Team 3 Project Spring 2022

Introduction: 

E-waste is a term that describes electronic devices, containing harmful toxins, that are dumped into landfills every year. The disposal of e-waste is a problem because the world produces 50 million tons of e-waste each year (Caballar, 2020). The proper methods for disposing of this e-waste have not been implemented across the world yet, and the negative effects are evident. In places like Agbogbloshie, lots of the electronic waste is burned in an attempt to recover the copper and other precious metals they contain. This has severe negative health effects for the people involved. As stated by Michael Ackah, “Lead (Pb) contributed greatly to non-carcinogenic ingestion hazard quotient for residents living near Agbogbloshie and Ashaiman e-waste recycling sites while arsenic (As) presented carcinogenic risks to children from the dismantling area topsoils” (Ackah, 2019). However, electronic waste does not have to be burned to cause harmful effects. The leaching of e-waste describes the process of discarded technology polluting the ground through the absorption of toxic materials. Technology, such as, phones, computers, and laptops, contain toxic elements and materials. When they sit in a landfill, water runs through them and strips these harmful elements, which then seep into the ground. This leaching occurred, again, in Agbogbloshie. In the burn area, “Geoaccumulation indices indicated that the topsoils of the burn area and dismantling areas of Agbogbloshie e-waste recycling site were strongly contaminated by Pb and uncontaminated by Cr, Fe, As and Ba” (Ackah, 2019). The world will not be able to continue down this same path without encountering significant negative effects in some form. Implementing a more sustainable method for recycling e-waste is imperative. 

Microbial Solutions: 

Many companies have already begun searching for solutions to the e-waste problem. One promising method is the use of microbes to remove valuable and environmentally harmful components of the waste. There is a wealth of scientific evidence to support the utility of microbes in the extraction of rare earth and precious metals. Ilyas et al. (2021) list several microbes / processes that will help create a circular economy. A team of researchers at Idaho National Laboratory found that the bacterium Gluconobacter oxydans produces an acid that binds to and dissolves rare earth metals, allowing them to be separated from the rest of the e-waste. (Kwok, 2019). Garzon et al. (2021) explore the use of Leptospirillum ferriphilum to oxidize iron in an environment impregnated with e-waste, also discussing optimal conditions for microbial growth, which may be applicable to other microbial processes. Because of their value, gold and silver have been the target of several companies / studies. Mint Innovation has developed a process that uses the bacterium Cupriavidus metallidurans to absorb gold from e-waste (after various chemicals are used to get the gold in solution). A benefit of this method is that the bacteria physically takes in the gold particles, collecting them in larger amounts that are easier to separate than some other methods (Kwok 2019; Caballar, 2020). Researchers at the

National University of Singapore are exploring a process using Chromobacterium violaceum to produce hydrogen cyanide, which binds to gold (allowing for separation). C. violaceum also converts leftover cyanide into nontoxic β-cyanoalanine, reducing the negative effects of the byproducts. Another microbe, Delftia acidovorans, is used to precipitate gold nanoparticles out of the cyanide solution. (Kwok, 2019). Pseudomonas balearica SAE1 tolerates toxic, e-waste filled environments well and can also be used to extract silver and gold through a bioleaching process. (Kumar et al., 2018) 

Experimental Approach: 

Microbes found by researchers Dr. Jason Whitham and Dr. Amy Grunden (NC State University Plant and Microbial Biology Department) were being studied. These microbes were isolated from their environmental source, pokeweed and acid mine drainage, by performing serial dilutions. This process allowed the microbes of interest to be separated from soil particles. Glycerol was added to the isolates for stabilization, allowing them to be stored at -80 oC. Samples were then plated on Tryptic Soy Broth (TSB) plates at room temperature by Brenna Bilodeau (an undergraduate working under Dr. Claire Gordy in the NCSU Department of Biological Sciences). First, the mixed culture was plated, and then individual pure cultures were streaked on separate plates. Bilodeau and Dr. Gordy used the isolated microbe cultures and cultured individual microbes by putting them in liquid cultures with TSA and putting them in a shaking incubator at 25°C. These samples were grown with the intention to perform cell lysis (cell death) in order to isolate strands of DNA. After the DNA was collected, it was tested for purity and quantity using a Nanodrop and a Tapestation. The isolated DNA was then run through the NanoPore MinION sequencer to get the whole genomic sequence. Taking the data from the sequencer, Dr. Goller then used the GLC Genomics Workbench to assemble the genomic data. Dr. Carlos Goller is a part of North Carolina State’s Biotechnology program. He prepared the DNA sample of our microbial genus Beijerinckia, and then used the NanoPore MinIon sequencer to sequence its entire genome. In order for this data to be useful, Dr. Goller then had to use CLC Genomic Workbench to assemble the data previously produced. By examining the genome, we will identify potential genes with applicability to the e-waste problem. 

Microbe Species: 

For our study, we are going to focus specifically on the microbial genus Beijerinckia. This microbe has a wide variety of useful properties and has been found pertinent in nitrogen fixation (Haque et al, 2020). It was also proven useful in creating a biofilm barrier. The purpose of a biofilm barrier is to reduce hazardous substances in soil into harmless substances, and Beijerinckia was incredibly successful in removing toxic chemicals from groundwater (Lim, Lee, and Lastoskie, 2010). This function is impactful in resolving negative effects of e-waste as it removes harmful waste from the environment that could have been secreted by e-waste. Another integral property of Beijerinckia is its ability to rely on lanthanides for methanol oxidation, which is essential in removing methane and methanol from the environment and atmosphere and

keeping Beijerinckia alive (Wegner et al, 2019). Based on this information, it should have the potential to sequester these rare earth metals, such as neodymium, lanthanum, and cerium. Being able to isolate lanthanides in this way would be pertinent to solving the e-waste problem as it would isolate them from the environment and prevent inevitable damage. 

Future Directions: 

In the future, research can be done to determine which (if any) metal-sequestration properties are present in novel microbes. Both Bejierinkia and Ensifer species were isolated from pokeweed and acid mine drainage as described above, and the genome of Ensifer assembled well. The assembled genome was searched for genes that may provide this strain with properties useful to researchers. Both the ‘Prokka ann’ and ‘Rast’ tools of KBase were used to conduct this search. When searching for ‘metal’ using the Prokka ann tool, results included Metalloprotease TldD, Metal cation efflux system protein CzcD, ATM1-type heavy metal exporter, ATP-dependent zinc metalloprotease FtsH, and Putative metal chaperone YciC. The same search using Rast returned Metal-dependent hydrolase, Metal chaperone (involved in Zn homeostasis), Metal-dependent amidase/aminoacylase/carboxypeptidase, Conserved membrane protein in copper uptake (YcnI / Copper metallochaperone PCu(A)C, inserts Cu(I) into cytochrome oxidase subunit II), and Hypothetical metal-binding enzyme (YcbL homolog). A follow-up Rast search for ‘copper’ returned Lead, cadmium, zinc and mercury transporting ATPase (EC 3.6.3.3) (EC 3.6.3.5) Copper-translocating P-type ATPase (EC 3.6.3.4), and Cytoplasmic copper homeostasis protein CutC. Finally, a follow-up Rast search for ‘cobalt’ returned Magnesium and cobalt transport protein CorA, and Cobalt-zinc-cadmium resistance protein. Based on these genes, research can be done into the interactions of metal with Ensifer. There are several future directions of particular note. Metal-dependent pathways and metal charperones may mean that Ensifer sequesters and requires metals in its environment. Several enzymes were identified that may lead to metal uptake, such as the conserved membrane protein in copper uptake, the hypothetical metal-binding enzyme, the lead, cadmium, zinc and mercury transporting ATPase, and the magnesium and cobalt transport protein. Depending on the direction of this particular ATPase, bringing metal into the cell could result in the production of energy for the cell in the form of ATP. The cobalt-zinc-cadmium resistance protein also gives researchers a reason to believe that this Ensifer species can survive environments with above average concentrations of these metals. More research in this area is needed to support or refute the potential functions proposed based on the genome.

References

• Ackah. (2020). Soil elemental concentrations, geoaccumulation index, non-carcinogenic and carcinogenic risks in functional areas of an informal e-waste recycling area in Accra, Ghana. https://www.sciencedirect.com/science/article/pii/S0045653519314948?casa_token=n3lz cPEJZNsAAAAA:QeIMbNDygrnTAscNeIuohDMt352orkuaZY4_rJ0RE7iTV6oFIBIWn r85cSVEN-DNFGlXlo49bw 
• Caballar, R. D. (2020, May 4). A New Zealand startup is using microbes to suck solid gold out of E-waste. Medium. https://onezero.medium.com/a-new-zealand-startup-is-using-microbes-to-suck-solid-gold-o ut-of-e-waste-48814d425eef 
• Garzon, J. A., Hubau, A., Joulian, C., & Guezennec, A. (2021, August 18). Bioleaching of E-waste: Influence of printed circuit boards on the activity of Acidophilic iron-oxidizing bacteria. Frontiers. https://www.frontiersin.org/articles/10.3389/fmicb.2021.669738/full 
• Gordy, C., Grunden, A., Whitham, J., & Bilodeau, B. Microbe samples. https://docs.google.com/document/d/1xqKYLeBJnFfC5c0DeZtS8vdNo6Oc7u-NgI4FvGJk og4/edit# 
• Haque. (2020). Facultative methanotrophs – diversity, genetics, molecular ecology and biotechnological potential: A mini-review. PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660913/ 
• Ilyas, S., Srivastava, R. R., Kim, H., Das, S., & Singh, V. K. (2021). Circular bioeconomy and environmental benignness through microbial recycling of e-waste: A case study on copper and gold restoration. Waste Management, 121, 175-185. https://doi.org/10.1016/j.wasman.2020.12.013. 
• Kumar, A., Saini, H.S. & Kumar, S. (2018). Bioleaching of Gold and Silver from Waste Printed Circuit Boards by Pseudomonas balearica SAE1 Isolated from an e-Waste Recycling Facility. Curr Microbiol, 75, 194–201. https://doi.org/10.1007/s00284-017-1365-0. 
• Kwok, R. (2019, January). How bacteria could help recycle electronic waste. Proceedings of the National Academy of Sciences. 116, 711-713. https://doi.org/10.1073/pnas.1820329116 
• Li, J., Zeng, X., Chen, M., Ogunseitan, O. A., & Stevels, A. (2015). “Control-Alt-Delete”: Rebooting Solutions for the E-Waste Problem. Environmental Science & Technology, 49(12), 7095–7108. https://doi.org/10.1021/acs.est.5b00449 
• Lim, Lee, & Latoskie. (2010). Ability of Beijerinckia indica to degrade. ProQuest | Better research, better learning, better insights. https://www.proquest.com/docview/1943818036 Singh, N., & Li, J. H. (2015). Bio-extraction of metals as secondary resources from E-waste. Applied Mechanics and Materials, 768, 602-611. doi:http://dx.doi.org/10.4028/www.scientific.net/AMM.768.602 
• Sua, X., Shena, H., Yaoa, X., Dingb, L., Yuc, C., & Shen, C. (2013). A novel approach to stimulate the biphenyl-degrading potential of bacterial community from PCBs-contaminated soil of e-waste recycling sites. Bioresource Technology, 146, 27-34. https://doi.org/10.1016/j.biortech.2013.07.028. 
• Valdes, A. M., & Walter, J. (2018, June 13). Role of the gut microbiota in nutrition and health. The BMJ. https://www.bmj.com/content/361/bmj.k2179 
• Wegner. (2019). Lanthanide-Dependent Methylotrophs of the Family Beijerinckiaceae: Physiological and Genomic Insights. ASM Journal Platform. https://journals.asm.org/doi/full/10.1128/AEM.01830-19 
• Wei, Liu, et al. (2016) “Nano-Hydroxyapatite Alleviates the Detrimental Effects of Heavy 
  Metals on Plant Growth and Soil Microbes in e-Waste-Contaminated Soil.” Environmental Science: Processes & Impacts, 18(6), 760–767. https://doi.org/10.1039/c6em00121a.