MICROBE 2018 recap - Tales of Biogas Core Microbial Communities: The Research, The Rant, The Learning Process

"Research is easy! You have your hypothesis, your starting point, experimentation, perhaps some optimizations and options you take along the way followed by results, analysis and whalah, you are done and have forever changed the world..."

... SAID NO ONE EVER!

Here's an example from a mathematical modeling of infectious disease class I took several years back while still in grad school.

The plan:

Everything goes smoothly...splendidly even, and we all take tea breaks as our analyses run seamlessly without errors or missing/wrongly formatted data leading to a totally understandible, interpretable results that confirms all our hypotheses...

Reality:

By the end, I think we all just wanted a stiff drink.

And this is research! 

The glory and terribleness of it - it's exciting, frustrating and above all a learning process.

The Research

Poster Session:

Impact of Inoculum and Starting Parameter of a Fermenter on Digesting Microbial Communities and their Biogas Production

PHR Mallinger, MC Melendrez (google scholar), RC Fink (google scholar)

St. Cloud State University, St. Cloud MN, USA

In 2017 I was brought in to St. Cloud State University, a primarily undergraduate institution, under a research grant acquired by Dr. Ryan Fink from Xcel Energy. My mission... bioinformatic analysis of microbial communities inhabiting a mesophilic anaerobic digester creating biogas. We had goals - (i) Who is there? (ii) what are they inferred to be doing (iii) prove that they are actually doing what you are inferring that they are doing and reconstruct the pathways (iv) can we toy with inputs to maximize biogas and digestate output and how does this impact methane pathways (v) can we define a core biogas community 'mixture'. The literature has been back and forth on all of these counts with few generalizations that can be made given the specificity of the experiments which, not surprisingly, dictate a lot of what will be in your microbial community and how much biogas is produced. Xcel's interest? Conversion of biogas to electricity of course.

Myself and our first year Master's student, Paige, set out to explore the world of microbial biogas communities...

Hypothesis: Inoculum ratios will impact biogas production and microbial community composition

Did the heavens open up and the doves fly down at the 'profoundness' of this hypothesis? Of course not, but when setting up downstream experiments aimed at looking at reconstruction of metabolic pathways associated with methane production in light of the microbial players as a function of digester set up, starting inputs and resulting biogas and digester outputs... this is a feasible master's project place to start.

Sometimes I feel that the 'basic' stuff... y'know the necessary experimentation that leads to the potential for 'sexier' science is pish-poshed more than it should be. It's all a process, this was ours.

Learning Objectives/Workflow:

• Student will be able to describe bacteria that contribute to fermentation pathways and methane production in anaerobic digester environments
• Student will be able to analyze microbial community composition over time using QIIME2
• Student will be able to describe how to conduct metagenomic sequencing for microbial community analysis at the 16S rRNA level
• Student will be able to hypothesize how the microbial community will change given different chemical or foodwaste inputs

First Reality Check: No anaerobic digester, hmmmm - looked like the install was delayed until we didn't know when!

Alternative that doesn't break the funding bank: Buy benchtop fermenters, not the perfect analog but allowing us to still explore the hypothesis and learning objectives at least until the digester is up and running...

"Rawr!" - http://www.oxebar.com.au/fermentasaurus/

Learning Objective add-on:

• Student will acquire and set up a benchtop fermenter system - Fermentasaurus
• Student will conduct experiments varying foodwaste and manure inputs and measure biogas and digestate chemical composition over time

What the student hears - 

"Buy a beer fermenter, 

put on some mucking boots 

and go shovel some cow manure...

have fun!"

Other research 'realities' we came upon:

• Limited availability of our GC-FID for measurement of methane composition of the biogas we had stored in biogas bags.
• Unexpected leakage of gas from biogas despite our efforts and sealing via duct tape and running control bags to see if that was truly the case or we didn't have any methane produced.

via Gfycat

• Logarithmic sampling design means 4 am lab trips (ah the life of a grad student) and hence needing building access.
• Super foaming in experiment #2 due to the food hall deciding we really must want all the oil and fat from their fryers so that's what we primarily got in our foodwaste buckets.

In the end...we got 3 experiments, the last one in replicate. We got chemical measurements via an agricultural soil testing kit, we got our longitudinal samples out for sequencing and we got our compositional analysis going...

From the Abstract: 

Biogas output in fermentors is determined by a diverse microbial community, with methanogens as the main methane producers. Methanogen establishment in the fermentor community depends on environmental conditions and size of the initial inoculum, where they typically reside. In this study, we manipulated organic waste composition and amount of initial inoculum to test the impact of these parameters on anaerobiosis onset and biogas output. Additionally, we investigated how sequencing experimental design impacts microbial composition outcomes. 

What did we do:

• Three experiments were performed over 120 to 198 hours in a benchtop fermenter system (Fermentasaurus®) using food waste sourced from St. Cloud State University and a manure inoculum from a local farm. 
• We used ratios of 1:10 or 1:5 of manure vs. feedstock in each experiment. 
• Digestate chemical composition was determined by soil chemical testing. 
• The microbial community was characterized using bacterial and archaeal specific 16S rRNA primers and high throughput sequencing with Illumina Miseq. 

What did we learn?

• Experiments E1 and E2 indicated food waste composition had the ability to impede microbiological production of biogas and the onset of anaerobiosis. 
• Ammonia production can inhibit biogas production and microbial community stability. Our E2 food waste was rich in fat resulting in higher ammonia content and lower biogas output (788 mL total biogas) 10-fold lower than E1 (3102 mL total biogas). 
• The increase in ammonia and higher fat content in E2 might have negatively impacted the microbial community thus affecting biogas production. 
• Experiment 3 (E3) produced 5242 mL and 4770 mL total biogas respectively (biological replicates conducted in E3). Assuming an electrical conversion efficiency of 35% where 1m3 = 2.14 kWh of electricity - the benchtop systems produced 11.22 Wh for fermenter 1 and 10.21 Wh for fermenter 2 of electric potential. Enough to power a single christmas light bulb (like the the ones that come on the string of lights...)

Move over actual electrical sockets, 

me and my beer fermenter got this!

via GIPHY

• Fluctuations in biogas output, potassium, magnesium and calcium support an inflection point of microbial community compositional transition followed by stabilization.
• Sequencing experimental design is important in obtaining an accurate reflection of the methane microbial communities within the fermenter systems. We ran 3 designs for sequencing, two traditional (V4 region and Prokaryotic primers) and primers that would still get the bacterial diversity but also pull out more archaeal diversity.
• While our 3rd design definitely resulted in a significantly increased representation of methanogens in the system - it will come at the cost of total species richness estimated. So you have to consider your question...where is your research focused? Are you still hitting your rarefaction plateaus? What is the cost/benefit of the design - and we are still exploring all of this.

• There was a delayed 'bloom' of Methanosphaera that popped up in our experiments suggesting that this organism may be involved in the reduction of biogas or metabolizing the accumulation of wastes (which cannot be removed in the batch fermenter system we had set up) as the system reached saturation.

Earth shattering?

Perhaps not.

Informative?

Well, yes.

Fodder for future experimentation and questions we can look at?

Absolutely.

The Rant
Don't worry...it's a short one.

Whether you agree with the experiment or not. Whether you like the mentor, advisor, student, project, grant, the lab mascot...whatever, or not. In the end you need to realize these projects are about learning, they take time, effort - many times unpaid effort on the part of students and faculty.

So when Paige mentioned to me that an older gentleman had simply come up, snarked at her poster, told her it was worthless and had already been done, then stalked away without allowing her to take him through it - I was livid.

There is always room for improvement and new ideas. That's the essence behind constructive critique. But flat out telling a student something they've spent a lot of time and effort on is absolutely worthless is simply unacceptable. For new students unseasoned in the world of scientific scrutiny - experiences like that can be scarring.

Thankfully that was the only unsavory experience she had with the vast majority of you offering helpful insight and constructive feedback!

The Learning Process

Aspects of this project were very frustrating for Paige, but she powered through. As with any graduate student program in research it's about learning to be comfortable with the unknown, unpredictable aspects of research and be flexible to what that means for your question, objectives and hypothesis. That sometimes we can get lost in the logistics, the details, the day to day manure mucking. I could see how it would be easy to lose sight of 'the point' of it all and what you are really trying to learn or accomplish when covered in manure. I was happy when, in the end, she rediscovered her interest in the work, the insights we were able to gain and where it could take us in the future.

Fear not...your manure mucking will not have been in vain!