Classifying the Genome of Bacillus Mobilis for Electronic Waste Recycling

Introduction:

 Electronic waste is any product containing electronic components that is at the end of its ideal usable life (2,3). Over the years, the world is beginning to accumulate more and more e-waste which is being dumped into mass landfills (2,3). Just in 2019 alone the world produced 53.6 million tons of waste, which is expected to double in the next 15 years (2). The main contributor to this issue is smaller electronics such as cell phones, which are being produced to have shorter lifespans and difficult to replace parts. With the United States being a hub for technological advancements and products of the future, it is also a major contributor to the electronic waste problem and exporting this waste to underdeveloped nations (5). Many companies and government institutions claim to recycle e-waste, but it has been found that 40% of what is said to be recycled is actually exported to developing countries (2). Although it does not seem like a concern for most people in these developed nations since they do not openly see in front of them the effects this has on the environment and public health, the implications are vast. There are many detrimental consequences as a result of improper electronic waste disposal. In terms of the environment, these heaps of e-waste have resulted in various toxins and heavy metals leaching into the soil via rainwater, causing metals like mercury and lithium to pollute the ground (2). Furthermore, these toxins released from the metals can bioaccumulate in the air when they are burned off, like dioxin gasses for example, thus creating a public health concern (5,4). As people increase their exposure to these toxins, it increases their risk for developing diseases such as cancer and neurological disorders (5). These predicaments could be managed and avoided with the implementation of better control systems to filter and recycle old electronics, however these methods can be cost effective. Since the cost of e-waste recycling is more than the revenue recovered from the wasted materials, it makes the environment and public health less of a priority for developing countries to implement a solution.

Microbial Solutions: 

Synthetic biology is one of the foundational elements of all microbial solutions. Synthetic biology focuses on altering organisms to be useful to us by giving them new or enhancing their abilities (10). And this is the most commonly used method when it comes to testing and using microbes for metal absorption (6). This is important because most of our studies on if microbes and bacteria can be used against waste and pollution involve it (6). An example of this being when Dr. Zhu and his group created bacteria specially designed and augmented to remove cadmium (1). These cells have the ability to remove cadmium with very high efficiency which is important as cadmium is lethal to humans and animals (1). Hence, synthetic biology is a crucial component of microbial solutions to e-waste. More examples of solutions using synthetic biology are explained below: 

• Extracting using stirred-tank reactors where certain bacteria are mixed in with objects to leach out the gold (1). 
• Mint Innovation’s use of a combination of chemical and biological resources to extract precious metals (i.e. gold, cobalt, copper, etc.) from e-waste. Mint’s uses a multistep process utilizing bacteria for biosorption and bioleaching metals to clump together the metals that were dissolved in the extraction process which uses their proprietary chemical solution (1, 7). 
• Bioprecipitation to remove metal from water streams instead of chemicals. This method has the potential benefit of not changing the pH of water and removing the bacteria fairly simply and easily (6). 

Experimental Approach:The microbes being studied were collected from an acid mine drainage site in collaboration withNC State researchers Dr. Amy Grunden and Jason Whitham. Soil was taken from areas of about room temperature where pokeweed plants grew and a culture of different microbes were isolated. From here, the microbes were cultured in Tryptic Soy Broth (TSB) and agar (TSA) plates and streaked in order to form singular colonies. The first characterization of the microbe was a Biolog GEN III phenotypic characterization. Upon the incubation of these plates at 28℃, microbes were selected from a singular location on the agar plate and placed into Biolog inoculating fluid. This was done so in the hopes that one species of microbe would be isolated for the study of its DNA. Correct concentrations of bacteria were obtained through the use of a turbidimeter set at 95% turbidity. From here, the microbe was placed into a Gen III MicroPlate from company BiOLOG (8) in order to phenotypically characterize the microbe. This is done through the use of a purple metabolic dye, which is used to assess the metabolic activity of the microbe over an incubation window. The turbidimeter was used in order to control for the microbial concentration per unit volume so that the results of the dye were not influenced by the number of microbes. The microbial solution was pipetted into various wells on the 96-well plate, each containing a different test for phenotypic characteristics. These tests include bacterial ‘food’ such as carbon, nitrogen, sulfur, and phosphorus sources, as well as various environments with ranging pH levels and chemical exposures (8). Examples of stressors in this assay include: 

• Sugars (fructose, galactose, sucrose, etc.) 
• Salts (various concentrations of sodium chloride, sodium lactate) 
• Acids (n-acetyl neuraminic acid, aspartic acid, mucic acid) 
• pH levels (5, 6) 

71 of the wells are used as carbon-source assays, 23 as chemical-sensitivity assays, and two as positive and negative controls. The degree to which the microbe could prosper in each well was determined through the strength of the purple metabolic dye (8). Next was the isolation of High-molecular Weight (HMW) DNA. Genomics DNA (gDNA) was isolated through the enzymatic lysis process using theNEB Monarch HMW kitfor tissues and cells. After lysing the cells, DNA was collected through the use of glass beads, which attract DNA by means of its electric charge. The DNA was washed several times before being collected in a solution and incubated in the lab. As another means of microbial identification, the 16S rRNA gene was sequenced by instructors Dr. Carlos Goller and Dr. Carly Sjogren. Compared to Nanopore sequencing, 16S Sanger sequencing is a lower throughput process that uses fluorophore techniques rather than measuring current. Similar to how the COVID-19 tests worked, Sanger sequencing relies on polymerase chain reaction (PCR) amplification of the gene sequence. Here, primers are used to define the region of DNA that is to be amplified. After amplification, the DNA was cleaned using the Qiagen DNA Clean Up Kit. The obtained 16S gene was then analyzed throughBLASTidentification. There were a few exact species that had close matches, but from this identification process the microbe was determined to be of the Bacillus genus. Following are the closest results for possible species: Bacillus mobilis, Bacillus toyonensis, Bacillus cereus, Bacillus thuringiensis, Bacillus wiedmannii. From here, long-read sequencing was performed on our previously isolated DNA using the Oxford Nanopore Technologies Minion Sequencer. In the Minion sequencer, strands of DNA pass through pores which can detect bases from changes in current. “Barcodes” are appended to the ends of DNA strands to allow for identification (11). In this trial, the DNA from all three groups were combined and sequenced simultaneously. The sequencer ran for a total of 72 hours and sequenced 7.58Gb of bases that passed quality control.However, Nanopore sequencing did not yield tangible results. This may be due to a lack of large read lengths (~10-100kb) for which the Nanopore-based assemblers are designed. Though we obtained a lot of raw data, the mean read length was ~750b, which is very small for Nanopore sequencing. The lack of large, contiguous pieces of DNA may have inhibited the assembly of DNA scaffolds and eventually a genome. The best course of action from here was to research the closest match to the 16S Sanger sequencing. A summary of the information gained from annotating the genome of Bacillus mobilis can be found in our KBase narrative(15).

Microbial Species:

Based on the environment our specimen was retrieved from, we know it can survive in low pH and warmer climates around 28 degrees Celsius. When isolated on an agar plate, the bacteria forms thick, cream colored colonies that look like little clouds. Based on the results from our Biolog experiment, we can see that our bacteria originally began by using gelatin, D-sorbitol, and L-arginine as carbon sources, all of which derive from either glucose or galactose. Eventually, it switched to metabolizing D-fructose, D-gluconic acid, and L-histidine, which are mostly glucose and fructose derivatives. This demonstrates how our bacteria primarily uses monosaccharides as an energy source before metabolizing more complex sugars.

In comparison with the known characteristics of Bacillus mobilis, the general information about being able to withstand acidic environments, being mesophilic, and forming white circular colonies correlates with the literature (9). Additionally, the metabolites our sample utilized are somewhat comparable to what is known about B. mobilis. According to the literature, B. mobilis is able to build acid from D-sorbitol and D-fructose, which is what was observed on our Biolog plates (9). However, they are also known to use arabinose, D-mannitol, L-fucose, and more (9). These results indicate that our Biolog sequencing was semi-reliable in helping to identify our organism, however there were some significant differences in what was metabolized since our microbe does not tend to use metabolites such as L-arginine and D-gluconic acid.

However, our group struggled significantly when it came to quantifying our DNA and sequencing the genome. Through Nanopore sequencing and BLAST identification, we were able to narrow down our potential species to be of the Bacillus genus. Nanopore sequencing likely did not produce large enough read lengths to be analyzed and assembled into creating our genome in Kbase. Thus, we analyzed a sequence from the National Library of Medicine, in order to still understand our 16S sequence (12).

Based on what was discovered about our species, it is evident that Bacillus mobilis is able to withstand harsh environments (9). This is a vital characteristic of electronic waste degrading organisms, since they must be able to survive in adverse conditions like low pH. Furthermore, since they are able to produce acid through metabolizing a variety of molecules, this contributes to their ability to degrade and breakdown waste. Based on the taxonomic classification of our organism, there is no official information regarding their ability to extract precious metals, like gold and copper, or specifically survive in those environments. However, since we know they are able to survive in low pH, both aerobic and anaerobic environments, and produce spores (9), we can assume that they would be able to withstand soil intoxicated with metals thus potentially contributing to the electronic waste recycling process.

Not only this, but substantial evidence regarding the ability of B. mobilis to exist in environments with heavy metals was found through genome annotation (13). Following are a sample of B. mobilis genes that are linked to resistance and transport of heavy metals: Cobalt/zinc/cadmium resistance protein CzcD, Lead, cadmium, zinc and mercury transporting ATPase. Arsenical-resistance protein ACR3, and Chromate transport protein ChrA. As you can see, these genes pertain to maintaining homeostasis in the presence of cobalt, zinc, cadmium, lead, arsenic, and chromium compounds.

This ability for Bacillus mobilis to live in areas with heavy metals is supported by the results of the BiOLOG phenotypic characterization, as well. After subtracting out the value of the negative control,there were only a few wells left with a significant amount of metabolic dye. One of these was the pH 6 well, which is a slightly acidic environment. Though the ideal pH for B. mobilis is pH 7, it is known to grow in pH ranges from 5-10 (16).

Lastly, other species in the family of B. mobilis have been found to remove heavy metals from the soil. For example, in a study by Ayangbenro and Babalola, B. Cereus was found to have “biosurfactant production” and the ability to remove heavy metals from the surrounding environment (14). This loosely suggests that B. mobilis may also have the ability to survive in the same environments and precipitate heavy metals.

Future Directions 

For future experimentation, we hypothesize that if Bacillus mobilis is introduced to printed circuit motherboards (PCBs), then the amount of metals present should decrease. The first step would be to gather the PCBs from an e-waste depositing site, process them by putting them in sodium hydroxide, which is used for metal cleaning and processing. Next, water should be run through the system to remove the sodium hydroxide, then leaving the metals out in the air to dry. Throughout the cleaning process, it is important to observe and collect any PCB residue so it can be analyzed for precious metals. Use this extracted residue to collect and measure the total metal concentration. And finally, introduce B. mobilis to the PCB residue and measure the metal concentration afterwards. Use a bacteria known to bioleach as the positive control and the first metal concentration measurement as the negative control. Now to see if B. mobilis effectively bioleached the PCB residue, subtract the new metal concentration from the old and divide by a hundred to get the percent of metal bioleached (13,14). 

Graphical Representation:

References

1. P. Han, W. Z. Teo, W. S. Yew. “Biologically Engineered Microbes for Bioremediation of Electronic Waste: Wayposts, Challenges and Future Directions” Special Issue: Engineering Biology in Environment and Sustainability. 6(1), 23-34, (26 February 2022).
a. https://ietresearch.onlinelibrary.wiley.com/doi/full/10.1049/enb2.12020
b. This source provides a definition of using microbes for bioremediation and various methods on how to use microbes for e waste recycling. It also has a practical table which organizes the advantages and disadvantages of microbes for recycling.
2. D. A. Bruun, P. Lein. “The toxicological implications of e-waste”. Open Access Government. (28 June 2021)
a. https://www.openaccessgovernment.org/the-toxicological-implications-of-e-waste/1141 39
b. This article gives a good overview of what e-waste is, a brief history of the Basel Convention, and some of the problems that e-waste poses.
3. A. K. Awasthi, M. Hasan, Y. K. Mishra, A. K. Pandey, B. N. Tiwary, R. C. Kuhad, V. K. Gupta, and V. K. Thakur. “Environmentally Sound System for E-Waste: Biotechnological Perspectives”. Current Research in Biotechnology. 1, 58-64, (November 2019).
a. https://doi.org/10.1016/j.crbiot.2019.10.002.
b. This is a paper that documents in depth the process of e-waste recycling through the use of biotechnology. There are a few good visuals that break down the complicated process into steps that are easier to follow.
4. R. Nithya, C. Sivasankari, and A. Thirunavukkarasu. “Electronic Waste Generation, Regulation and Metal Recovery: A Review”. Environmental Chemistry Letters. 19(2), 1347-68, (20 October 2020). a. https://doi.org/10.1007/s10311-020-01111-9.
b. This source further describes using microorganisms for metal recovery and some specific methods used to do so. It also includes information about the factors required to perform this technique.
5. M. Khurrum S. Bhutta, Adnan Omar, Xiaozhe Yang. “Electronic Waste: A Growing Concern in Today’s Environment” Hindawi Publishing Corporation. Economics Research International. 2011 (15 June 2011).
a. https://www.hindawi.com/journals/ecri/2011/474230/
b. This source provides general information about electronic waste and why it is a problem worldwide. It discusses the specific implications of electronic waste and why people should be more aware and care about the subject.
6. A. Işıldar, E. D. van Hullebusch, M. Lenz, G. Du Laing, A. Marra, A. Cesaro, S. Panda, A. Akcil, M. A. Kucuker, and K. Kuchta. “Biotechnological Strategies for the Recovery of Valuable and Critical Raw Materials from Waste Electrical and Electronic Equipment (WEEE) – A Review”. Journal of Hazardous Materials. 362, 467-81 (January 2019).
a. https://doi.org/10.1016/j.jhazmat.2018.08.050.
Kaylie Maynard, Versace Prew, Gabe Thompson BIT 295 Experimental Design Project
b. This gives a more in depth look at the different processes involving biotechnology and the recycling of e-waste, including ‘heterotrophic bacterial bioleaching of metals’ and ‘fungal bioleaching’ and some of the chemistry associated with both. Included is also a figure on how different metals are distributed through technologies.
7. “What We Do”. Mint Innovation. (2022).
a. https://www.mint.bio/what-we-do.
8. “Gen III MicroPlate Instructions for use.” BiOLOG.com. 1-8 (October 2016).
a. https://www.biolog.com/wp-content/uploads/2020/04/00P_185_GEN_III_MicroPlate_IF U.pdf
b. This document gives information about using Biolog plates and what the test demonstrates about the organism.
9. Y. Liu. “Bacillus mobilis 0711P9-1”. BacDive. 2017.
a. https://bacdive.dsmz.de/strain/140964
b. This source provides basic characteristics about our species, Bacillus Mobilis, which is used to help understand our results.
10. NHGRI. “Synthetic Biology.” Genome.gov, (13 March 2019)
a. www.genome.gov/about-genomics/policy-issues/Synthetic-Biology.
b. This website discusses synthetic biology and the ethics behind using this technology to experiment on the human genome.
11. Oxford Nanopore Technologies. “MinION”. Oxford Nanopore Technologies. (2022) a. https://nanoporetech.com/products/minion
b. This website provided information about using Nanopore sequencing and how the technology works.
12. USA.gov. “Bacillus mobilis”. National Library of Medicine.
a. https://www.ncbi.nlm.nih.gov/genome/?term=Bacillus+mobilis
b. This website is where we retrieved the genomic information for our species and what we applied in Kbase in order to run the tests and receive taxonomic information.
13. J. Bai, W. Gu, C. Liao, W. Yuan, C. Zhang, J. Wang, B. Dong, K. SHih. “Bioleaching for Extracting Heavy Metals From Electronic Waste Sludge.” Industrial and Municipal Sludge. 525–51 (2019) a. https://doi.org/10.1016/b978-0-12-815907-1.00023-4
b. Helped give insight on how to make an experiment on Bioleaching.
14. G. Venugopal, M. Kaari, M. P. Ramakodi, R. Manikkam. “Bioleaching of Heavy Metals From e-Waste Using Actinobacteria.” Methods in Actinobacteriology. 705–08 (2022)
a. https://doi.org/10.1007/978-1-0716-1728-1_102
b. Helped give insight on how to make an experiment on Bioleaching.
15. Thompson, Maynard, Prew. Classifying the Genome of Bacillus Mobilis for Electronic Waste Recycling, KBase Narrative, https://kbase.us/n/131578/90/ (2022)
a. A link to our Kbase Narrative
16. L. C. Reimer, J. S. Carbasse, J. Koblitz, C. Ebeling, A. Podstawka, J. Overmann. “BacDive in 2022: the knowledge base for standardized bacterial and archaeal data.” Nucleic Acids Research. 50(D1), 741-746 (29 October 2021).
a. Nucleic Acids Research; database issue 2022.
Kaylie Maynard, Versace Prew, Gabe Thompson BIT 295 Experimental Design Project
b. Information on Bacillus mobilis species and growth conditions