Planetary Protection (Reverse Contamination)

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This is the third of three posts about the planetary protection workshop I attended at NASA Ames from March 24-26, 2015. The first is here.

I mentioned, in my last post on forward contamination, that reverse contamination is the primary concern for Planetary Protection (PP). In this context, reverse contamination refers to the transport of Martian life to Earth. Although, I did hear a totally different definition from Jen Law (the current flight surgeon for the ISS,) which I will get back to later.

I totally get why the primary mission of Planetary Protection is to, well, you know, protect OUR planet. After all, life on Earth has a very long history of ensuring the survival of life on Earth. But, from my perspective, it seems like less of a concern than forward contamination, which all seem to agree is inevitable. It’s tempting, as someone whose job is NOT to contribute to policy on this topic, to roll my eyes when people in the room suggest that astronauts returning from a Martian collecting trip might show signs of illness due to infection by a Martian microbe. First of all, see previous post for the extreme survival skills required to get by on Mars and then ask yourself: what are the odds that something adapted to life on Mars is likely to become a human pathogen upon first contact? Not very? Really, no one has idea. But, let’s assume that a Martian microbe actually has infected a human astronaut, and we have a sick astronaut on a long flight home, then ask: how would we attribute the illness to the Martian microbe? Are we going to apply Koch’s Postulates during the return voyage? Are we going to do some high-throughput 16S rDNA surveys or metagenomics? If we do, will we find evidence of familiar human pathogens. Of course we will. Wait, do Martian microbes even have DNA? It must be the case that other Planetary Protection workshops will be addressing these issues, because every time I brought them up people were like, “Yeah, yeah, we know that stuff is important and difficult.” But, no talks on these subjects at this workshop. Maybe next time!

So, with respect to avoiding reverse contamination, the main objective is to, as they say, “break the chain” (BTC) of contact with Mars. Bob Gershman discussed the technology under development to meet this objective for a robotic Martian sampling trip. According to the NASA procedural requirements (see NPR 8020.12), “Samples returned from Mars by spacecraft should be contained and treated as though potentially hazardous until proven otherwise.” How contained is contained? The PP Office has a “draft” requirement of < 0.000001 probability of inadvertent release of a single unsterilized Mars particle to the Earth’s biosphere. I MEAN. How the hell do you make that calculation!? What’s really cool is that they will actually figure that out.

So, anyway, there needs to be the sealing of compartments and the external sterilization of components and the withstanding of very unpleasant Earth re-entry conditions, including extreme heat and force. Gershman presented a very detailed, technical account of various sealants and containment materials and reentry technological developments. I was relying pretty heavily on my audio recorder for this talk, because it was too information-dense for me to take useful notes, but it died. So, I’ll share with you an excerpt from his abstract.

“Sealing modalities being investigated include brazing, explosive welding, bagging, and conventional o-rings. Sterilization modalities include heat, pyrotechnic paint, plasma, and hydrogen peroxide; but it should be noted that NASA has not yet considered which of these – if any – could be certified for sterilizing Mars material. Also, technology is needed to assure (with an unprecedented degree of confidence) that the Earth entry vehicle would withstand the thermal and structural rigors of Earth atmosphere entry and that the sample container and its seals would survive Earth entry, descent, and landing. Concepts for a new Earth entry vehicle that could satisfy the stringent MSR reliability requirements have been under study for several years, including some preliminary technology development activities.”

So, basically, we’ve thought a lot more about forward contamination because that draws upon our understanding of sterilization and contamination in the context of human health and microbial monitoring, a la CDC, DHS, DOD, etc. The procedures and technologies for dealing with safely bringing Marian life to Earth are very much under development.

I mentioned that the flight surgeon in the room was using a different definition for reverse contamination. She was referring to the fact that, once the astronauts return to Earth, they are likely to have compromised immune systems and altered microbiomes. They may be at risk from exposure to common Earth microbes. I thought this was an interesting take on “reverse contamination.” Also, it brings up the topic of microbiomes, which is why I was in the room. I was there to talk about the microbiology of the built environment and share some of the results from Project MERCCURI’s microbial ecology of the International Space Station. The ISS represents an extreme built environment for a number of reasons, microgravity, high radiation levels, very little exposure to Earth air, very little flux of Earth microbes. The spacecraft that carries humans to Mars will experience extremes of those extremes. How will the microbiomes of the astronauts and their homes respond? Don’t worry, NASA is on that, too.

Planetary Protection Workshop (Forward Contamination)

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This is the second of three posts about the planetary protection workshop I attended at NASA Ames from March 24-26, 2015. The first is here.

Forward contamination, in the context of planetary protection, refers to the transport of microbes from Earth to Mars. The title of the workshop, and many talk titles refer to “human extraterrestrial missions,” but really, we’re talking about sending astronauts to Mars to walk on the surface of Mars, drill holes in Mars, scoop up dirt from Mars, and then returning the astronauts to Earth. There was almost no talk about human habitation on Mars. First things first, I suppose.

So, John Rummel kicked things off with a brief history of planetary protection. The gist of it is that, because we at some point deemed the moon devoid of life and uninhabitable, we didn’t think much about planetary protection until we started exploring Mars. In 1991, the stance on planetary protection was basically, “Viking didn’t find life on Mars, therefore no big deal, let’s go explore.” In 2000, gullies were found on Mars, suggesting the presence of water, leading to the Pingree Park Workshop in 2005, which addressed the question: Can we explore Mars without contaminating it?

(I did not know this, but I learned from Gerald (Jerry) Sanders that there is water on Mars in the atmosphere, in hydrated soil, in permafrost, in icy soils, in recurring slope linnea (hypothesized briny water), and in aquifers that are suspected to be >1km below the surface. So, there are defined “Special Regions” on Mars that are more likely than others to be able to support Earth life, and those are to be avoided or treated super-special so as to avoid forward contamination.)

The forward contamination discussions fall into two broad categories: superbugs and human-associate microbes. First, there are some superbugs that could hitch a ride on the surface of spacecraft and find a place to grow on Mars. These are more likely to find a home on Mars, but it’s worth discussing ways to remove them from surfaces. Second, human-associated microbes are far less-likely to do well on Mars, but the general consensus is that we cannot avoid contaminating Mars with them. There seemed to be a little bit of concern that they would interfere with the search for life (by providing false-positives,) but most people seemed familiar enough with evolution to agree that we are not likely to mistake something with a 99% 16S rDNA sequence identity to Staphylococcus aureus for Martian life.

Paulino-Lima isolated a strain of Geodermatophilus that is extremely resistant present-day Martian UV radiation with LD10 at least 33 times greater than Deinococcus radiodurans. Read all about it here. He described some experiments in a “Mars chamber” that’s been/being built in Brazil. Interestingly, Brazil has a long history of astrobiology, reviewed here. He suggested that a big knowledge gap that we need to address is that everything we know about radiation resistance comes from cultured isolates, and that we should be doing more environmental work. Several UV-exposure experiments (e.g., by Shuerger, Mancinelli, and Paulino-Lima) showed that dust can provide a very effective shield against UV radiation. Dust particle size, and depth of coverage both have significant effects.

Marcco Mancinelli from SETI gave the keynote on the second day, discussing some experiments in Mars-like places:

Terrestrial analogs of Mars include the dry valleys of Antarctica and the Atacama desert. University Valley is cold and dry, with not much organic carbon. -20 deg C seems to be the limit for microbial activity, even in cold and dry-adapted sandstone endolithic communities there. The Atacama desert is the oldest, continuously dry place on Earth (which was in the news for flooding the day after this talk!) There you find endolithic communities in these salt-pillar-looking things (halites). The surface of the rock blocks UV radiation, but is translucent enough to allow photosynthesis.

There are also “space environments” orbiting the Earth. ESA has the BIOPAN, which is a little laboratory attached to a Russian satellite and EXPOSE, a research platform attached to the outside of the ISS. We’ve actually been “throwing everything into space” since the 60s, and most everything dies instantaneously. Bacillus subtilis is the exception. On NASA’s Long Duration Exposure Facility (LDEF) B. subtilis spores were viable after a six-year stint in space. Mancinelli also showed that halophiles (like those living in halites) could survive space exposure for 2 years.

How well-suited is Mars for Earth life? Not very.

Andrew Schuerger gave us a (ranked) list of 17 biocidal factors on Mars, that I think is worth presenting in full.

  1. solar UV irradiation
  2. extreme dessiccation
  3. low pressure (1-4mbar)
  4. anoxic CO2 atmosphere
  5. extremely low temperatures (global average of -61 deg C)
  6. solar particle events
  7. galactic cosmic rays
  8. UV-glow discharges from blowing dust
  9. solar UV-induced oxidants
  10. globally distributed oxidizing soils
  11. extremely high salt levels in surface soils at some sites
  12. high concentration of heavy metals in soils
  13. likely acidic conditions in regolith (I had to look that one up)
  14. perchlorates in at least some soils (although people were constantly shouting about microbial perchlorate metabolism)
  15. lack of defined energy sources freeof UV irradiation
  16. no known source of available nitrogen or carbon
  17. no obvious redox couples for microbial metabolism

That sounds like a pretty nasty place to make a living. Schuerger was also frequently the voice of “well, duh.” For example, his simulations suggest that if you’re standing on Mars, and you want to sterilize a piece of equipment, then you can just expose it to the sun for a few minutes-hours and you’re going to nuke everything. He also showed a cool study where they took the Moon-1 planetary rover on a drive over pristine snow on Arctic sea ice. When they stopped to camp along the traverse, he took samples from the floor of the rover and from the snow surface at points 10m away from the rover (ahead, behind, upwind, downwind) and plated them. Inside the rover was really diverse, outside the rover was nothing. This seemed like an interesting example of a study where culturing methods might actually be more sensitive than molecular-based methods. Unless you believe that unculturable things are less likely to be dispersed than the things that grew on the rover plates, this approach does a nice job of avoiding the issues associated with low-biomass PCR-based studies. So, as the rover moved over the ice, it wasn’t spewing forth microbes (well, duh.)

I had dinner with Amy Ross (the geologist) one night, she talked a lot about geology and caving and answered a ton of questions about NASA bureaucracy. And then the next day, she gave a talk as the ARCHITECT OF HUMAN EXPLORATION SPACE SUITS. I don’t how you don’t bring that up at some point in the conversation! The most interesting single fact I learned about forward contamination is that space suits are leaky. The current Mark III spacesuit has 50 leakage paths. How big are the leaks? What is leaking out? We don’t know. Seriously. So, that’s a knowledge gap.

Thinking about forward contamination is really fun, but the primary concern for planetary protection is the reverse contamination. I’ll post about that next!

Experimental design for microbiome research in space.

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Thanks to Project MERCCURI, I’ve been fortunate to develop a relationship with Sharmila Bhattacharya, a researcher at NASA Ames who is doing Drosophila research on the ISS. Her group is developing a “Fruit Fly Lab” for any researcher to run experiments on the ISS. She’s sending up her own experiments on several upcoming SpaceX flights, and when possible, she is providing me with samples of fly carcasses, dissected fly guts, and/or fly feces obtained from swabbing the interior of the enclosures. She is interested in (among other things) the affect of spaceflight on immune function. Dovetails nicely with my interest in microbiome research, right!?

So, the first experiment (HEARTFLIES) I got two groups of fly dissected guts, enclosure fecal swabs, and fly carcasses. I did 16S rDNA PCR sequencing from them and found a HUGE difference between the flies that went to the ISS and the flies that stayed on the ground. That was pretty exciting until I realized that the space fly gut microbiomes looked basically like regular old fly gut microbiomes and the ground fly microbiomes contained something like 99% of a single OTU of Lactobacillus.

As it turns out, the media for the ground flies dried up. The flies were very dehydrated (which affected their heart assays as well.) “Never fear,” they told me, “we are going to re-do those ground controls.” Huh? It’s been more than a month since the space flies were sent into space. A new batch of flies cannot be used asa control for those space flies. It doesn’t matter that they are the same genotype, maintained in the same conditions. They are a totally different population now with respect to their gut microbiomes.

For these ISS experiments, they always use this type of “asynchronous control.” Why? Because they cannot synchronously mimic on the ground the conditions that the flies are experiencing in space. Why not? Because ambient conditions on the ISS fluctuate and they don’t learn about those fluctuations on the ground until well after they’ve already occurred. Makes sense. So, in subsequent experiments, they have been running “best guess” synchronous ground controls in addition to their asynchronous controls.

Tomorrow, I’m finally going to get to tour her lab at NASA Ames. We’re going to chat about these kinds of issues. For one thing, flies can swap microbiomes, but not genotypes. Do their experimental enclosures separate the genotypes? I have no idea. We’ll see tomorrow. I wonder if anyone who has piggybacked on experiments in animals like this has any wisdom to share?

Workshop on Planetary Protection Knowledge Gaps for Human Extraterrestrial Missions (Intro)

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http://www.nasa.gov/ames/events/ppw2015workshop/#.VRMLLJPF8mU

I’m at a NASA Ames workshop this week. The goal is to have a discussion about planetary protection with respect to human spaceflight, in particular to Mars, mostly during a “sample and return” mission and a little bit about human habitation on Mars.

I’m tweeting with #planetaryprotection. There’s also live streaming here: https://ac.arc.nasa.gov/planetaryprotection/

The broad goal of planetary protection is to make sure that we don’t contaminate the universe with our Earth stuff and vice versa, forward and reverse contamination, respectively. The first half of the first day was an exhaustive review of previous workshops on the subject. The reports are available here. James Johnson conducted an extensive literature review, which will soon be published in Advances in Space Research Special Issue: New Challenges for Planetary Protection, so keep an eye out for that. The Outer Space Treaty of 1967 was also brought up by many times. Apparently (according to Cassie Conley) if you can decipher the language, Article IX indicates that the “highest priority for planetary protection is to protect the Earth.” Of course, planetary protection was a concern during the Apollo missions to the moon, but the protections (especially with respect to reverse contamination) were pretty flimsy. They splashdown in the ocean, change into a new suit, hop into a helicopter, get paraded past hundreds of people, and then enter quarantine for 21 days.

120718020623-apollo-capsule-peace-signs-color-horizontal-large-gallery

At some point, it was decided that there was no life on the moon, so reverse contamination dropped off the radar. As far as forward contamination is concerned, well, there not just flags and golf balls, but also many, many bags of trash and human waste sitting on the surface of the moon right now.

The second half of the first day consisted of talks on the subject of Microbial and Human Health Monitoring. I’ll post about those later.

IPython notebook for basic microbial ecology analysis using QIIME

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This is an IPython notebook I created to provide my colleagues and collaborators with a copy of my basic analysis workflow for analyzing microbial community sequence data using QIIME. This workflow starts with quality-filtered, de-multiplexed 16S rDNA sequences and a mapping (metadata) file, and ends with some basic diversity analysis output.

I hope this is useful, and I’d love feedback, especially of the “why the hell are you doing it like that?” variety.

I am hosting it on GitHub (a first for me.)

http://jennomics.github.io/QIIMEbyJennomics/

Disclaimer: I tested my notebook using the files that I’ve provided, and it worked for me. I use QIIME (macqiime) and IPython notebook for my research, and I used online tutorials to figure out how to use these tools. I am not a software developer or an IT support person or even a trained computer scientist.

Beginners can get started with these:

http://www.wernerlab.org/software/macqiime

http://nbviewer.ipython.org/github/ipython/ipython/blob/2.x/examples/Notebook/Index.ipynb

http://nbviewer.ipython.org/github/biocore/qiime/blob/1.9.0/examples/ipynb/illumina_overview_tutorial.ipynb

 

What the fungi do I do with my ITS library (Part 2)

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Previously, I expressed some concern about size variation in my environmental fungal ITS PCR libraries. I’m still concerned about that, but I have an additional concern. The ITS region can’t be aligned, and I’m partial to phylogenetic approaches to pretty much everything. So maybe ITS is not for me?

So, I asked Twitter again…

[View the story “What the fungi do I do with my ITS library? (Pt. 2)” on Storify]

In summary, I don’t think that I can use ITS given the size variation that I see, and I’m not sure that I want to, given the fact that you cannot align it to do phylogeny-based analyses.

28S (or LSU) is a reasonable alternative to ITS that has two big downsides: 1) the reference database is much smaller than the ITS reference database and 2) it does not provide the fine-scale taxonomic resolution that ITS does.

Rachel Adams referred me to Amend et al,  in which they use both. I’ll have to look into this approach…

What the fungi do I do with my ITS library?

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It’s been about 8 years since I started working on my first 16S rRNA PCR survey (of Drosophila gut microbes). At that time, I was occasionally asked, “what about Archaea or what about microbial Eukayrotes?” Then, and ever since, my reply has been that it’s hard enough to get a handle on what’s going on with the bacteria – I don’t need to make my life more challenging by broadening my scope.

But, finally, this month, I’m making my life more challenging. As part of my new Seagrass Microbiome Project, I’ve decided to tackle the fungi. As far as I can tell, ITS is the “barcoding” marker of choice for fungal types. For many reasons, it’s best to follow the herd when doing this sort of thing: 1) someone else has already designed, tested, and published results with these primers, 2) there is a reasonably large database of ITS sequences available to compare my sequences to, and 3) I lack the interest and personnel to explore an alternative approach.

So, I just plunged right in. At first, I tried some new primers designed by Nick Bokulich, but he warned me that they were “finicky” and he was correct. I got no amplification with my seagrass samples, and the positive control I had only worked about half the time. I know some other fungi people, well, I know Jason Stajich (@hyphaltip), so I asked him which primers I should use, and I decided to go with the primers set used in a cool paper by Noah Fierer’s lab, in which they looked at fungi in rooftop gardens in New York City.

Those worked, and a few days ago I got word that my sequencing run was in. It looks like crap. We typically get about 12 million sequences from our MiSeq runs, but this time, I only got 4 million. I was also told that the reverse reads looked much better than the forward reads.

So, now, in addition to working with a new “barcode,” I have to troubleshoot a crappy sequencing run. In many ways, it’s nice to have undergrads and a technician in the lab who do all of my lab work for me these days, but it sucks when it’s time to troubleshoot because I’m so far removed from the bench that I have no idea what’s going on anymore.

So, the first thing I asked for was the Bioanalyzer trace that’s always run before the library goes on the machine. It looks like this:

Bioanalyzer trace for my first fungal ITS MiSeq run

Bioanalyzer trace for my first fungal ITS MiSeq run

I had been told that there was size variation. I had even seen some of the PCR gels. But, still this is not what I expected to see. Upon seeing this, I am concerned about two things. 1) If there is strong preferential amplification of smaller DNA molecules during the bridge PCR on the flow cell, then will I even see DNA from those larger peaks? 2) With our 300bp reads, for sure the amplicons in the peaks <400 will have overlapping forward and reverse reads, but for sure the 676bp amplicons will not. What effect will these two things have on my analysis? How do I accommodate this size variation? One of the reasons to follow the herd with these methods is that other people have probably already encountered and dealt with exactly this issue, so I turned to Twitter…

There are some great resources suggested here. I know what I’ll be reading this weekend…

[View the story “What the fungi do I do with my ITS library?” on Storify]

MicroBioBlitz

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Image

As soon as I was introduced to the concept of a BioBlitz, I immediately emailed the boss man Jonathan Eisen saying that we should arm these people with microbial sampling kits. He, apparently having received the same BioBlitz announcement that I did, was seconds away from sending the same email to me!

We contacted Jack Gilbert at the Earth Microbiome Project and Janet Jansson at LBL/JGI/JBEI, and George Phillips, the organizer of the Mount Diablo BioBlitz, and they were all on board to help make a MicroBioBlitz happen.

A few days ago, I received another announcement about the 2nd annual UC Davis SEEDS BioBlitz. So, yesterday, I got the go-ahead from those organizers, last night I threw together 20 swab kits, and today I took them to the Putah Creek Riparian Reserve for a test run of the MicroBioBlitz concept. It was So. Much. Fun! The BioBlitzers were super-excited to start swabbing their organisms of choice. I gave them no guidance, and I can’t wait to see what they decided to swab! Before I left, they had swabbed lizard butts and oak galls, and they were on the lookout for otter scat!

I couldn’t stay for the whole event because Vivian needed a lunch and a nap, but before we left, I had her swab a Valley Oak for us! She loved helping, but cried when I took the swab away from her, so I gave her a swab to play with, and she spent the next 20 minutes swabbing everything she could reach!

MBL #3 – The Volta Experiment

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Before you even have a chance to really get to know your classmates, you are led on a dark walk to a stinky Cedar Pond, where the brave venture chest-deep into the cold, sulfuric muck. They stomp around in the soft, slimy, decomposing leaf litter and sediment, releasing bubbles of methane gas. The methane is trapped by one person (Dan Buckley, in this video) in a stopped funnel. On the count of three, Dan removes the stopper and Steve Zinder holds a flame as the methane shoots out of the funnel. Voila! Volta!

MBL # 2 – Jorg Overmann and my pet phototrophs

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I mentioned in my last post that one of the many wonderful things about the Microbial Diversity Course at the MBL is the quality of the invited speakers. Another favorite of mine was Jorg Overmann, who wrote the book on Molecular Symbioses and is the director of the DSMZ (I did not know that!) He introduced us to his favorite bugs, the consortium known as Chlorochromatium aggregatum.

A really nice summary of what we know about them is here on the Small Things Considered blog. Basically, there is a flagellated Beta-Proteobacterial rod that is encrusted with non-motile green sulfur photosynthetic symbionts (epibionts, to be precise, because they are attached to the surface.) Their relationship is complex, but what’s overtly cool about it is that the rod can chauffeur the epibionts around in search of the right conditions for photosynthesis. One can (and we did) see their cool scotophobic behavior in a light microscope. It looks like this (run towards the light!):

Source

Jorg said that I should be able to find these consortia in the nearby Trunk River, so hello, of course I will try to enrich for them! However, he strongly discouraged my reliance on their enrichment for my independent research project in the last half of the course, because they are quite elusive and grow very slowly.

So, let me backup a little here. The first three weeks of the course you attend lectures in the morning and then you are in the lab until late at night. Those weeks are spent attempting to culture a diversity of isolates from environmental samples that you collected in the first few days. You are also introduced to all of the super-cool equipment housed at the lab, to which you will have full access. For the last three weeks of the course, you draw upon your new-found knowledge, the support of those around you (students, instructors, TAs), and the abundant resources at the MBL to work on an independent research project. Most people work on AN independent research project, but I apparently am not most people. I worked on FOUR. This is the first (and favorite) that I will describe in a series of blog posts.

Setting up the enrichment is easy. You just take some sediment from the river, put it in a bottle with some spring water supplemented with sodium sulfide, making sure that there is no head space and no way for oxygen to get in. Then, incubate it at room temperature with a light-dark cycle and wait.

Then you wait. I waited for 23 days. Some of them had purplish top layers of sediment and some had greenish top layers. Jorg said that Chlorochromatium aggregatum will form a biofilm on the surface of the glass. A biofilm formed! On the surface of the glass! It was purple.

Then you play with your biofilm. First of all, before I proceed, I did NOT find any consortia, but I hope you will not be too disappointed, because what the hell was I going to do with them in the week that I had left anyway? My biofilm consisted primarily of two cell types (see below.) You can see big, oval cells that contain highly refractive sulfur granules, and you can see some smaller, dark rod-shaped things.

I decided to use one of our cool toys to see if I could physically isolate the presumed purple sulfur bacteria (encircled in yellow) from those other guys. I wanted to isolate them 1) to get a 16S PCR product to identify them and 2) to see if I could transfer them from the biofilm into new culture medium and grow them.

I used the PALM CombiSystem which is a laser dissection/optical tweezer type microscope. Basically, I could spread out my biofilm on a slide covered by a very thin membrane, then use a laser to cut out a circle around one or more cells on the membrane, and then use some mysterious quantum force to catapult the membrane+cell(s) directly into the cap of an Eppendorf tube (filled with culture medium or whatever.) What the… what!?!? That was supercrazycool! Somewhere I have a movie of it, but you can check this out to get an idea:

So, I was able to isolate these guys (they were Chromatiaceae, per RDP) for 16S PCR and I was able to get them to survive the transit into the Eppendorf tube. After two days, they were still metabolically active!

These figures are from my final report. I must admit that I wrote the bulk of it, while drinking beer, and during an all-nighter. So, I wont vouch for it’s quality and can’t recommend that you read it. These reports, regardless of the quality, are good to have around because current students pore over old reports for inspiration. Reading it myself after 3 years, I felt a strange combination of “Wow, I did that?” and “Eww, I wrote that?” But, my goal here is just to share a little bit of my MBL experience with the world, and I hope you enjoy it.