Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway

Abstract

Many studies have reported microorganisms as efficient biocatalysts for colour removal of dye-containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR-1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox-active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin.

Figure 1

Effect of different electron donors on dye reduction by S. oneidensis MR‐1R. Decolorization activity is indicated as per cent of unreduced dye that remains in solution, with formate (●), lactate (○), pyruvate (▪), acetate (◆), citrate (◊), glucose (□), no donor (▴) or no cells (▵). Error bars represent the data range for triplicate cultures.
A. Decolorization of 1% LRB.
B. Decolorization of 1% RBB.

Figure 2

Differences in decolorization rates of (A) 0.1% LRB and (B) 0.01% RBB by MR‐1R and insertion mutants. The legend next to (B) also applies to (A). M3174 is not included in (A) as its phenotype was exactly the same as for MR‐1R. For clarity, the following colour code is used: black, MR‐1R controls; grey, T2S; red, cytochrome cmaturation; green, periplasmic and OM proteins; blue, CM components, and orange, other. Error bars represent the data range for triplicate cultures.

Figure 3

Comparison of the decolorization rates of (A) 0.1% LRB and (B) 0.01% RBB between MR‐1R (●) and mutants MTRF (□), OMCA (▪), OMCB (○), MTRF/OMCB (▴) and OMCA/OMCB (▵).

Figure 4

The contribution of melanin in the reduction of RBB. The melanin‐free (mutant 4HPPD) and melanin‐producing MR‐1R cells exhibit reduced decolorization rates compared with the melanin‐overproducing strain. The latter was pre‐grown in the presence of excess tyrosine (2 g l⁻¹).

Figure 5

Model for the decolorization mechanism by S. oneidensis. The arrows indicate the possible routes of electron transfer to the extracellular dye RBB resulting in colourless reduction products. The thick arrows represent the principal pathway starting from formate to OmcB via formate dehydrogenase (FDH), menaquinone (MQ), CymA and MtrA. All cytochromes c are shown in red. The type II secretion system is not included in the scheme. The contribution of the hypothetical protein encoded by SO_1913 is unknown. See text for details.