phenazines were known for a long time as pigments and antifungal/antibacterial compounds (Schoental 1941; Haynes et al. 1956; Mann 1970).

Chemical synthesis of phenazines renders toxic chemicals including, aniline, azobenzoate, lead oxide, o-phenylenediamine which are potentially toxic as well as this process, (1) shows relatively less productivity, (2) needs harsh reaction process, and (3) produce toxic by-product. Whereas, selective phenazine synthesis can be possible using bacterial fermentation with added advantage of noncytotoxicity and growth limiting potential for eukaryotes, hence can be used as effective therapeutic agent for eukaryotic organisms (Laursen and Nielsen 2004). Also natural phenazine derivative have proven to be the more effective biocontrol agent than synthetic one (Nansathit et al. 2009). Thus, natural phenazine can always be a choice of selection for wide applications.

Phenazine production has been studied in different bacterial strains including florescent Pseudomonas (Mavrodi et al. 2006; Maddula et al. 2008; Li et al. 2008; Shanmugaiah et al. 2010), Streptomyces sp. (Gebhardt et al. 2002; Clinton et al. 1993; Zendah et al. 2012; Ohlendorf et al. 2012; Fotso et al. 2010; Kondratyuk et al. 2012), Bacillus sp. B-6 (Kim 2000; Li et al. 2007), Brevibacterium (Podojilt and Gerber 1967), Burkholderia (Mavrodi et al. 2006), and archae Methanosarcina mazei Gö1 (Abken et al. 1998; Beifuss et al. 2000) and few others. Among all phenazine producers Pseudomonas sp. and Streptomyces sp. have been studied at metabolic and genomic level. It was observed that the structural complexity of phenazines increase from Pseudomonas sp. to Streptomyces sp. (Saleh et al. 2009) and Methanosarcina mazei (Beifuss et al. 2000) as long side chain where it served as final electron acceptor in electron transport chain (Abken et al. 1998; Beifuss et al. 2000). The change in structure related to change in phenazine properties.

The extra ordinary potential of phenazines is due to their physicochemical properties, i.e., oxidation–reduction (redox) and their bright pigmentation, which changes with pH and redox state (Pierson and Pierson 2010). These prosperities have been used for biotechnological applications such as (1) biocontrol agent (Rane et al. 2007a, b), (2) microbial fuel cell (Torres et al. 2010; Sanderson et al. 1987), (3) organic light emitting devices (OLED) like phenanthroline-fused phenazine (Chen and Xiao-Chang 2004), (4) antitumor agent (Laursen and Nielsen 2004; Mavrodi et al. 2006; Kondratyuk et al. 2012), (5) biosensor like glucose sensor (Ohfuji et al. 2004), H2O2 (Santos et al. 2005), (6) biocolor for dyeing silk fabrik (Saranya et al. 2012), (7) mineral reduction (Hernandez et al. 2004), (8) oil degradation (Norman et al. 2004), (9) anticandidal (Morales et al. 2010), (10) food colorent (Saha et al. 2008) etc.

5.2 Phenazine Production

Broad range of bioactivity and applicability left impact on researcher to increase the productivity of phenazine from laboratory to fermentation scale production using potential phenazine producer. A wide variety of phenazines are biosynthesized by bacteria are given as follows.

5.2.1 Pseudomonad Phenazines

Pseudomonas is foremost phenazine producing bacteria with almost one-third of all known phenazines. Among all bio-chemo origin phenazines, pyocyanin was the first isolate one by Fordos in the 1850s from isolated Pseudomonas. Till date, fluorescent pseudomonads are the best studied phenazine producer which includes Pseudomonas aeruginosa, P. fluorescens, and Pseudomonas chlororaphis. Production profiling of P. aeruginosa, include (1) phenazine-1-carboxylic acid (PCA) (Rane et al. 2007a, b; Mavrodi et al. 2006); (2) phenazine-1-carboxamide (PCN) (Shanmugaiah et al. 2010), (3) pyocyanin (PYC) (Ra’oof and Latif 2010; Kaleli et al. 2006); (4) 1-hydroxyphenazine (1-OHPHZ) (Kerr et al. 1999), (5) Aeruginosin A and B (Holliman 1969; Herbert and Holliman 1969), etc. While, P. chlororaphis was reported for production of (1) PCA, (2) orange-colored 2-Hydroxyphenazine-1-carboxylic acid, (3) brick red-colored 2-Hydroxyphenazine. More than one phenazine derivative can be produced by P. aeruginosa and P. chlororaphis depending upon the genetic and environmental makeup (Mavrodi et al. 2006). P. fluorescens have found to produce only PCA production (Mavrodi et al. 2006). P. aeruginosa is the only known species capable of producing the very distinctive water-soluble pigment pyocyanin (Gohain et al. 2006), however pyocyanin negative P. aeruginosa strains are also reported (Mavrodi et al. 2006).

5.2.2 Streptomyces Phenazines

A vast diversity of phenazines was noticed in Streptomyces sp. like endophenazines (Gebhardt et al. 2002), diphenazines (Ding et al. 2011), phenazinomycin, D-alanylgriseoluteic acid (Giddens and Bean 2007), Geranylphenazinediol (Ohlendorf et al. 2012), esmeraldin, and saphenamycin (Clinton et al. 1993), also been studied till the date.

Apart from these major groups’ phenazines like pelagiomicins A/B/C, myxin, PCA have been reported from Pelagiobacter variabilis, Sorangium sp. and Bacillus sp. respectively.

5.3 Phenazine Regulation and Environmental Factors

Phenazine biosynthesis is based on the phz gene expression, which turn on or off and allowing control of phenazine production (Linares et al. 2006). Different nutritional (carbon and nitrogen), metal (iron and phosphate), and environmental/process (pH, oxygen) parameters were found to regulate phz gene expression (Slininger and Jackson 1992; Slininger and Shea-Wibur 1995; Siddiqui and Shaukat 2004; van Rij et al. 2004).

Many reports had suggested, growth rate and phase-dependent phenazine production in pseudomonads, i.e., maximum phenazine production at late exponential and early stationary growth phase, while comparatively less product accumulation in early and mid-exponential phase (Chin-A-Woeng et al. 2001). With most important concern, quorum sensing, cell density dependant genome regulation are the most influencing factor for PCA and PCN production in P. aureofaciens (Pierson et al. 1995) and PCN in P. chlororafis (Chin-A-Woeng et al. 2005), respectively. The PCA molecule is thought to be the precursor for all other phenazine derivative (Gohain et al. 2006). Addition of exogenous PCA, as a precursor molecule in fermentation medium showed enhanced phenazine production in P. chlororaphis GP72, postulating that exogenous PCA may act as final electron acceptor and autoinducer providing more energy for bacterial growth and metabolite production (Huang et al. 2011).

5.4 Nutritional Requirement for Phenazine Production

Media components of production media specify the productivity of the metabolite during fermentation. Pierson and Pierson (2010) had suggested phenazine production is depending upon the nutritional condition. Hence, qualitative and quantitative effect of nutrients in production medium has to be optimised during process optimization studies. Depending upon the type of phenazine producer nutrient condition was found to be changing. Although phenazine production is aged process, however, the assessments of its nutritional as well as fermentation parameters for its production are still not well documented.

5.4.1 Nutritional Factor for Pseudomonad

Different media were studied earlier for pseudomonad phenazines production like (1) Pigment Production Medium D (Kluyver 1956), (2) Synthetic medium (Chang and Blackwood 1969), (3) Alanine medium (Frank and DeMoss 1959; Meyer and Abdallah 1978), (4) 1 % Casamino Acids-salts medium (Whooley and McLoughlin 1982), etc. Detailed of these media (Table 5.1) suggests requirement of sodium and potassium metal requirement during phenazine fermentation. Similarly, except 1 % Casamino Acids-salts medium other media comprising glycerol nutrition for phenazine production.

Table 5.1 Phenazine production media and producer
Full size table

Yuan et al. (2008) showed glucose and soytone as influencing factors for PCA production in Pseudomonas sp. M-18q using Plackett Burman design (PBD) where, increasing the glucose concentration and decreasing the soytone concentration result in increasing the accumulation and secretion of PCA in growth medium (Yuan et al. 2008). Similarly, effect of carbon and nitrogen source on gacA-deficient Pseudomonas sp. M18G mutant suggest the glucose as influencing carbon source; likewise yeast extract (0.28 %) was found most influencing factor for growth and soy peptone for maximum PCA production (He et al. 2008). The PCA biosynthesis at optimised conditions in Pseudomonas flurescence 2–79 have found to be accelerated by glucose as carbon source with unnoticeable influence of nitrogen source (Slininger and Shea-Wilbur 1995). Similarly, zinc sulfate, ammonium molybdate, and cytosine as micronutrition had shown increased PCA biosynthesis in P. flurescence 2–79 (Slininger and Jackson 1992).

Effect of individual amino acids on PCN production studied in P. chlororaphis PCL1391 illustrated the influence of aromatic amino acids and casamino acids (van Rij et al. 2004). In another study, Fusaric acid, a self defensive molecule of Fusarium oxysporum was shown to suppress the PCN production which can be overcome by presence of phenylalanine in P. chlororaphis PCL1391 (van Rij et al. 2005).

Labeyrie and Neuzil (1981) showed enhanced growth rate as well as enhanced pyocyanin secretion of P. aeruginosa A237 in amino acids (tyrosine and phenylalanine) supplemented media. The concentration of glycerol and paraffins was found to stimulate the production of pyocyanin and phenazine derivatives in P. aeruginosa (MacDonald 1967). The selective and increased production of PCA and PCN was achieved by Byng et al. (1979) using m or p-aminobenzoic acid as selective inhibitor of PYC specifically inhibit of phenazine methylation.

5.4.2 Nutritional Factors for Streptomyces

As like pseudomonad phenazines various media were exploited for Streptomyces phenazine production which mainly includes, (1) soybean–mannitol medium (Ding et al. 2011), (2) GOT medium (Fotso et al. 2010), (3) M2 medium (Zendah et al. 2012), (4) SPD medium (Ohlendorf et al. 2012), (5) TSB 10, modified Trypticase Soy broth (Mitova et al. 2008), (6) GYM1 (Mitova et al. 2008), (7) GYM2 (Mitova et al. 2008), (8) M1 (Mitova et al. 2008), (9) LB (Mitova et al. 2008), (10) MB (Mitova et al. 2008). In all studied media (Table 5.1) a complex protein source was used like casein peptone, soy protein digest, yeast extract, and oatmeal. The change in streptophenazine biosynthesis in presence of antibiotics like, i.e., tetracycline and bacitracin in fermentation medium suggest two- to threefold increase in synthesis (Mitova et al. 2008). In marine Streptomyces sp., subinhibitory concentrations of antibiotics were found to enhance and modulate the production of new phenazines, i.e., streptophenazines A–H (Mitova et al. 2008).

The phenazine production media for Bacillus sp. as mentioned in Table 5.1 by Kim (2000) and Marine broth (Li et al. 2007) stipulate iron (Ferrous) requirement for phenazine secretion apart from other nutrition factors. Phenazine production using Bacillus strain 39 deep sea sediment isolate, has also been studied in marine broth by Li et al. (2007).

Very few reports have been seen in case of methanophenazine (Abken et al. 1998; Beifuss et al. 2000) where a complex media (Table 5.1) was tried for phenazine production from Methanoscina mazei Gö1.

5.5 Fermentative Conditions of Phenazine Production

Environmental and process parameters, i.e., temperature, pH, and dissolved oxygen, respectively affect growth and hence secondary metabolites secretion. Change in pH condition, i.e., from neutral to slight acidic or alkaline minimizes the phenazine production while phenazine production at pH 7 has given considerable phenazine yield, while optimum pH-control during late phase of fermentation has been studied with effective PCA production (Li et al. 2010). pH and temperature sensitive PCA production was detected in P. flurescence 2–79, where optimum productivity was recorded at pH 7 and 25–27 °C with greater cell density (Slininger and Jackson 1992).

Influenced of abiotic environmental factors, i.e. pH, O2 exchange and temperature on PCN production in P. chlororaphis PCL1391, showed PCN productivity was increased at 1 % oxygen and at low magnesium concentrations, while noticeable decrease observed at pH 6 and temperature 16 °C. Also, production of autoinducer during cell growth in pseudomonas is influenced by cell density, which directly affects the PCN production positively by increasing the PCN yield during fermentation process (Rij et al. 2004; Mavrodi et al. 2006).

In current scenario insufficient known knowledge is available about DO requirement for phenazine production during fermentation. However, reports claimed that the higher productivity of PCA fermentation yielded at 20 % of DO with optimized agitation and aeration condition. However, increased in DO (50 %) drastically decrease the PCA yield during fermentation, which might be due to cell lysis caused by increased agitation condition and unsuitable pH (Li et al. 2010).

5.6 Phenazine Productivity

PCA production in Pseudomonas sp. M18G gacA mutant showed 30-fold increase productivity from 0.02 to 0.6 gL−1 compared to the wild-type strain (Ge et al. 2004). With optimum glucose and yeast extract in fermentation media increased PCA production from 673.3 up to 966.7 μgmL−1 in Pseudomonas sp. M18G gacA mutant (He et al. 2008). The same strain at optimized fermentation conditions after 60 h of incubation showed 1.89 gL−1 of PCA production (Li et al. 2008). Rane et al. (2007a, b) had recovered 18 gm of crystalline PCA at large-scale fermentation (125 L working volume in synthetic medium) from P. aeruginosa ID 4365. Phenazine production in Bacillus sp. B-6 yielded 400 μgmL−1 PCA in chemically defined medium (Kim 2000).

5.7 Conclusion

Characteristics of phenazine and its derivative opened its applicability in different biotechnological segments. Current research in evaluation of new properties and its exploitation of same warrants economic fermentative production of phenazines. In the same regard, this chapter rationalizes the factors influencing phenazine fermentation. To a great extent, nutritional and environmental factors supporting growth as well as phenazine production have been discussed in different Pseudomonads and Streptomyces strains. The future debate on alone phenazine gene expression will certainly lead to techno economic production of selective phenazines.