Rhodococcus erythropolis: An Environmental Microbe Isolation Study


Microbes are an omnipresent living force found in every environment and habitat on the planet, from deep below the ocean in the crushing darkness at hydrothermal vents, to within human bodies and floating in the air around us. The diversity of microbes, especially bacteria, is staggering and constantly shifting through the tremendous evolutionary prowess and genetic flexibility of these organisms.

For the purposes of this research, I chose to investigate the microfauna of my aquarium biome. I have worked to culture a realistic and comfortable habitat for the organisms I keep, which has involved obtaining live plants, cultivating algae, using a heater to maintain constant temperature, and using a LED lamp to mimic sunlight. Nitrogen fixing and ammonia-oxidizing bacteria are necessary for a healthy aquarium environment (Urakawa et al., 2008)(Zehr et al., 2000), and bacteria considered pathogenic to fish and aquatic plants are perpetually present in the water (Baran et al., 2008)(Smith et al., 2012).

My goal was to isolate and identify a bacterial strain from my aquatic microbiome, then determine the likely role it plays within the system I have worked to cultivate. To do so, I sampled the aquarium water, allowed the bacteria to grow colonies from which to extract pure culture, and isolate a pure culture from which to run both genomic and physiological tests upon. I theorized the microbe I isolated is a form of nitrogen-fixing bacteria, as my tank is a healthy system with no diseased fish or plants, and has been thriving with minimal water changes, a sign of nitrogen cycling within the ecosystem.

However, the microbe I identified was Rhodococcus erythropolis, a versatile and occasionally pathogenic bacteria associated with aquatic and soil environments. This was somewhat surprising due to the predicted relative rarity of R. erythropolis in comparison to other microbial populations, such as nitrogen fixers in aquatic environments, but considering the methods I used, the bacteria I chose had already been through a selective process once I decided to isolate.



Sampling and Growth

The aquarium biome was sampled by submerging a sterile swab of cotton into the water and gently swiping it across a Betta splendens (Betta fish). The swab was then wiped across a plate of sterilized SA agar and left to incubate at room temperature for 6 days. Bacterial growth appeared after 72 hours in three small colonies. The SA plate had initially been intended to select for fungal growth, but I chose to isolate and study the acidophilic bacteria growing on the plate instead.

These bacterial colonies were isolated into pure culture by the quadrant streak method (Lab 2 Handout). Four successive streak plates were made over the course of 3 weeks, and were incubated at 37 °C during the growth phase of the bacterium.



When the isolate was sufficiently purified, a Gram stain was conducted to determine if the strain is Gram-positive or Gram-negative. This test was conducted according to the methods of Lab Handout 4.

The isolate was also tested for a variety of physiological abilities. A fluid thioglycollate test was used to determine the strain’s oxygen class, a catalase test establishes whether a bacterium contains the catalase enzyme, and a oxidase test to see if the isolate contained cytochrome c oxidase. An API 20E test strip was additionally used to test the isolate’s capacity for fermentation of various sugars, the presence of specific enzymes, and decarboxylations of amino acids, with 20 tests in total. All physiological tests were completed according to the methods of Lab Handout 6.

To obtain a more specific idea of the bacterium’s identification, DNA extraction and whole genome sequencing was conducted on the organism. Extraction was completed with a freshly streaked isolate and the PowerSoil Isolation kit according to the methods of Lab Handout 5. Genomic sequencing was completed in the Illumina MiSeq DNA sequencer at the University of Alaska-Fairbank’s DNA Core Lab, and analyzed in BaseSpace with SPAdes Genome Assembler and Kraken metagenomics according to the instructions of Lab Handout 6. These tests would determine the closest genetic match to the extracted DNA, and how many contigs, functional genes, and tRNA regions are identified, as well as what each section coded for, when relevant.

Unfortunately, after genomic extraction, the original isolate was lost. I attempted to reisolate the same bacteria again through similar sampling methods and selection of a colony of visually identical microbes, but was unsuccessful. Therefore, any antibiotic resistance by my original strain remains unknown, as well as its ability to ferment lactose or sugars, as would have been tested in Labs 8 and 9.



This isolate was observed to grow most quickly at 25 °C. Colonies were matte white and circular with defined edges and a convex shape. This bacteria was able to grow under both refrigeration and at 37 °C, though slowly.

Gram staining of the isolate showed the microbe as a gram positive bacillus. The rods most commonly appeared clustered in pairs (diplobacilli), bent at an angle so as from a distance they appear to be one spiral bacteria. The individual bacilli are approximately 0.5μm long.


Figure 1. Isolate gram-stained and magnified at 1000X. This organism is clearly gram-positive and is clustered in pairs with few exceptions.


In physiological tests, the isolate was positive in the catalase and oxidate tests. (Acharya, 2015). Complete results for the API 20E test strip are listed in Table 1. The ONPG test (tests for β-galactosidase enzyme) was inconclusive due to an unfixable air bubble that hindered results, and is not included.


Test Result
ADH- determines decarboxylation of the amino acid arginine by arginine dihydrolase Negative
LDC- determines decarboxylations of the amino acid lysine by lysine decarboxylase Negative
ODC- determines decarboxylations of the amino acid ornithine by ornithine decarboxylase Negative
CIT- detects utilization of citrate as only carbon source Negative
H2S- detects production of hydrogen sulfide Negative
URE- detection of enzyme urease Positive
TDA- detection of the enzyme tryptophan deaminase Positive
IND- detection of production of indole by the enzyme tryptophanase Positive
VP- determines if fermentation of glucose by bacteria utilizing the butylene glycol pathway is being utilized Positive
GEL- tests for the production of the enzyme gelatinase Negative
GLU- detects fermentation of glucose (a hexose sugar) Positive
MAN- detects fermentation of mannose (a hexose sugar) Positive
INO- detects fermentation of inositol (a cyclic polyalcohol) Positive
SOR- detects  fermentation of sorbitol (a alcohol sugar) Positive
RHA- detects fermentation of rhamnose (a methyl pentose sugar) Positive
SAC- detects fermentation of sucrose (a disaccharide) Negative
MEL- detects fermentation of melibiose (a disaccharide) Negative
AMY- detects fermentation of amygdalin (a glycoside) Negative
ARA- detects fermentation of arabinose (a pentose sugar) Negative

Table 1. A complete listing of the results from my API 20E test strip for my isolate (Acharya, 2015).


In the fluid thioglycollate test, the isolate proved able to survive in all areas of oxygenation, but preferred to grow at the surface, making it a facultative aerobe. Growth was thickly concentrated at the surface and directly below the surface of the agarose, but a thin line of microbial growth was visible extending from the surface to the base of the tube.

Genomic tests indicated only approximately 70% of genetic data was usable, as the other data was not even classified as bacteria in the analysis. However, within the usable information, the confidence level of the identified sequences were 95% or greater. Complete percentages of taxonomic rank are illustrated in Table 2, indicating a high level of precision in the identification of the isolate as Rhodococcus erythropolis (BaseSpace, 2017).


Kingdom Phylum Class Order Family Genus Species
Bacteria Actinobacteria Actinobacteria Actinomycetales Nocardiceae Rhodococcus erythropolis
99.11% 98.41% 98.41% 98.34% 95.99% 96.05% 95.91%

Table 2. The confidence levels of identified sequences within the bacterial genome and the identified classifications. Note all of these values are derived from ∽70% of total data, but the remaining 30%  of data was completely unidentifiable, even to the kingdom level, and as such has been disregarded in this interpretation.


Only one contig of 1420bp was identified. Additionally, only one tRNA was observed, and 169 coding regions.

Lastly, BLAST analysis was used as a secondary classification system to check the accuracy of results from Kraken metagenomics. In BLAST, the result for nucleotide analysis on contigs came back as 97-94% match for R. erythropolis, confirming the Kraken results (National Center for Biotechnology Information, 2017).



The results of the genomic tests led me to believe the strain I isolated was a variety of R. erythropolis, despite having relatively small amounts of genetic data to work with. The lack of contigs, functional genes, and tRNA leaves room for there to be a considerable amount of error in the identification, but the certainty of identification for what is present from the sample is considerably confident.

Additionally, if we are to analyze the characteristics of R. erythropolis, it is a Gram-positive, mesophilic, aerobic rod bacteria. Interestingly, it was recently observed to be a human pathogen under specific circumstances, causing septicemia in immunocompromised individuals (Park et al., 2011). Ironically, a fish in the tank I sampled died of septicemia earlier this year, though there is no means to determine if R. erythropolis was responsible for the death.

These organisms are commonly found in both soil and aquatic environments, as well as eukaryotic cells. In wild environments, they are known for utilizing a wide variety of organic compounds. Different strains have demonstrated the ability to undergo oxidations, dehydrogenations, epoxidations, hydrolysis, hydroxylations, dehalogenations and desulfurization in order to process the resources of their habitat (de Carvalho, 2005). Due to this diversity, Rhodococcus sp. are considered important industrial organisms, utilized in production of bioactive steroids and fossil fuel biodesulfurization, and is also regarded as the most commercially successful microbial biocatalyst (McLeod, 2006).

Comparatively, my isolate demonstrated several traits associated with Rhodococcus species. My isolate was gram-positive and grew in rods. The diplobacilli arrangement noted in my isolate was never mentioned as a distinguishing factor of R. erythropolis or other Rhodococcus species. I collected my sample from an aquatic environment, and though the environment I collected it from was warm, I found the bacterium grew much more successfully at room temperature. Additionally, my bacteria was established as tolerant of acidic environments from the initiation of the experiment. R. erythropolis is not characterized as an acidophile, so I believe it is an ability it possesses, but as demonstrated by growth on TSA plates and TSB, not the preferred environment of the organism.

Physiological tests revealed my bacteria sample can process urea, produces the enzymes tryptophan deaminase and tryptophanase, and can ferment a variety of simple sugars, but does not appear to have the dramatic abilities some R. erythropolis strains, though some were never tested. Although my isolate thrived in an aerobic environment, a characteristic of Rhodococcus sp., it also showed the capacity to persist in all oxygen zones, something not all Rhodococcus sp. can do.

Due to the genomic and physical evidence presented, I believe my isolate is a variety of Rhodococcus, though it may be debatable whether it is R. erythropolis, considering the lack of additional genomic data, and the diversity of the Rhodococcus genus. It is commonly acknowledged that Rhodococcus is environmentally widespread, and many more species may be present than currently documented in journals or databases (Park, 2011).

Several tests were conducted in lab research on other isolates which were not possible to do on my bacterium, due to its unfortunate and premature loss from the lab samples. As such, there can be no further research on this specific organism, though if there were, would like to investigate its abilities in several other ways.

I especially would have liked to analyze antibiotic resistance of my isolate. As it is an environmental microbe, especially one which can compete in the same pH zone as fungi, I would imagine it would have resistance to fungal antibacterials so as to better contend with fungi living in the same niche. Additionally, since it has potential to be pathogenic, it would have been beneficial to see what resistance or susceptibility it may have.

Though it would have been interesting and insightful to test my isolate’s ability to process petroleum, considering the use of R. erythropolis as a biodegradation, this was an opportunity not presented to me in this study. Another experiment I would have liked to complete in future research is analysis of the pathogenic potential of my specific strain. As there are multiple accounts of R. erythropolis causing septicemia in patients, perhaps analyzing growth on blood agar would show how quickly an infection of my isolate could become threatening.

In conclusion, my bacterium sampled was not a nitrifying organism, as I had predicted. Rather, it is a decomposer with the capacity to degrade a variety of environmental compounds, likely taking full advantage of the waste products and other organic matter present in the aquarium biome I took it from. There is a wide range of further experiments I would have liked to have conducted on this organism, but due to the follies of lab, I have, for the moment, lost the ability to further study this opportunistic organism. However, this entire study was a reinforcement of the staggering amount of diversity within the microbial world and the startlingly prolific world just out of our sight at all times.



Archarya, T. (2015). API 20E Test System: Introduction, Procedure Results and Interpretations. Retrieved from https://microbeonline.com/api-20e-test-system-introduction-procedure-results-interpretations/ on April 2, 2017.


Baran V., Dvorska L., Matlova L., Pavlik I., Svastova P. (2008). Distribution of mycobacteria in clinically healthy ornamental fish and their aquarium environment. Journal of Fish Diseases 29: 383—393.


de Carvalho, C.C.C.R., da Fonseca, M.M.R. (2005). The remarkable Rhodococcus erythropolis. Applied Microbiology and Biotechnology 67: 715—726.

Illumina BaseSpace. (2017). SequenceHub. Retrieved from https://basespace.illumina.com/dashboard on April 2, 2017.


McLeod, M.P., et al. (2006) The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proceedings of the National Academy of Sciences of the United States of America 103: 15582—15587.


National Center for Biotechnology Information. (2017). BLAST Nucleotide Sequence. Retrieved from  https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome on April 2, 2017.


Park, S.D., Uh, Y., Jang, I.H., Yoon, K.J., Kim, H.H., Bae, Y.J. (2011). Rhodococcus erythropolis septicaemia in a patient with acute lymphocytic leukaemia. Journal of Medical Microbiology 60: 252—255.


Smith K., Schmidt V., Rosen G.E., Amaral-Zettler L. (2012). Microbial Diversity and Potential Pathogens in Ornamental Fish Aquarium Water. PLOS One: https://dx.doi.org/10.1371/journal.pone.0039971.

Urakawa H., Tajima Y., Numata Y., Tsuneda S. (2008). Low Temperature Decreases the Phylogenetic Diversity of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofiltration Systems. Applied and Environmental Microbiology 74: 894-900.


Zehr J.P., Carpenter E.J., Villareal T.A. (2000). New perspectives on nitrogen-fixing microorganisms in tropical and subtropical oceans. Trends in Microbiology 8: 68-73.



(This is a collaborative project by Grace Lund and Connor Ito)





Cuddly Enough to Kill is the most scientifically accurate plush microbe collection on the market! Featuring Ebola virus, Giardia lamblia (Giardia),  Mycobacterium tuberculosis (Tuberculosis),  Bacillus anthracis (Anthrax), and  Escherichia coli (E. coli)!

First up is Ebola!

Our Ebola plushes feature glycoprotein studding, represented in soft and huggable polyester! These Ebola also show twisted tube shape of the actual virus with none of the painful, life-threatening effects of the real thing!

Next up, one of the most common causes of waterborne disease- Giardia!

 Giardia are extremely transmittable microscopic parasites which cause gastrointestinal distress and can be lethal in the case of children, the elderly, or the immunocompromised! We have represented the shell of Giardia with machine washable fleece and their signature flagella in blanket yarn!

Now, Tuberculosis, affectionately known as TB!

Our TB are rendered in plush red fleece to represent the unique waxy cell coating, which must be dyed with acid fast stains, resulting in their red color! These microbes also feature Plasmid Pocketsâ„¢! Collect and share plasmids with all your microbe friends, transferring antimicrobial resistance, virulence factors, and more!

Now featuring Bacillus anthrasis, known commonly as Anthrax!

These cuties are the only obligate pathogens of the genus Bacillus! In our plushies, the wonderful purple color represents their Gram-positive cell coat! Now featuring Spore Sachelsâ„¢, the best way to store your spores in the case of poor growing conditions! If you or your friends both have Anthrax, you can form chains of the rods with the new feature  Streptobacilli!

Now, everyone’s favorite: E. coli!

 These fluffy little fellas are Gram-negative gastrointestinal bacteria covered in pili! Each one comes with four flagela that it’ll use to swim it’s way right to your heart! Or intestines! But be careful, these microbes have been known to be dangerous!


No, really, we have a surplus of microbes! They’re replicating so fast, we can’t keep up with them! And we want to pass these pathogens on to you! Come look at our selection and take home a microbe today!


(All silliness aside, we set out to create accurate representations of microbes with the features of each organism in a hands-on method that could be used to demonstrate how diverse the microbial world can be. We thought about how to appeal to all ages, yet still be as accurate as possible while on a college budget, and decided to make plush microbes. Each microbe has its own unique feature, and we tried to cover a variety of different anatomies. All in all, this project took a long time to complete, with our final microbe count at 28.)

Previously Undiscovered Carbon Cycle in the Poles

Title: Polar glaciers may be home to previously undiscovered carbon cycle (April 12, 2017)

Link:  https://www.sciencedaily.com/releases/2017/04/170412105910.htm via the National Science Foundation

Summary:  New research on the carbon cycle of glacial ecosystems has revealed a new source of overlooked  carbon. Previously, it was thought carbon in glaciers was the result of organic matter from ancient ecosystems or more recently trapped carbon, such as soot.

However, a new study revealed that in “supraglacial” stream environments, streams that flow on glaciers, most of the carbon was produced by photosynthesizing microorganisms.

While glacial streams may seem like a small ecosystem, the writers of the paper state polar glacial streams “represent an important component of the global carbon cycle.” Approximately 11 percent of the Earth is covered in ice, and therefore valid area for supraglacial systems to exist. These streams are some of the largest glacial ecosystems, but until now, their contribution to the global carbon cycle had not been considered. As the global climate warms, and these supraglacial systems grow, the microbial output of carbon might also increase.

At this point, more research will be necessary to determine the exact extent of these microbial systems and their impacts on the global climate.

Connections:  We have looked at different carbon cycles and how the global environment is affected by fluctuations in the system. This is a slightly different ecosystem than what is typically discussed, but truly demonstrates the versatility of microbial life.

Critical Analysis:  Personally, I think it is odd that nobody has looked into glacial stream systems for microbial life until now. We know microbes can exist happily in space, so what would stop them from happily existing in polar streams? Or maybe we did know there were microbes, but not that they photosyntesized.

This outlines what I think is a great shortcoming of this article. While it explains the findings and their relevance well, it does not give much background information, either about the environment, prior discoveries, or even the carbon cycle.

I believe in this case, this article was written to grab the attention of the reader and give them a small fragment of a fact to make them interested in reading the entire paper, or at least that’s what it did for me. It left me feeling like I didn’t have the whole picture.

Questions:  How else do microbes contribute in polar ecosystems? Are there other organisms in these supraglacial ecosystems?

Painting with Microbes

I chose relatively basic microbe combinations for my paintings, but wanted to see what the different mediums would do to the microbial growths. I was extremely upset by the lack of E. coli for the presence of the deep metallic green I had seen in images of this project.

I cannot upload the images of my microbial plates, but I created a fish, a flower, and a stylized ‘S’.

My intent was to create three different pieces using the same microbes, and see how differently they could all look. I didn’t look up how the microbes would interact with each other, or how they would grow on the different agars, because I thought it would be a more interesting artistic process to set the microbes down on medium and see how they went from there. Something of a Deistic process, where once things have been set in motion, they are not interfered with, regardless of if the result is what I had initially pictured or not.


A Microbe Hunter Plies Her Trade In Space

Title: A Microbe Hunter Plies her Trade in Space

Date: March 14, 2017

Link: https://www.npr.org/sections/health-shots/2017/03/14/511891419/a-microbe-hunter-plies-her-trade-in-space

Summary: Microbiologist Kate Rubins has been investigating the unique microbiome of the International Space Station and establishing a microbiology lab in space. There has been 16 years of accumulation of the microbes brought by varying crews up from Earth, and microbes tend to stick around. While most of the microbes are harmless, some do have the potential to cause problems for crew, varying from infection to mold growing on the wall panels of the Space Station.

Microbiology in space is incredibly important to the future of space travel, be it for identifying alien life, preventing disease while in orbit, or simply improving the quality of life for spacefaring explorers.

Connections: We have been working to identify microbes from various environments and biomes, just as Rubins has, albeit from a less prestigious location. Being able to quickly and accurately identify microbes in all scenarios is something emphasized through the course, and Rubins is putting what we are learning to practical application whilst in orbit.

Critical Analysis: This article was actually a transcript of an NPR broadcast, and so read a little bit differently than most traditional news articles. It was filled with interview blurbs from Rubins, recollections of her time in space, and easy-to-follow explanations of her work. I know NPR works to tell stories as well as news, and this article was an interesting and engaging story. However, if you want the hard details about what Rubins was doing, or the specific microbial work done in the International Space Station, this is not the article for you. This is, more than anything, a story, and is not meant for conveying details. However, as a springboard for ideas, or an interesting illustration that your job can sometimes take you in strange and exciting directions, this is a good read or listen.

Questions: What other positions has NASA been searching for since it has stopped focusing on pilots? Rubins is a microbiologist, but what other branches of the sciences or other professions have gone to space?

Discovery of an HIV reservoir marker: New avenue toward eliminating the virus

Date: March 15, 2017

Source: CNRS (Délégation Paris Michel-Ange)

Summary:  A protein marker has been discovered that allows cells carrying dormant HIV viruses to be distinguished from healthy cells. This will allow the isolation, and hopefully the destruction of such HIV reservoir cells in order to make remission possible.

Link: https://www.sciencedaily.com/releases/2017/03/170315144033.htm

Connection: We have recently been discussing the reproductive cycles of viruses and how some varieties have the ability to go dormant for periods of time and remain undetected by the host.The dormant viruses inside of reservoir cells may reemerge at any given time.In the case of HIV, the ability of the virus to do so is the reason why patients must receive treatments for the rest of their lives in order to suppress the virus.

Analysis: This article is relatively short, but informative. The writing is easy to follow in layperson terms, but still conveys the discovery effectively. The journal is cited at the bottom of the page, so if one wanted to read the entire discovery in scientific terms, they would be able to. It appears to be scientifically sound, and considering it was published in Nature, that is another mark of its credibility. The fact that something like this has been discovered poses great possibilities in eventually curing HIV, and it was exciting to see this when I was looking up articles.

However, they did have a very small study group, and I hope to see this expanded upon in future studies. Only 12 HIV-positive individuals were checked for the marking protein, and while it was found in all the individuals, I think that for good  science it should have more verified successes before considering it a solidly proven fact.

Question: How long has this research been in progress? It states that the idea of identifying reservoir proteins has been around since 1996, so has this research been in progress for the last 21 years?

Assignment 1: Introduction

Icefishing a couple weeks ago when I was back home!
Here’s a picture where you can actually see my face. I was counting caribou from a bush-plane for a summer job!

Hello, all. My name is Connor Ito, my major is General Biology and my minor is in the Arts! I’m currently a junior. Most of my interests are directly related to my current academic pursuits; I am an avid outdoorsperson and enjoy sketching, painting, and other creative endeavors.

This is actually my second time studying microbiology with Dr. Leigh. I was a RAHI Research student and worked for a short time in her lab investigating a fungal remedy for the sulfolane contamination in North Pole.