Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Summary

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Abstract

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

Introduction

Melanins are polyphenol compounds whose monomeric unit is the indole ring. Melanins are responsible for most of the black, brown, and grey colorations of plants, animals, and microorganisms¹. The most common melanins are eumelanins, which are dark brown or black pigments widely distributed in vertebrates and invertebrates². Cephalopods, plants, and microbes are the major sources of melanin³. Melanin obtained from microorganisms has several advantages over melanin from cephalopods and plants. They are fast in growth and do not cause any problems of seasonal variations. Also, they modify themselves according to the growth medium and the operating conditions provided. Considering all these facts, the microbial sources are exploited as a major source of melanin with potential commercial applications^4,5,6. Guo et al. used recombinant Streptomyces kathirae SC-1 to produce melanin up to 28.8 g/L in an optimized medium⁷. Lagunas-Muñoz et al. Obtained about 6 g/L of melanin in recombinant E. coli⁸. Wang et al. obtained about 4.2 g/L of soluble melanin after medium optimization⁹. Researchers also investigated marine cultures to produce melanin. Kiran et al. produced melanin from marine Pseudomonas species¹⁰. Kumar et al. isolated Pseudomonas stutzeri HMGM-7 from marine seaweed, which produced melanin in nutrient broth. All the reported melanin producers require medium supplements, yeast extract and/or peptone for melanin production, which may hinder the scale-up of the bioprocess. Raman et al. investigated melanin production from Aspergillus fumigatus AFGRD105, which did not require costly additives¹¹.

A marine bacterium known as Pseudomonas stutzeri, isolated from seaweed, was selected for melanin production. This microorganism does not require additional supplementation of tyrosine and yeast extract for the production of soluble melanin and grows in seawater-based media, thereby reducing the potable water footprint and medium cost. Thaira et al. reported an optimized bioprocess using coconut cake meal for melanin nanopigment production using this marine bacterium in a previous publication¹². The produced melanin was used for the highly efficient removal of heavy metals from the synthetic groundwater. However, during medium optimization, plausible feedback inhibition of melanin on the growth and product formation was identified. Therefore, in this protocol, feedback inhibition of melanin is investigated and quantified using the generalized Han-Levenspiel model. A simple and effective adsorptive bioprocess protocol is developed to reduce feedback inhibition to the maximum extent.

Protocol

1. Growth and maintenance of Pseudomonas stutzeri culture. Obtain the bacteria Pseudomonas stutzeri from MTCC Chandigarh (supplied as freeze-dried ampoule). Disinfect the surface of the ampoule with alcohol (70%). Mark the ampoule near the middle of the gauge and break at the marked area. NOTE: Care should be taken in opening the ampoule as the contents are in vacuum. Take precautions while breaking the ampoule, as a snap opening will draw the cotton plug…

Representative Results

Figure 1A,B demonstrates growth and melanin production under the influence of different initial melanin concentrations. The cell growth and melanin production decreased substantially with the increasing initial concentration of melanin. At 500 mg/L initial melanin concentration in the medium, the biomass and melanin reduced to almost 50% of the maximum value obtained in control. At 1000 mg/L of initial melanin concentration, there was marginal melanin production. The biomass…

Discussion

Product inhibition is a major bottleneck in bioprocessing which leads to reduced productivity. Several methods exist to reduce product inhibition, such as continuous bioprocess and in situ product removal techniques. However, these options require a complete overhaul of the existing bioprocessing facility^{23,24,25,26,27,28<…}

Materials

Alumina+A15A26A3:A13A3:A16A3:A17	HiMedia	GRM1909
Activated carbon	HiMedia	PCT1001
Artificial Sea Water Medium	HiMedia	M1942
Bradford reagent kit	HiMedia	ML106
Celite	HiMedia	GRM226
Centrifuge	REMI	CM-8
Centrifuge tubes	HiMedia	PW1207
Dinitrosalicylic acid	HiMedia	GRM1582
Erlenmeyer flask	Borosil	4980021
fuller's earth	HiMedia	GRM232
Glucose	HiMedia	MB037
HCl	HiMedia	AS004
L-tyrosine	HiMedia	RM069
Microsoft Excel	Microsoft
Nanoparticle analyzer	HORIBA Scientific	Nanopartica SZ-100
Nutrient Agar medium	HiMedia	M001
Petri plate	HiMedia	PW054
Phosphate buffer	HiMedia	M1452
Sodium hydroxide	HiMedia	MB095
Spectrophotometer	Thermofisher	Genesys 10
Stirred tank bioreactor	Scigenics	Bioferm LS
TE Buffer	HiMedia	ML060
Transmission Electron Microscope	JEOL	JEM-2100
Zeolite	HiMedia	GRM3834