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Biofuel matchmaker: Finding the perfect algae for renewable energy

Streamlined process speeds up algae strain selection, could lower biofuel production costs

A dozen glass cylinders containing a potential payload of bright green algae are exposed to hundreds of multi-colored lights, which provide all of sunlight’s natural hues. The tiny LEDs brighten and dim to mimic the outdoors’ constantly changing conditions. To further simulate a virtual cloud passing overhead, chillers kick in and nudge the algae a little cooler.

A new, approximately $6-million collaborative project is using this unique climate-simulating laboratory system as part of a new streamlined process to quickly pare down heaps of algae species into just a few that hold the most promise for renewable fuels.

Discovering which algae species is best suited to make biofuel is no small task. Researchers have tried to evaluate algae in test tubes, but often find lab results don’t always mirror what happens when green goo is grown in outdoor ponds.

PNNL’s Laboratory Environmental Algae Pond Simulator system, also known as LEAPS, mimics the frequently shifting water temperatures and lighting conditions that occur in outdoor ponds at any given place on earth. This allows researchers to test multiple algae strains with the conditions at different places, but without the cost and time needed to actually grow them at those locations.

The Algae DISCOVR Project — short for Development of Integrated Screening, Cultivar Optimization and Validation Research — is trying out a new approach that could reduce the cost and the time needed to move promising algal strains from the laboratory and into production. At the end of the three-year pilot project, scientists hope to identify four promising strains from at least 30 initial candidates.

“Algae biofuel is a promising clean energy technology, but the current production methods are costly and limit its use,” said the project’s lead researcher, Michael Huesemann of the Department of Energy’s Pacific Northwest National Laboratory. “The price of biofuel is largely tied to growth rates. Our method could help developers find the most productive algae strains more quickly and efficiently.”

The project started this fall and is led by PNNL, out of its Marine Sciences Laboratory in Sequim, Washington. The project team includes three other DOE labs — Los Alamos National Laboratory, National Renewable Energy Laboratory and Sandia National Laboratories — as well as Arizona State University’s Arizona Center for Algae Technology and Innovation.

Algae can be turned into renewable biofuel, which is why scientists want to discover an inexpensive, fast-growing strain of algae. Scientists at Pacific Northwest National Laboratory have developed a system to speed up this search. The unique climate-simulating system uses temperature controls and multi-colored LED lights to mimic the constantly changing conditions of an outdoor algae pond. By simulating outdoor climates inside the lab, the system saves researchers time and expense.

Step by step

The project’s early work relies on PNNL’s Laboratory Environmental Algae Pond Simulator mini-photobioreactors, also known as LEAPS. The system mimics the frequently shifting water temperatures and lighting conditions that occur in outdoor ponds at any given place on earth. The system consists of glass column photobioreactors that act like small ponds and are placed in rows to allow scientists to simultaneously grow multiple different types of algae strains. Each row of LEAPS mini-photobioreactors is exposed to unique temperature and lighting regimens thanks to heaters, chillers and heat exchangers, as well as colored lights simulating the sunlight spectrum — all of which can be changed every second.

The first phase of the team’s multi-step screening process uses PNNL’s photobioreactors to cultivate all 30 strains under consideration and evaluate their growth rates. Algae strains with suitable growth will be studied further to measure their oil, protein and carbohydrate content, all of which could be used to make biofuels. The algae will also be tested for valuable co-products such as the food dye phycocyanin, which could make algae biofuel production more cost-effective. The first phase will also involve evaluating how resistant strains are to harmful bacteria and predators that can kill algae.

Next, the team will look for strains that produce 20 percent more biomass, or organic matter used to make biofuel, than two well-studied algae strains. The top-performing strains will then be sorted to find individual cells best suited for biofuel production, such as those that contain more oil. Those strains will also be exposed to various stresses to encourage rapid evolution so they can, for example, survive in the higher temperatures outdoor ponds experience in the summer.

Outside the box

After passing those tests, the remaining strains will be grown in large outdoor ponds in Arizona. Researchers will examine how algae growth in the outdoor ponds compares with the algal biomass output predicted in earlier steps. Biomass will also be harvested from outdoor-grown algae for future studies.

Finally, the team will further study the final algae strains that fare best outdoors to understand how fast they grow in different lighting and temperature conditions. That data will then be entered into PNNL’s Biomass Assessment Tool, which uses detailed data from weather stations and other sources to identify the best possible locations to grow algae. The tool will crunch numbers to help the team generate maps that illustrate the expected biomass productivity of each algae species grown in outdoor ponds at any location in the U.S.

Data and strains will be made public in the hopes that algae companies and other researchers will consider growing the most productive strains identified by the project.

This project is supported by DOE’s Office of Energy Efficiency and Renewable Energy.

Potential future work not included in the current project could include converting harvested algae into biofuels, examining operational changes such as crop rotation to further increase biomass growth, and assessing the technical feasibility and economic costs of making biofuel from algae selected through this process.

Source: PNNL

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Study refutes how fruit flies developed alcohol tolerance

The common fruit fly, the tiny insect drawn to your beer or wine, has evolved to have an impressive tolerance for alcohol.

More than two decades ago, in one of the first papers using gene sequences to find signatures of natural selection, scientists hypothesized that a molecular change in an enzyme gave the Drosophila melanogaster fruit fly species its superior ability to metabolize alcohol. Scientists concluded that the change they found in the Alcohol dehydrogenase (ADH) protein could be the adaptation that allowed D. melanogaster to colonize ethanol-rich habitats in rotting fruit better than its nearly identical relative, Drosophila simulans.

Kristi Montooth, an associate professor in the School of Biological Sciences, displays a bottle containing fruit flies that are the subject of her research. She is a co-author of a new scientific paper investigating the evolutionary causes of fruit flies’ tolerance for alcohol. Image credit: Craig Chandler

It seemed a logical conclusion that the gene sequence changes that altered amino acids in an enzyme that breaks down alcohol would be the mechanism of natural selection.

However, the authors of a new paper published online Jan. 13 by Nature Ecology Evolution say they have now refuted that hypothesis.

Their findings indicate that intuition and signatures of selection in gene sequence may not be enough for scientists to conclusively solve the puzzles of molecular evolution. Tests also are needed to check how the changes function in organisms.

Using genetic engineering, scientists resurrected the fruit flies’ ADH protein from ancestral species to compare whether the amino acid changes that have occurred in D. melanogaster’s ADH enzyme actually improved the fruit flies’ ability to tolerate alcohol. The answer, conclusively, was “no.”

“This paper takes advantage of modern molecular biology and genetic approaches to test some of those hypotheses,” said University of Nebraska-Lincoln biologist Kristi L. Montooth, a fruit fly expert who co-authored the new study. “It doesn’t dispute that this species has a high ethanol tolerance. But it suggests that the molecular changes that have led to that high tolerance are not in this protein. They must be in other genes in the genome.”

The search for new pathways in fruit flies could offer new clues about alcohol tolerance in humans, Montooth added.

Montooth and University of Chicago researcher Mohammad A. Siddiq conducted experiments with living transgenetic flies in her Lincoln laboratory during October 2015. They found the ADH amino acid changes made no discernible difference in the species’ ability to survive while being fed increasingly heavy doses of alcohol. The flies had been genetically engineered by University of Wisconsin researcher David Loehlin so that their only genetic difference was in the ADH protein sequence.

The experiments with living fruit flies supported the findings of other experiments investigating the enzyme’s biochemistry.

With molecular evolution, scientists seek to identify the specific molecular changes underlying the trait changes that shape the evolutionary family tree.

“We’re generating so much sequence data right now, from so many species, that it’s relatively straightforward to look for signatures of selection in genes and to find good candidates for adaptations,” Montooth said. “But those are just candidates. You have to functionally test them if you want to say those are the variants favored by natural selection.”

Siddiq and Montooth began working together in 2008 after Siddiq took the freshman biology course Montooth taught while on faculty at Indiana University. He began working in her laboratory and co-authored research with her while he was an undergraduate student.

“Despite now being in my fifth year of graduate school, I’m still publishing papers with the professor that I met on my first day of college,” Siddiq said.

Siddiq, who is the study’s first author, is now pursuing a doctoral degree at the University of Chicago, where he is studying with co-author Joseph W. Thornton, a professor who studies the molecular mechanisms of evolution. David W. Loehlin, a post-doctoral researcher at the University of Wisconsin-Madison, also participated in the study, creating the transgenic flies used in the experiments.

Siddiq came to Lincoln to work with Montooth on the fruit fly study, he said, because of her experience and expertise in evolutionary physiology and because of her influence on his career.

“I took her introductory biology class (as a college freshman) and loved it because her passion for evolution and its role as a unifying principle in biology really came through,” Siddiq said. “My appreciation for studying traits not only in the test tube, but in the level of organisms, comes from the experiments I did during my undergraduate years. “

The new study is the first demonstration of an approach combining ancestral sequence reconstruction with the making of transgenic organisms to directly test hypotheses at multiple biological levels, Siddiq said. It’s a broadly applicable approach that in theory could be used with other organisms that lend themselves to transgenic engineering.

“One of the neat things about this paper is that it looks at the effects of this gene’s mutations at the level of how the enzyme functions in the test tube, how it functions in the fly and then the consequences for how the fly deals with ethanol,” Montooth said. “It’s looking at effects of mutations across different levels of biological organization. Each of the co-authors played a role by investigating a different level of organization.”

The study received funding through the National Science Foundation and the National Institutes of Health, including an NIH training grant and NSF fellowship for Siddiq and an NSF CAREER award for Montooth. Loehlin received support from a Howard Hughes Medical Institute postdoctoral fellowship.

Source: University of Nebraska-Lincoln


Researchers Quantify in High Speed a Viper’s Strike in Nature for the First Time

Feeding is paramount to the survival of almost every animal, and just about every living organism is eaten by another. Not surprisingly, the animal kingdom shows many examples of extreme specialization — the chameleon’s tongue, fox diving into snow, cheetah sprinting — for capturing prey or escaping predators.

What factors determine the success/failure of a strike or escape in predator-prey relationships? Image credit: Higham Lab

The antagonistic predator-prey relationship is of interest to evolutionary biologists because it often leads to extreme adaptations in both the predator and prey. One such relationship is seen in the rattlesnake-kangaroo rat system — a model system for studying the dynamics of high-power predator-prey interactions that can be observed under completely natural conditions.

Curiously, however, very little is known about the strike performance of rattlesnakes under natural conditions. But that is now about to change because technological advances in portable high-speed cameras have made it possible for biologists like Timothy Higham at the University of California, Riverside to capture three-dimensional video in the field of a rattlesnake preying on a kangaroo rat.

See video. Links to other videos under “Related Links” below.

“Predator-prey interactions are naturally variable — much more so than we would ever observe in a controlled laboratory setting,” said Higham, an associate professor of biology, who led the research project. “Technology is now allowing us to understand what defines successful capture and evasion under natural conditions.  It is under these conditions in which the predator and prey evolve.  It’s therefore absolutely critical to observe animals in their natural habitat before making too many conclusions from laboratory studies alone.”

A question Higham and his team are exploring in predator-prey relationships is: What factors determine the success/failure of a strike or escape? In the case of the rattlesnake and kangaroo rat, the outcome, they note, appears to depend on both the snake’s accuracy and the ability of the kangaroo rat to detect and evade the viper before being struck.

“We obtained some incredible footage of Mohave rattlesnakes striking in the middle of the night, under infrared lighting, in New Mexico during the summer of 2015,” Higham said. “The results are quite interesting in that strikes are very rapid and highly variable. The snakes also appear to miss quite dramatically — either because the snake simply misses or the kangaroo rat moves out of the way in time.”

Many studies have examined snake strikes, but the new work is the first study to quantify strikes using high-speed video (500 frames per second) in the wild.

Study results appear in Scientific Reports.

In the paper, Higham and his coauthors conclude that rattlesnakes in nature can greatly exceed the defensive strike speeds and accelerations observed in the lab. Their results also suggest that kangaroo rats might amplify their power when under attack by rattlesnakes via “elastic energy storage.”

“Elastic energy storage is when the muscle stretches a tendon and then relaxes, allowing the tendon to recoil like an elastic band being released from the stretched position,” Higham explained.  “It’s equivalent to a sling shot — you can pull the sling shot slowly and it can be released very quickly. The kangaroo rat is likely using the tendons in its lower leg — similar to our Achilles tendon — to store energy and release it quickly, allowing it to jump quickly and evade the strike.”

To collect data, the team radio-tracked rattlesnakes by implanting transmitters. Once the rattlesnake was in striking position, the team carried the filming equipment to the location of the rattlesnake (in the middle of the night) and set up the cameras around the snake. The team then waited (sometimes all night) for a kangaroo rat to come by for the snake to strike.

“We would watch the live view through a laptop quite far away and trigger the cameras when a strike occurred,” Higham said.

Next, the researchers plan to expand the current work to other species of rattlesnake and kangaroo rat to explore the differences among species.

The research was supported by start-up funds from UC Riverside, the National Science Foundation and the Desert Legacy Fund from the Riverside-San Bernardino Community Foundation.

Higham was joined in the study by Clint E. Collins, a former graduate student in his lab and now a postdoctoral scholar at the University of Idaho; and Rulon W. Clark, Malachi D. Whitford and Grace A. Freymiller at San Diego State University, Calif. Higham and Clark have been interested in predator-prey interactions for more than a decade.

Researchers Quantify in High Speed a Viper’s Strike in Nature for the First Time

Feeding is paramount to the survival of almost every animal, and just about every living organism is eaten by another. Not surprisingly, the animal kingdom shows many examples of extreme specialization — the chameleon’s tongue, fox diving into snow, cheetah sprinting — for capturing prey or escaping predators.

What factors determine the success/failure of a strike or escape in predator-prey relationships? Image credit: Higham Lab

The antagonistic predator-prey relationship is of interest to evolutionary biologists because it often leads to extreme adaptations in both the predator and prey. One such relationship is seen in the rattlesnake-kangaroo rat system — a model system for studying the dynamics of high-power predator-prey interactions that can be observed under completely natural conditions.

Curiously, however, very little is known about the strike performance of rattlesnakes under natural conditions. But that is now about to change because technological advances in portable high-speed cameras have made it possible for biologists like Timothy Higham at the University of California, Riverside to capture three-dimensional video in the field of a rattlesnake preying on a kangaroo rat.

See video. Links to other videos under “Related Links” below.

“Predator-prey interactions are naturally variable — much more so than we would ever observe in a controlled laboratory setting,” said Higham, an associate professor of biology, who led the research project. “Technology is now allowing us to understand what defines successful capture and evasion under natural conditions.  It is under these conditions in which the predator and prey evolve.  It’s therefore absolutely critical to observe animals in their natural habitat before making too many conclusions from laboratory studies alone.”

A question Higham and his team are exploring in predator-prey relationships is: What factors determine the success/failure of a strike or escape? In the case of the rattlesnake and kangaroo rat, the outcome, they note, appears to depend on both the snake’s accuracy and the ability of the kangaroo rat to detect and evade the viper before being struck.

“We obtained some incredible footage of Mohave rattlesnakes striking in the middle of the night, under infrared lighting, in New Mexico during the summer of 2015,” Higham said. “The results are quite interesting in that strikes are very rapid and highly variable. The snakes also appear to miss quite dramatically — either because the snake simply misses or the kangaroo rat moves out of the way in time.”

Many studies have examined snake strikes, but the new work is the first study to quantify strikes using high-speed video (500 frames per second) in the wild.

Study results appear in Scientific Reports.

In the paper, Higham and his coauthors conclude that rattlesnakes in nature can greatly exceed the defensive strike speeds and accelerations observed in the lab. Their results also suggest that kangaroo rats might amplify their power when under attack by rattlesnakes via “elastic energy storage.”

“Elastic energy storage is when the muscle stretches a tendon and then relaxes, allowing the tendon to recoil like an elastic band being released from the stretched position,” Higham explained.  “It’s equivalent to a sling shot — you can pull the sling shot slowly and it can be released very quickly. The kangaroo rat is likely using the tendons in its lower leg — similar to our Achilles tendon — to store energy and release it quickly, allowing it to jump quickly and evade the strike.”

To collect data, the team radio-tracked rattlesnakes by implanting transmitters. Once the rattlesnake was in striking position, the team carried the filming equipment to the location of the rattlesnake (in the middle of the night) and set up the cameras around the snake. The team then waited (sometimes all night) for a kangaroo rat to come by for the snake to strike.

“We would watch the live view through a laptop quite far away and trigger the cameras when a strike occurred,” Higham said.

Next, the researchers plan to expand the current work to other species of rattlesnake and kangaroo rat to explore the differences among species.

The research was supported by start-up funds from UC Riverside, the National Science Foundation and the Desert Legacy Fund from the Riverside-San Bernardino Community Foundation.

Higham was joined in the study by Clint E. Collins, a former graduate student in his lab and now a postdoctoral scholar at the University of Idaho; and Rulon W. Clark, Malachi D. Whitford and Grace A. Freymiller at San Diego State University, Calif. Higham and Clark have been interested in predator-prey interactions for more than a decade.

Source: UC Riverside