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In Part 1 of this series, I briefly discussed sampling at MASSTC.  In Part 3, I’ll be writing about when septic systems go bad, and in Part 4, I’ll be writing about why we know so little about them.

Twenty two percent of US households, and most of the toilets installed in the developing world, utilize Onsite Wastewater Treatment Systems (OWTS) to remediate household wastewater.  Yet we know almost nothing about the microbial ecology of OWTS.   In this post, I’m going to give a summary of what is currently known about the microbiology of OWTS.

Why Use an OWTS?

Shouldn’t we replace these all with municipal wastewater treatment plants?  Not so fast.  OWTSs are used to treat household water in areas where a municipal plant isn’t cost effective.  They are relatively inexpensive, and, after installation, they typically deliver years of problem-free service without needing maintenance. The tank needs to be pumped, but depending on usage, that might only need to be done every five years or so.  While they can and do contaminate groundwater, they are best utilized in areas with low population density.

Important Processes in Wastewater Treatment

The household wastewater is first collected in the tank, where solids settle out.  We presume it’s partially digested there by fermenters, but hey, we just don’t know, but I’ll get back to the state of our knowledge later.  After that, it travels through a series of pipes into the leach field, and from there percolates into the soil.

At the interface of the soil and the pipes, a black, slick biofilm called the “biomat” forms.  The biomat is important because we think these organisms are responsible for degrading organic carbon….  but they can build up rapidly, and the biomat has low hydraulic conductivity.  If a biomat overgrows, the septic system will clog and won’t function properly.

After the biomat, the leachate flows by gravity into the soil, where nitrification occurs.  We can take a few guesses on processes and organisms involved in nitrification based on what we know about nitrification in soils and in wastewater treatment plants.

In a municipal wastewater treatment plant, one of the first steps in treating wastewater is nitrification, or ammonia oxidation.  This is a process where ammonia is used as an electron donor, and oxygen is used as an electron acceptor.  Since the nitrification tanks have to be kept aerated, wastewater treatment plants spend vast amounts of energy making that happen, both by pumping in air and stirring the sewage.

We used to think we knew everything about nitrification in wastewater treatment plants, but that’s not even the case.  If you remember your own micro class, you probably remember learning about nitrification by Nitrosomonas and Nitrospira in wastewater, and they are certainly there….  but that information comes from enrichment cultures and predates the revolution in culture-independent microbiology.  It also predates the discovery of ammonia oxidation by Archaea, and now that researchers have gone looking ammonia oxidizing archaea in wastewater treatment plants, they’ve found them  (1).

In OWTS, nitrification occurs in the leach field, but is it Nitrosomonas and Nitrospira?  Or it could be ammonia oxidizing Archaea, because those are abundant in soils (2) except for when they’re not (3).  One thing George Heufelder at MASSTC told me is that he frequently observes robust nitrification in his systems when the pH is too low for Nitrosomonas and Nitrospira… which leads me to think that the Archaea might be dominant (4).

There’s yet another process that can remediate ammonia in wastewater, and that’s anammox, or anaerobic ammonia oxidation.  This is a fascinating process carried out by bacteria in the phylum Planctomyces.  A handful of municipal plants are running using the anammox process (5), and we think in at least one instance, scientists have observed anammox in an OWTS (6).

In municipal wastewater treatment, following nitrification, the wastewater moves to an anaerobic denitrification tank.  Denitrification may or may not occur in a leach field.

State of knowledge about OWTS microbial ecology:

These systems have been left behind the culture independent revolution in microbiology.  Here’s the sum total of all the data we have on OWTS:  A 16S clone library (~400  amplicons) from an OWTS was published in 2009 (7) .  A study by Robertson and colleagues, published in 2011, identified anammox as a dominant process in a septate plume by sequencing 18 DGGE bands (6).  Jose Amador’s group at the University of Rhode Island constructed 16S clone libraries of 100 amplicons on OWTS mesocosms, published in 2013 (8) . Alex Vickers, one of my undergraduate students, constructed a 16S clone library from MASSTC leachfields of approximately 200 amplicons.  No previous study investigates Archaeal community composition, and to the best of our knowledge, nobody has probed an OWTS for functional genes.  That’s not a lot of information!

Next blog post is going to be about what happens when OWTSs fail, and how we might be able to improve OWTS with a bit more knowledge.

References:

1.         Sauder LA, Peterse F, Schouten S, Neufeld JD. 2012. Environmental Microbiology 14: 2589-600

2.         Leininger S, Urich T, Schloter M, Schwark L, Qi J, et al. 2006. Nature 442: 806-9

3.         Di H, Cameron K, Shen JP, Winefield C, O’Callaghan M, et al. 2009. Nature Geoscience 2: 621-4

4.         Yao H, Gao Y, Nicol GW, Campbell CD, Prosser JI, et al. 2011. Applied and environmental microbiology 77: 4618-25

5.         Kartal B, Kuenen J, Van Loosdrecht M. 2010. Science 328: 702-3

6.         Robertson WD, Moore TA, Spoelstra J, Li L, Elgood RJ, et al. 2011. Ground Water: no-no

7.         Tomaras J, Sahl JW, Siegrist RL, Spear JR. 2009. Appl. Environ. Microbiol. 75: 3348-51

8.         Atoyan JA, Staroscik AM, Nelson DR, Patenaude EL, Potts DA, Amador JA. 2013. Water 5: 505-24

 

Did you know that 22% of households in the USA use septic systems, also known as Onsite Wastewater Treatment Systems (OWTS), to handle their wastewater?  And did you know we know almost nothing about the microbial ecology of these critical systems?

Shocking and shameful, really.

Why is understanding OWTS microbial ecology so important?  First, OWTS represent a significant source of greenhouse gas emissions in the US.  Secondly, microbial processes in OWTS protect our groundwater from contamination.  When OWTSs fail, drinking water can be compromised, and the contaminants can end up in lakes and estuaries, which can hurt the fish.

Watch Mike Rowe from Dirty Jobs work with a Septic Tank Technician!  http://dsc.discovery.com/tv-shows/dirty-jobs/videos/septic-tank-technician.htm

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The grass is always greener over the septic tank! Really, they mean the leach field.

Most people think of the septic tank as the main part of the OWTS, but the part I’m most interested is the leach field.  See those dark green stripes in the grass, on the picture on the right?  Those are above perforated pipes buried in the soil, and liquids flow out of the tank through those pipes.

That’s where the microbes are working their magic.  One of the most important processes that occurs in the leach field is nitrification.  That’s the conversion of ammonia, which is highly toxic, into nitrate, which is much less toxic.  There’s several types of microbes that can perform nitrification, and they are all incredibly fascinating, but we don’t know which ones are active in leach fields!  There is some evidence that, in some of these systems, a process called anammox is occurring.  That’s conversion of ammonia not to the less toxic nitrate, but directly to nitrogen gas.  In wastewater treatment plants, anammox releases fewer greenhouse gases than nitrification, so it would be fantastic if we could manipulate septic systems to favor anammox as well.

That’s why I went to visit the Massachusetts Alternative Septic System Testing Center out on Cape Cod.  Sure, the classic microbiologist Cape Cod field trip is Sippewissett Marsh, but MASSTC is way more interesting.

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This is George Heufelder who runs MASSTC.  MASSTC is one of a few facilities in the world that is equipped to test experimental septic systems.  He works with the National Sanitation Foundation and private contractors that manufacture and market septic systems.

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MASSTC receives sewage from the Massachusetts Military Reservation, and George Heufelder and Keith Mroczka can use the sewage to test up to 20 different systems at once.

They can also climb down these lysimeters to sample the water that is coming out of the leach field, which would presumably mimic the liquids that would end up in the groundwater.

IMG_0186The picture underneath is what the lysimeter looks like from the top, and the picture above  is George descending into the lysimeter.

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I went out to MASSTC to get samples from the leach field, the tank, and the effluent, and I’m hoping to be able to get metagenomic data.  With metagenomic data, I should be able to make some educated guesses on which microorganisms are responsible for nitrification.

It’s been a wonderfully gratifying season in the Wiatrowski lab!

I just received word that PhD student Yingjiao Wang and master’s student Tyler Robison’s work has been published online in the journal Biometals.  You can find the article here for now: http://link.springer.com/article/10.1007%2Fs10534-013-9679-2  DOI 10.1007/s10534-013-9679-2

The paper is titled “The impact of ionic mercury on antioxidant defenses in two mercury-sensitive anaerobic bacteria”.

What is this research about?  Well, many bacteria resist ionic mercury, Hg(II), using a mercury resistance operon (mer).   When bacteria have mer, in the presence of Hg(II), they transport Hg(II) into the cell and reduce it to Hg(0).  Hg(0) is less toxic, and plus, is a gas – so it evaporates and leaves the bacteria’s immediate environment.

We asked the question, “What happens when bacteria that don’t have mer are exposed to Hg(II)”?  It turns out, we know how Hg(II) effects antioxidant defenses in mammals, but not in bacteria!  Yingjiao and Tyler set to work characterizing the impact of mercury on glutatione, thioredoxin, and lipid peroxidation in the bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA.

A big surprise for us was the observation that Shewanella is more resistant to Hg(II) under conditions when more Hg(II) enters the cell.  We though it would be the other way around!  We think that when Hg(II) enters the cytoplasm, it may be detoxified by binding to glutathione.

I’m pleased to announce that my lab has a paper out in Archives of Microbiology!

Here you can find a link to the paper.

The title of this paper is Identification of a possible respiratory arsenate reductase in Denitrovibrio
acetiphilus, a member of the phylum Deferribacteres.

This body of work began when members of my 2009 First Year Seminar class, Annotation of a Microbial Genome, identified a number of possible complex iron-sulfur molybdoenzymes (CISMs) in the genome of a strain of Denitrovibrio acetiphilus.  These enzymes can be used by organisms to “breathe” compounds such as selenate, arsenate, perchlorate, dimethyl sulfoxide, and nitrate.  We were working on this genome through the Joint Genome Institute’s  Interpret a Genome for Education Program.  I’ve had students collaborating with this project ever since, and can’t say enough wonderful things about the program.

Based on our in silico results, we hypothesized that Denitrovibrio acetiphilus would be able to use arsenate, selenate, and dimethyl sulfoxide as terminal electron acceptors.  Kyle Denton demonstrated that the organism could grow with these terminal electron acceptors provided, and demonstrated reduction of arsenate to arsenite in growing cultures.  He then determined that the gene identified by our first year students as a possible arsenate reductase was induced in the presence of arsenate.

This has really been a fun project to work on, because I’ve been able to see all my students develop.  Kyle used this as part of his master’s thesis, and did such a great job writing it that I barely had to bug him for details during the writing process.  He’s working on a PhD at the University of Connecticut right now.

It was also a joy to work with the first year students, and engage entering students in “real” research.  Many students from my cohort have gone on to do research in other faculty members labs, and I think about half of them are currently in our master’s program.

I’m so proud of everyone involved in this project!  Thanks, folks!

 

Three students in the Wiatrowski Lab presented their work last week at the Boston Bacterial Meeting.

This meeting is a great opportunity for microbiology students in New England. There were close to 600 people in attendance this year.   Except for the Keynote, which this year was given by Bonnie Bassler, all of the oral presentations are given by students or postdocs, and the entire meeting is run by postdocs and students.  This makes it a great opportunity for our students to present their work to microbiologists in a welcoming setting.

Can’t wait to go back next year!

Yingjao Wang at the Boston Bacterial Meeting

Yingjao Wang at the Boston Bacterial Meeting

Alex Vickers at the Boston Bacterial Meeting

Alex Vickers at the Boston Bacterial Meeting

Songnian Liu at the Boston Bacterial Meeting

Songnian Liu at the Boston Bacterial Meeting

 

 

 

I just set up Twitter for the Biology Department and the BCMB program.

You can find us at @ClarkUBiology and @ClarkuBCMB. Please follow us! We’ll be tweeting seminar announcements and department and program news. I bet you’re wondering why this is better than the e-mail lists. Well, there’s a problem with the e-mail lists, especially if you’re an undergraduate student here at Clark. These e-mail lists are ripped from the registrar, and have to do with your declared major. The Biology Department only sends announcements to Biology majors, and thus, BCMB majors never find out about the events taking place at Lasry. Similarly, Biology students are not on the BCMB majors e-mail list, which is managed through the Chemistry Department, and thus never find out about anything going on north of Maywood Street. If you happen to be a sophomore and haven’t declared a major, then you don’t get any of the e-mails at all.

If you happen to be doing research or outreach this summer, Tweet about it and mention us (put @ClarkUBiology) in your tweet. I’ll happily retweet for the rest of the community to see!

I’ve just organized a fun club for any biologists at Clark interested in learning a bit about computer programming. We’re getting together in the Lasry Lobby this summer, on Tuesdays and Thursdays, at 1:30 PM to start to learn a language of our choice.

We’re going to be using the tutorials at Codecademy.

Here’s a pic of our first meeting!
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We’re all going to work at our own pace, and the purpose of the club is really just to remind us to work on our skills, and provide a supportive environment. Come on down if you are interested. You don’t have to commit to the whole summer.

I’ve started to learn Python. If a bunch of us make progress, I’m going to see about running through the Biopython tutorial.

What can you do with BioPython? Well, a lot, really. You can trim sequences, query ExPASy, download and upload stuff from NCBI, make trees…. I think it’s going to be really fun!

I’m just adding a few pictures from the 2013 Academic Spree Day here at Clark.

Colin Rutner and Erin Thayer are working on the mer operons in Xanthobacter autotrophicus Py2.
HW2013ColinR

Alex Vickers has characterized microbial communities in septic leach fields from the Massachusetts Alternative Septic System Testing Center.
HW2013SpreeAlex1

And Amanda Barbosa has been continuing Kyle Denton’s work with the possible respiratory arsenate reductase from Denitrovibrio acetiphilus.
HW2013SpreeBarbosa1

Lab update:
Wow! I can’t believe that it’s been over a year since the last lab blog update! So much has happened in the lab!

First of all, Corri Stewart defended her Masters Thesis, in May. Congratulations Corri!

We also both said hello and goodbye to Amanda Petrus. Here is a picture of Amanda sampling at the Massachusetts Alternative Septic System Testing Center:

Alex Vickers has been working on a clone library from the leach fields. Guess what lives in the leach fields? I’m not telling just yet – but it was a bit more interesting than we initially thought.

Colin Rutner (BCMB 2013) just got accepted into the BCMB 5th year Masters Program. Congratulations, Colin! Here’s to a productive 2013 in the Wiatrowski lab!

The Wiatrowski Lab would like to extend a heart congratulations to Kyle Denton on the successful defense of his Masters Thesis today!

Kyle and I at his post-defense party!

Kyle is one of the first students to graduate from Clark University’s Biochemistry and Molecular Biology 5th year BA/MA program. Kyle gave a spectacular seminar, and wrote a wonderful thesis. His leadership in our laboratory is already greatly missed.

Kyle will be continuing on for a PhD in the University of Connecticut Health Center’s Genetics and Developmental Biology Graduate Program. Best wishes on your further studies, Kyle!

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