Sequencing Pseudomonas kunmingensis and Its Potential For E-Waste Reduction

Project Audio Recording



Electronic waste is defined as the electronics that have lost most of their use due to old age, damaged or being outdated and obsolete and as a result are thrown out as waste products (Gao). Some examples of electronic waste are televisions, monitors, batteries, circuit boards, and plastic casings (Gao). E-waste generation has become a large problem as a result of the fast-paced world we live in right now and the high demand of new, faster technologies (Andeobu). “44.7 million metric tonnes generated in 2016, up 8% from 2014” (UNU). And since e-waste is hard to recycle, most of it ends up in landfills, mostly in impoverished areas in Africa and Asia. Some countries and companies send their e-waste under false pretenses to third-world countries to save money on recycling, making e-waste accumulation worse (Toxic E-Waste Dumped).

One of the main problems with e-waste is that the toxic metals and chemicals in e-waste can leach into the local environment from the landfills, potentially poisoning water sources and the soil around it (Andeobu). The leaching can cause disastrous effects on the local wildlife as they might ingest hazardous chemicals, which can bioaccumulate inside them (Canada). And eventually, all these dangerous chemicals end up in the human body system when we consume animals that contain dangerous levels of chemicals and metals due to bioaccumulation. Additionally, the common disposal method of burning creates ingestion via smoke accumulating as neurotoxins in the brains of workers disposing of e-waste. According to an article by CNR “The burning of e-waste is associated with lung and neurological diseases as well as many types of cancer” (Adogla-Bessa, 2018). Additionally, “combustion from burning of e-waste creates fine particulate matter, which is linked to pulmonary and cardiovascular disease (Jin et al., 2015; McAllister, 2013).

Some microbes that live in harsh environments, such as acid mines or electronic waste sites, can break down metals. This natural process is known as “bioleaching” (Kwok). These microbes can be harnessed to create a solution to electronic waste. One way that microbes break down metal is that they “chemically modify the metal, setting it free and allowing it to dissolve in a microbial soup, from which the metal can be isolated and purified (“These Microbes Are Being Used to Clean Up E-Waste”). “One microbe that has been used to process e-waste is C. violaceum. Microbes have different ways of breaking down metal, from leaching metals to binding and dissolving them (Kwok). Overall, certain microbes have important properties that can be used in solutions to minimize the dangers of electronic waste. 

This microbe species was collected in a metal-rich environment from Dr. Amy Grunden from NC State. After collection, the bacteria was grown on agar plates to be prepared for streaking and isolation of a colony. It was then isolated by streaking on tryptic soy agar plates and later, into BUG agar to be prepared for sequencing. To begin sequencing this microbial organism a review of pipetting skills through the usage of color-dyed water was a recommended tool. Additional pre-sequencing information crucial to the sequencing experiment was how to streak on a plate with agar. We practiced streaking and then deposited a selection of our microbe onto an agar plate to be dispersed. The desired outcome from the streaking of our microbial species on the plate was to isolate colonies to create a better idea of an individual species. This formation of discrete colonies was achieved through multi-directional streaking. After waiting for a while our microbe species started to flourish on some of the plates. Afterwards, some of the individual colonies were put into Biolog Gen III Microplates to measure the phenotypic metabolic rate through observing the intensity of the purple color in each well of the Biolog plate. The next step was to lyse open the bacteria to isolate DNA. This was accomplished via the enzyme lysozyme and through purifying the genetic information using a High Molecular Weight DNA Extraction Kit from the New England BioLabs. After being stored on ice and adding a buffer the HMW (High Molecular Weight) gDNA binding and elution is achieved by using: DNA Capture Beads, Isopropanol, gDNA Wash buffer, and gDNA Elution Buffer in collection tubes that are to be centrifuged. Next, the goal was to wrap the DNA in a solution in a plastic tube around glass beads. After removing the liquid and adding a buffer wash through repetitions, the beads and DNA were separated. We then centrifuged and incubated the solution to prepare for the sequencing of the species’ genome. Next, we needed to assess the size of the DNA and quality. We did this using a Nanodrop Spectrophotometer, Qubit Fluorometer, and TapeStation Gel Electrophoresis System. These devices all gave us data that allowed us to assess the integrity of our DNA. Next, our instructors did a PCR amplification of the 16S gene. This allowed them to identify the bacterial species of our microbe. Lastly, we sequenced our bacteria using the Oxford Nanopore Technologies (ONT) MinION. We also used the ONT Rapid Barcoding Kit so we could sequence multiple DNA samples in one flow cell. We connected the flow cell to MinKNOW software. The MinKNOW software came back and told us that the total length of reads for our isolate was 5,274,261 base pairs. 3 contigs were identified once the sequencing was complete. 

Microbe 102 was sequenced and identified as Pseudomonas kunmingensis. Pseudomonas kunmingensis has been studied before and identified as an exopolysaccharide-producing bacterium that had been extracted previously from a phosphate mine and studied (Xie, 2013). This gram-stain-negative, aerobic bacterium has previously been used in wastewater treatment as an agent to remove ammonia nitrogen which is increasingly being used in practices like agriculture resulting in eutrophication. This microbe species produces yellow-orange colonies with a smooth surface (Zhang, 2015).  Our colony was collected in a metal-rich environment from Dr. Amy Grunden from NC State. This metal-rich environment was an acid mine drainage and pokeweed site. After sequencing the genome apps used to assemble the genome were minKNOW, Unicycler, Prokka, GTDB-Tk and Basic Local Alignment Search Tool also called BLAST.

Genome sequencing required the usage of the Ligation Sequencing Kit (SQK-LSK112) with ONT Rapid Barcoding Kit, ONT Flow Cell Priming Kit, minION FLO-MIN 106 R9.4.1. Flow cells, sequencing auxiliary kit. Furthermore, genome sequencing involved DNA repair, end-prep, adapter ligation and cleanup before loading into the minION flow cell which then ran the samples in the flow cells. These steps were achieved using a P200 pipette mixed with 30 microliters of thawed and mixed flushed tether with a new tube of thawed and mixed flush buffer and fortex. (Goller Sjogren, 2022). The solution was inserted using a P1000 pipette into a priming port. 800 microliters of mixed FB and FLT were pipetted. 200 microliters of the priming mix was placed into the flow cell via the priming port. The library was loaded next which involved using 400 nanograms of genomic DNA and nuclease-free water, fragmentation mix and was incubated at 30 degrees celsius for 1 minute then 80 C for one minute in the thermocycler. The size of microbe 102 was determined to be 5,274,261 base pairs with 3 contigs. 1 contig is the length of 4,545,348, 2 is 438,347 and the last one is 290,566 base pairs. There are 5929 genes predicted with 5848 protein coding genes. 

The microbe pseudomonas kunmingensis, could attach to copper and zinc. This was found using the Annotate Assembly and Re-annotate Genomes with Prokka. This allowed for it to be deduced based on its ability for ATP to allow this microbe to metabolize copper and zinc for their benefit. They can survive in cobalt, magnesium, and cadmium-rich environments. This suggests these microbes can grow in environments with waste products with cobalt, magnesium, and cadmium. Furthermore, it is suggested that if in an environment with amounts of copper and zinc these elements are presumed to be used as sources for ATP production. Therefore these elements are consumed and able to be utilized as reduction agents of the compounds in environments they accumulate and negatively affect ecosystems in. For example, microbes in areas of car waste and motor oil would be presumed to have zinc processing abilities due to the presence of that element and the survival of the microbe. This idea is applicable in waste production that Pseudomonas kunmingensis processes coding proteins for. For example, Pseudomonas kunmingensis possesses a cobalt-zinc-cadmium resistance protein CzcA which suggests to us the ability for resistance and utilization in environments with these elements. An area that possesses copper as a byproduct is metal mining. Therefore, we believe that this microbe could survive, reproduce, and reduce the presence of copper. This idea is also replicated for cadmium reduction in sewage sludge waste. Pseudomonas kunmingensis is believed to have genes for processing these elements like ATP-dependent zinc metalloprotease FtsH, putative copper-importing P-type ATPase A and Magnesium and cobalt efflux protein CorC.

Graphical Abstract

KBase Narrative Link

KBase Narrative

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