Benthic algae: the iPhone 4S of algae?

Introduction: the next generation of-next-gen biofuels
Algae are often billed as "next-gen" biofuels, because they're expected to surpass the productivity and envitronmental sustainability of first-generation biofuels based on corn and soy.  However, new research published in the journal AMB express suggests there's an even newer generation of biofuel on the horizon.  Will biofuels soon outpace Apple and its iPhones in terms of product cycles?  Only time will tell.

The problem: too much water
One major roadblock for producing algal biofuel is separating algae from the water in which they grow. To collect algae from current systems strategies like filtration, centrifugation, gravity, and evaporation are used.  Each of these approaches has its drawbacks, which result in increased cost of production.  Filters are expensive and need to be cleaned.  Centrifugation takes a lot of energy.  Gravity and evaporation take time and space that could be used for growing more algae. 

It takes a lot of energy to separate the algae from the water in this raceway system.

The new kid on the block: benthic cyanobacteria

Not all algae, however, grow as single cells in the water that need to be scooped up in some way.  Some algae scoop themselves up into large biological mats.  In July, three researchers from Cawthron Institute in New Zealand investigated three different species of benthic cyanobacteria that harvest themselves!  What are benthic cyanobacteria, you ask? Benthic organisms come from the seafloor or lake bed (the benthos). Cyanobacteria used to be known as blue-green algae until someone realized that they're in fact bacteria and not algae at all.  That said, they have very similar properties to eukaryotic algae.  For instance, they can turn CO2 into sugar and lipid using sunlight.  They can store other nutrients for use.  They are a potential feedstock for biofuels.

What makes these particular algae unique is their ability to grow in easily collectible mats.  However, as I've mentioned in previous posts, many wild organisms that look useful don't necessarily grow in culture.  Indeed most organisms (over 99%) don't grow in culture.  The challenge before these authors was finding the culture conditions that would produce the maximum amount of bacteria.

A mat of benthic cyanobacteria from New Zealand that has collected on a rock

The hunt for the substrate: what's handy?
The main challenge before the authors was finding a substrate for the bacteria to grow.  In order to figure this out, they literally just looked around the lab.  As you can see from the figure below, the authors just stuck common lab items into bioreactor bags and hoped something would stick.

The various conditions researchers tried to grow this new form of algae.  Panels A and B are bioreactors held horizontally or vertically.  Panel C is a bioreactor bag with silicone lab tubing.  Panel D is clearly just a bottle brush in a bag.  Panel E is another piece of silicone tubing folded in on itself.

The result: great green glop!
Surprisingly, the authors were able to find optimal growth conditions just by grabbing what's around.  It turns out the looped silicon grew 2 times more cyanobacteria than the other growth strategies.  That said, the final yield even from the best bioreactor was 7 times lower than growing normal algae in an open pond without CO2 amendment and far lower than growing normal algae in a photobioreactor.  I guess that's what makes this organism a next-generation next-gen biofuel.  If further optimization can occur (through improved culture conditions, genetic manipulation, further bioprospecting, or more), perhaps this cyanobacteria will outperform todays best algae.  

The cyanobacterium Phormidium autumnale after 36 days of growth in a photobioreactor in and out of water (a and b).

Shedding some light on dark organisms

INTRO: dark organisms defined

Physicists have dark matter.  Biologists have what I like to call dark organisms.  These are the myriad organisms that cannot grow in culture, and thus remain relatively unstudied. Consider that some estimates suggest that only 1 in 1000 bacteria can be successfully grown in the lab right now.  That leaves a lot of work to do to to uncover the literally millions of organisms producing antibiotics, anti-cancer therapeutics, and biofuels of tomorrow.

COAL-BED METHANE: a biological process in need of refinement

Since the 1980's, it's been known that some of the natural gas found in coal deposits came from bacterial organism (Schoell 1980).  Of late, companies like synthetic genomics and Luca technologies have developed chemical mixes that can stimulate the bacteria living in coal deposits and get them to eat more coal and turn it into methane.This is a huge potential source of (relatively) clean energy from coal.

Consider, however, that many extreme organisms converting coal into cleaner methane die when moved from the nutrient-poor depths of a coal mine to the nutrient-rich surface, or to the lab. 

We need to understand and grow the organisms producing our future methane, if we want more of this energy.

A GENETIC LOOPHOLE: why culture the organism, when you can get the sequence anyways?

A recent paper in the Journal of Bacteriology turned me on to a new solution to the problem of studying unculturable bacteria.  Why not just sequence the bacteria where they live?

Now that we have next-generation sequencing technology and the complete sequence of almost every model organism, it is possible to rapidly sequence a small amount of starting DNA collected from the wild.

In this JB paper.  The authors collected about a gallon-and-a-half of water from an offshore oil field, isolated the DNA, and assembled the genome of an entirely new species found growing in that oil field. They compared the DNA sequences they collected to a reference organisms sequence grown in the lab.  when they assembled all of their DNA sequencing data, they found a 1.6 million base sequence representing a new strain of the species Methanococcus maripaludis called X1. 

While the authors found some inconsistencies and point mutations in their data, they suggest these results do not indicate multiple strains in their water, but rather technical errors they were able to sort out manually.  Either way, the data from strain X1 will lend insight into the various genes present in nonculturable strains of this organism.

THE NEXT STEP: using these genes in culture

It's an exciting step to see a new strain of an organism sequenced without culturing it first.  It's a big step forward to shedding light on these dark organisms.  The next step will be when we successfully use the genes from these organisms to produce new antibiotics, therapuetics, and fuels. 

REFERENCES

 Schoell M. 1980. The hydrogen and carbon isotopic composition of methane from natural gases of various
origins. Geochim. Cosmochim. Acta 44:649–61

Wang X, Greenfield P, Li D, Hendry P, Volk H, Sutherland TD. Complete Genome Sequence of a Nonculturable Methanococcus maripaludis Strain Extracted in a Metagenomic Survey of Petroleum Reservoir Fluids. J Bacteriol. 2011 Oct;193(19):5595. PubMed PMID: 21914896.

Who doesn't love protein and fat?

INTRO: from 50,000 feet to angstroms

I've begun this blog about bioenergy research with two articles seemingly unrelated to the field of bioenergy research.  I did this to begin with a 50,000 foot view of the field of bioenergy research and look at its challenges and prospects more broadly.  As a graduate student myself, I know what it's like to spend a lot of time down in the deep dark depths of a problem without coming up for air for some time.  So, it's been refreshing to begin this project looking from 50,000 feet up rather than in its depths.

However, today I'd like to zoom in from 50,000 feet to delve into the depths of bioenergy research with a paper that just came out focused on algal biology.  I don't think I'll get too lost in the depths because this paper does a good job of linking its research to the big problem it's trying to solve.

THE BIG PICTURE: veal algae?

One way to get more biodiesel from algae may be to get each individual algal cell to produce more lipids -- perhaps to even fatten them up like some kind of veal calf.  Indeed, under certain conditions, many algae will store energy as lipids in specific intracellular structures called oil bodies.  Some algae can reach a lipid content of 70% by dry weight.

In a recent paper, appearing in the journal Proteomics, Hoa M. Nguyen and others set out to discover the protein machinery that make up the oil bodies in the hopes that some day this knowledge will translate into a way to fatten up individual microalgae to produce high-yielding biofuel.

Scanning electron micrograph image of the microalgae C. reinhardtii

THE STRATEGY: classic purification with new analysis

 It has been possible to purify oil bodies using classic, established biochemical techniques since the mid-nineties.  However, a detailed profile of all the proteins found in these purified oil bodies has yet to be published.  Nguyen and others decided to grow a mutant strain of the model microalga C. reinhardtii under conditions known to produce oil bodies. They then processed the algae through 7 purification steps to recover pure oil bodies.  They tested these oil bodies and found a relatively pure fraction containing high levels of the lipid triacylglycerol (TAG) as well as a known component of oil bodies (major lipid droplet protein).

Once they knew they had a respectably pure sample, they ran the peptide versions of their proteins through a mass spectrometer and identified the proteins present in their sample.

Summary of Chlamydomonas oil body purification and proteomic analysis. Nile red staining labels oil bodies.  SDS-PAGE stain reveals purification of oil body fraction. Abbreviations: M, protein marker; MLDP, major lipid droplet protein; TLC, thin-layer chromatography; TAG, triacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine. Note: Numbers below the gel lanes correspond to the major steps of oil body purification as indicated in the figure. (From Nguyen et al. 2009)

THE RESULTS: well, what do we have here?

The authors found 330 relatively abundant proteins in their oil body purification. To see the top proteins identified check out table 1 from the paper.

To get further understanding, the authors put these proteins through a series of bioinformatic programs to figure out what their functions might be. As a result, they found that many of the proteins they discovered were involved in metabilism, transport, vesicle trafficking, and redox. Many of the highly abundant proteins are known to be involved in lipid metabolism.

THE ENTICING FUTURE: what to do with this new knowledge

One aspect of this paper I enjoy is that it's a dataset brief, meaning it's very high quality, but preliminary data.  In the end, its findings yield more questions for the field than answers, and also some enticing possibilities.

One such question is, how can we manipulate the proteins found in oil bodies to increase the oil content of algae? To this end, the authors provide an enticing possible answer.  For instance, the authors point out "Oil body-associated lipases could be involved in TAG degradataion processes occurring concurrently to TAG synthesis. Down regulation of such lipases might boost oil accumulation under non-stress conditions".  It would be exciting and promising, indeed to see if knockout of these newly-identified lipases could promote oil body formation.

In the end this paper offers an enticing beginning to a stream of new questions.  What is the role of highly abundant proteins of unknown function found in the oil bodies?  What proteins are found in oil bodies in the context of growing oil bodies vs shrinking oil bodies? Can the proteins found in oil bodies be manipulatied to yield more oily algae?

I look forward to follow the research of these authors and others as they continue the basic biological research into algal oil production processes. Hopefully someday soon, we will have more answers than questions!

REFERENCE:

Nguyen HM et al. "Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: With focus on proteins involved in lipid metabolism" Proteomics (2011) issue 11. PMID 21928291.

Laser algae?

A recent paper got me thinking about a far-out solution to a major challenge to industrial-scale algae production

PROBLEM: shallow light
For algae production, one major barrier between bioenergy yields that can be reached in theory (or even in the lab) and the yields achieved in the field is the penetrance of light into a pond. In the lab, a dense culture of algae gets exposed to more light through the turbulence of a constantly swirling flask. In a pond, only the algae at the surface get exposed to light and produce energy.
.

light-blocking effect of photosynthetic algae. NPQ is nonphotosynthetic quenching; P is photosynthetic energy. (Melis 2009)

Industrial yields could improve if more algae could get access to more light.

THE PRACTICAL SOLUTION: cut the chlorophyll

About two years ago, Anastasios Melis proposed a counterintuitive, yet elegant, solution to this problem. He proposed to reduce the chlorophyll content in each algal cell. You see, a lot of light ends up hitting chlorophyll overloaded with photons. If the amount of chlorophyll is reduced, light can go deeper into a culture and charge more algae, possibly improving overall yield.

light-blocking effect reduced with less chlorophyll. NPQ is nonphotosynthetic quenching; P is photosynthetic energy. (Melis 2009)

THE FAR-OUT SOLUTION: laser algae
A recent article appearing in Nature photonics just totally blew my mind. They turned human cells into lasers! The authors simply over-expressed green fluorescent protein (GFP) in cells, pumped up the GFP with pulses of light, and the cells fired off laser light as a result. The authors and others have proposed to use this technology to get light to previously inaccessable places in human tissue, and to potentially activate light-sensitive drugs.

However, why not use this technology to get light deeper into an algal culture?

A human cell emitting laser light in a dish. (Gather MC and Yun SH 2011)








THE FAR-OUT PROBLEM: too much equipment
In order to get cells to fire like a laser, you can't just overexpress GFP. You need a big apparatus to complette the laser. In the original paper, cells were plased in a resonator chamber and the GFP was activated with pulses of light to pump up the laser and cause it to fire. This is obviously a little too equipment intensive for game time.

However, there are ways to get single cells to produce light. Indeed single cells do this readily in nature!

The entire apparatus for creating a cell-based laser. Bottom, a microscope pulses light at a cell (blue arrows) The cell (placed between rectangular resonators) emits laser light (green arrows). d=20 micrometers. (Gather MC and Yun SH 2011)








BACK TO EARTH: the lesson here

While writing this post, I realize that the idea of cell-based lasers is way too ridiculously crazy to ever be used to produce algae based biofuel. However, I think another lesson can be drawn from this paper.

These authors used commonly available biological reagents to accomplish extraordinary tasks. For instance, basically any cell biology lab can create a GFP cell like the one described in this paper, and many do every day. Yet, the authors were able to take a common reagent that people have used for almost 20 years and do something extraordinary with it.

Perhaps, there is hope for some existing reagent to do something incredibly cool with algae. Could expressing the luciferase gene make algae glow like fireflies and get more light to more cells? Could a common mitogen enhance their growth? What other common reagents are available that could boost the yield of industrial algal biofuels?

REFERENCES
Gather MC and Yun SH. "Single Cell Biological Lasers". Nature Photonics. volume 5 (2011): 406-410.

Melis A. "Solar energy conversion efficiencies to photosynthesis: Minimizing the chlorophyll antennae to maximize efficiency". Plant Science. volume 177 (2009): 272-280.

A new yeast and a new hope

INTRODUCTION: What does beer have to do with anything?

I came across this recent PNAS paper during my weekly bioenergy search on pubmed. It confused me because the topic of this paper has NOTHING to do with bioenergy. However, I want to present this paper because (1) I think this paper’s findings have important implications for the field of bioenergy and (2) the main topic of this paper focuses on another favorite topic of mine – BEER!

HISTORY: The domestication of beer yeast

Before I get into the great finding in this paper, I’d like to backtrack a little bit to the history of beer production to introduce the problem.

It’s well understood that crops used to make beer, bread, and the like were domesticated by humans during the agricultural revolution, and that as we cultivated those plants, they changed. What is less understood is how the microbes we used in the brewing process were unwittingly cultivated by our ancestors, long before the discovery of microbes themselves.

Early in our brewing history, wild yeast fermented our grains for us. These yeast still exist today, and still brew for us. These yeast are called ale yeast, and (obviously) produce ale-type beer. You could go to many groceries (or basically any specialty beer store) and find some of my favorite ales like Redhook ESB, Fat Tire Ranger IPA, and La Fin Du Monde Belgian-style ale. These yeast ferment grain into alcohol at near room temperature (about 65 degrees F), grow at the top of the fermentation vessel , and either are, or directly evolved from the baker’s yeast Saccharomyces cerevisiae.

Up until the 15th century, ale yeast were the only game around. No problem for me, those are my favorites. But, if you’re like most people, you prefer lager beer. These beers, which were first brewed around modern Germany, ferment grain at a much colder temperature than ale yeast (45 - 60 F), live at the bottom of the fermentation vessel, and have not been found naturally in the environment – only in the brewing environment. These yeast are named Saccharomyces pastorianus. More commonly, it’s known as lager yeast, and it produces some of the most popular beers in the world, including Budweiser, Coors, and PBR. Some of the existing European ancestors to these American giants include Stella Artois, Heineken, and Budvar.

PROBLEM: Whence the lager?

We know that lager yeast arose in the 15th century from historical accounts. We also know some about its biological history. Two completely separate species of yeast (or more) can combine their genetic codes in a process known as hybridization. We know that lager yeast has the genetic code from multiple wild yeast inside of it. Part of its code comes from the ale yeast S. cerevisiae. Some of its code matches up with a yeast known to contaminate the brewing process known as S. bayanus. However, the origin of much of its genetic code remains unknown. It has been hypothesized that lager yeast is made of a hybrid of ale yeast, the contaminant yeast, and some third lager yeast, or possibly it is a hybrid of ale yeast and some yet undiscovered strain of contaminating S. bayanus yeast. The authors of this paper set out to find the origin of the lager yeast species.

THE HUNT: Know your oaks

The S. bayanus contaminants can be found naturally in the wild. They like to grow in the bark of oak trees in the northern hemisphere and beech trees in the southern. How do these yeast manage to contaminate beer? Well, brewing equipment was often made of sturdy oak in the days long before sterilization. It stands to reason that our modern contaminants arose from oak trees and oak casks millennia ago.

The authors chose related beech trees to look for the parent of the lager yeast. Others have looked in the oak forests of Europe to find the lager yeast. These authors chose to look someplace else. Because lager yeast are a cold tolerant species, the authors decided to look in the consistently cold beech forests of Patagonia, in Peru. Lo and behold these authors found and named a new species (S. eubayanus for true bayanus) all the way in the forests of South America. So, now we can thank South America for potatoes, tomatoes, and lager beer!

This is the first article I’ve ever read with a new species classification in it. I highly recommend skipping to the end where the species is classified in both English and latin. Pretty classy stuff, science!

HOW THEY DID IT: The yeast needle in the beech haystack

You can’t just stroll around the forests of Patagonia with a magnifying glass expecting to find lager yeast. Here’s what these authors did to see the forest of microbes in the trees. The authors took 133 samples of bark and soil that grew around three different species of beech trees as well as yeast that grow on fungi on top of beech trees. From these 133 samples, the authors isolated yeast species and purified the DNA from individual strains. They then chopped up the DNA using a restriction enzyme and compared the resulting banding pattern of the new yeast with the known cold-tolerant yeast S. bayanus (that old contaminant) and S. uvarum (a yeast involved in cider production). The authors were able to group their selected yeast strains into two populations (Pop. A and Pop. B)using this analysis. Finally, they found the lager yeast ancestor by sequencing the complete genome of one representative strain from populations A and B, and comparing those sequences to the modern lager yeast, the S. uvarum cider yeast, and the S. bayanus brewing contaminant yeast. The authors found that their Patagonian species A and B differed from each other in sequence by about 6-8%. They also showed these species are different species experimentally by showing their spores are highly inefficient at hybrid crosses. They found that species B only differs from S. uvarum cider yeast by 0.52%. Interestingly, they found that the sequence of species A almost perfectly matches the non-ale-yeast portion of S. Pastorianus lager yeast (a divergence of only 0.44% by sequence). Thus the new species S. eubayanus was identified and named.

CONCLUSIONS: What on earth does this have to do with biofuel?

Half a millennium ago, some brewers must have unwittingly brewed their favorite ales in Patagonian beech casks. Their haphazard use of dirty, yeast-ridden equipment resulted in a blockbuster yeast strain that produces some of the most popular beers in the world.
We are now in the process of actively domesticating microorganisms like oil-producing algae, methane-producing bacteria, and alcohol-producing yeast. Many of these organisms can be hybridized with their cousins and genetic modification can make even more precise changes and include even more diverse genes from far-distant organisms. However the question remains: will we be able to rationally beat what our ancestors were able to do by chance? Are we limiting ourselves by using clean equipment? Maybe we need to roll the dice, throw some muck into our experiments, and see what happens.

My mission

This blog seeks to discuss some of the big new findings in the field of Bioenergy research, to make connections between other biological discoveries and questions in the field of Bioenergy research, and to frame them in terms of the larger challenges the field faces.

I'm new to bioenergy and coming from biomedicine. I'm hoping this blog will be an opportunity for me to start exploring the world of bioenergy and make connections between it and other fields of biology.