Description

Biodegration and degradation of methylene blue dye by S. elongatus

Inspiration

We were inspired by our previous study on the phytoremediatory abilities of aquatic plants native to Hong Kong. P. fluitans is a free-floating aquatic plant with multiple roots, which is a popular product for aquarium maintenance locally. We observed that the leaves and roots of P. fluitans growing in our school lab became dyed with colour after a few days placed in a dye solution, indicating that it did not possess the biodegradative ability of chemical dyes. Thus, we were intrigued by the prospect of investigating and improving the phytoremediatory capabilities of other aquatic plants and bacteria species.

Background

The synthetic dye industry is a important driving force behind a diverse variety of fields, such as the textile, light, and chemical industries. In particular, the textile industry is a main consumer of these synthetic dyes - 10,000 tons out of the 7 × 107 tons of synthetic dyes produced worldwide are used by textile industries [1].

China is widely regarded as the “world's factory”, and the name holds true for its role in the dye and textile industries. In terms of annual production, export, and retail, China firmly holds first place in the world textile industry. The sector has experienced consistent economic growth over the last decade, now producing nearly 65% of the world’s clothing [5]. In the dye industry, its fuel production and trade volume rank first in the world [9].

However, this economic prosperity has a dark side. The amount of waste water discharged by the textile industry is as high as 900 million tons per year, accounting for the sixth place in industrial discharge [12]. Untreated textile wastewater contains a high concentration of toxic chemical dyes and heavy metals discharged as effluent, which contaminate local waters, causing serious pollution and posing a serious risk to aquatic ecosystems and human health.

Current treatment methods for industrial wastewater include physical methods, such as filtration, flocculation and adsorption; or chemical methods, like oxidation and ozonation. However, the water-soluble and stable properties of synthetic dyes make them difficult to be removed from wastewater using simple treatment methods [11], and the possible generation of toxic intermediates raises concerns on ecofriendliness. These methods also require expensive machinery and chemicals, which are unaffordable for many small scale factories and companies [11].

Current treatment methods for industrial wastewater

Bioremediation is a burgeoning field of research that has emerged as a promising biological method to treat wastewater. Using various organisms and microbes such as plants, algae, and bacteria, contaminants of soil and water are detoxified or eliminated.

Project

Methylene blue (MB)

Is a thiazine dye under polymethine dyes. It is the most common and highest-consumed dye in the textile industry, and is considered one of the most popular clothing colourants [12]. Worryingly, it is toxic, carcinogenic, mutagenic, and can be fatal to humans at concentrations over 5 mk/kg [11]. It is also water-soluble and forms a highly coloured sub-product in water, which decreases sunlight transmittance and oxygen solubility in aquatic environments. This affects the photosynthetic and respiratory activity of aquatic life, and has dire impacts on biodiversity. It is difficult to degrade by traditional means as it is thermal and light-stable.

S. elongatus

S. elongatus is a species of cyanobacteria, which have long been considered promising candidates for microbial cell factories. They rely on a cost-effective minimal growth requirement; CO2, light, water, and minimal nutrients. Their photosynthetic nature also makes them an attractive green option for the atmospheric reduction of CO2 on top of biosynthesis of the proteins of interest. S. elongatus also possesses a high salt and metal tolerance, allowing them to be the optimum chassis for microbial remediation. We hope to improve its bioremediative ability by engineering it to express homologous ligninolytic isozymes characterized in other species.

Enzymes

Laccases (Lac) belong to the enzyme family of multi-copper oxidases (MCOs) and can be obtained from a wide variety of species such as bacteria [12], brown and white-rot fungi [14], and various plant and algae species [2]. Together with lignin peroxidase (LiP) and manganese peroxidase (MnP), this enzyme combination has displayed the ability to catalyze the breakdown of various inorganic and organic substances such as wood, plastic, paint, and jet fuel into nutrients in many species (Viswanath et al., 2014), and have been proven to be the main enzymes responsible for catalyzing the degradation of MB [12].

System

We aim to design plasmids with inserts of homologous variants on these three genes from species such as B. thuringiensis [12] and T. pubescens [14] . The plasmid will first be transformed into DH5a E. coli for cloning, then electroporation for homologous recombination-based genomic insertion will be performed based on a protocol by Omayra et al. (2022) into the genome of S. elongatus. Expression of our vectors will be confirmed by PCR and MB-degrading activity will be quantified by spectrophotometric analysis.

MB degraded metabolites' toxicity has been found to be lower than that of the parent compound [12]. To investigate the toxicity of the degradation products, which have been proposed to be aromatic amines, raw and treated EC50 values will be used for comparison

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