José Olivares and Dick Sayre at Los Alamos National Laboratory

José Olivares and Dick Sayre at Los Alamos National Laboratory

by David Schwartz

As the National Alliance for Advanced Biofuels and Bioproducts, NAABB, comes to the end of the second year in its three-year lifecycle, it’s looking more and more like Aquatic Species Program: The Next Generation. Armed with a vast array of scientific resources and brainpower, this broadly based, government funded, research program into the functional future of algae, shows great potential to deliver the goods that the ASP determined were possible, or at least not impossible.

The nearly 40-member consortium, connecting national laboratories with university research departments with private industry, targets major objectives in Algal Biology, Cultivation, Harvesting/Extraction, and Conversion to fuels. Coproduct development is included in the mix, as well as Economic/energy-balance modeling and resource management components to establish the technologies’ viability for overall sustainability, as well as profitability and flexibility during the evolution of the biofuels markets.

NAABB members are developing coproducts in large scale marketable livestock and mariculture feeds, through the production of lipid-extracted micro and macro-algae (LEA). The industrial Coproducts program also involves the development of synthetic natural gas production, thermal energy from LEA, bioplastics from proteins and lipids and feedstocks for the nitrogen chemical industry.

Conducting this orchestra of discovery is Los Alamos National Laboratories’ (LANL) director of biofuels research, Dr. José Olivares, who has shepherded this project since the proposal stage. Working closely with him to coordinate the gargantuan effort is the esteemed biologist, Dr. Richard Sayre, who recently relocated to LANL from his lead role at the Donald Danforth Plant Science Center, in St. Louis, MO.

We spoke recently with José to get his first hand account of the progress so far, and what to expect going into the third and final year of this landmark project.

We are approaching the end of the second year of the NAABB, the largest government-funded algal research consortium to date. How would you assess the progress so far?

We’ve had a lot of progress. In the last two years we’ve met a number of milestones and deliverables in all of the different areas of research we have going. For example, we hit a major milestone in delivering a report to the DOE in which we isolated and screened over 500 algal isolates from nature. And by February of this year we had actually isolated about 1300 strains of algae from prospecting of natural sources around the U.S., and screened about 900 of those for their productivity as far as growth and lipid production.

Of those 900, we went into a second tier screening with 60 of those isolates. And of those 60, we identified a number of them that look extremely promising, with productivities that are much higher than our gold standard, Nannochloropsis salina. These are new isolates of other organisms, three of which we are taking to outdoor cultivation at our Pecos site, at Texas A&M. We are hoping they are as productive outside as they are in the lab.

Test candidate strains for growth in different media Brooklyn College

Test candidate strains for growth in different media Brooklyn College

Are you ready to reveal what those superstrains are at this point?

No, I’m not. They are being very closely protected, as you can imagine. We will make them available once Brooklyn College, the owner of that intellectual property, allows us to make them public. These are natural strains, so the IP is in knowhow, and we want to make sure that our partners get a jump-start on them.

Eventually what we hope for is that we can take them on to some engineering development, but that I suspect is going to be outside of the current NAABB funded project. We only have one more year to go, so we’re focusing primarily on developing transgenic tools for other organisms that are more popular, such as Nannochloropsis salina and Chlorella protothecoides.

Another set of milestones in the algal biology side dealt with developing transgenic tools for three species: Chlamydomonas reinhardtii, which is our model species, Botryococcus braunii and Chlorella protothecoides. And we delivered a report to DOE earlier this year that showed that we were developing a nice systems biology pipeline towards understanding the genome and transcriptome of these organisms.

We have developed a number of tools that will help us transform these organisms into more productive, more robust, candidates. In the process of doing that we’ve also identified a number of other organisms that were contaminants to these original ones, and that presented us some nice new sources of information, for example, Nannochloris and the Tetraselmis species of algae, both of which have very different and very particular biological characteristics that we’re trying to exploit for the program.

We are developing a strategy to transform Chlorella and Nannochloropsis species towards more productive and more robust organisms. It’s been hard going but we’ve worked out several pathways by primarily trying to understand how to engineer the chloroplast of the organism and by understanding the metabolic function of a number of enzymes, and this will help us design new genetic tools into these organisms. This part of the project is happening here at Los Alamos National Laboratory, as well as at Texas A&M, University of Arizona, Phycal and Targeted Growth. There are also minor partners working on new molecular developments in Chlamydomonas reinhardtii from UCLA, Washington University St. Louis, and the Danforth Plant Science Center.

In the non-transgenic area, we’re developing a couple of methods with our partners at the University of Arizona to adapt current organisms to become better producing and more robust.

In the area of adaptive evolution we’ve been working with Chlorella protothecoides primarily. The University of Arizona has developed methods to have it grow faster under limited phosphate conditions. And, as we know, phosphate is going to be a limiting nutrient for any kind of agriculture. This work has been a series of adaptive evolutions under a chemostat selection series that reduced the phosphate availability of the organism and selected for the best growing isolates of the culture, regrowing them with even more limited availability of phosphate, while keeping their growth rate and productivity very high and effective.

At LANL we are using flow sorting and selecting subpopulations of algae that have different lipid properties; we can choose either end of a bell curve where we can select algae with very high or very low concentrations of lipid. When we select the top part of that curve and regrow it we end up with an isolate of algae that has a 50% increase in lipid content compared to wild type. The culture stays healthy and can be maintained for many rounds beyond that.

We had very good lipid productivity through these procedures, so we tried similar methods looking at Nannochloris sp. and selecting for fast growing species, and we’ve actually been able to almost triple the productivity of this organism. This process we’ve developed looks like a very nice new technology for selection and adapting of species under natural conditions, which would not need to include any transgenic modification of algae.

Mini-raceways at Pacific Northwest National Laboratory

Mini-raceways at Pacific Northwest National Laboratory

What sorts of developments have you had on the cultivation side?

In cultivation we had a major deliverable in showing we could use photobioreactors to achieve growth rates of 5 grams dry weight per liter, with a minimum of 50% lipid content, and in some cases close to 70%. The work was primarily done at New Mexico State University and at Solix Biofuels, two of our partners. They demonstrated that these levels of productivity are achievable under appropriate conditions. The tests were performed continuously over a year and a half period of time.

Probably the last major deliverable that we had earlier this year was a down selection of our harvesting and extraction technologies. We’ve had seven different technologies for harvesting and extraction in development throughout our partnership – five harvesting and two extraction. We went through a very quantitative and rigorous method for comparing these against each other, against benchmarks such as centrifuge techniques and wet extraction techniques that are more popular in the outside world right now. We then downselected to four harvesting and extraction technologies that would continue on to Phase II (scale up).

Of these four technologies, three of them are harvesting technologies, including an electrolytic flocculation technology out of Texas A&M, membrane harvesting technology from Pacific Northwest National Laboratory, and the acoustic harvesting technology at Los Alamos National Laboratory. And then we have a very special extraction technology using mesoporous nanoparticles, out of Iowa State University, targeting very high value chemicals within the oils of the algae, especially polyunsaturated fatty acids and special compounds such as chotopherol (Vitamin E) – which is produced by some algae in fairly high quantities.

So far those are NAABB’s major deliverables and accomplishments. There is a new set of deliverables being worked on right now for month 24, which is this month (April), and those are primarily addressing areas of conversion technologies and some animal feed research and sustainability issues.

How much of this information has been published since the beginning of the project?

In the past two years we have had over 50 technical publications accepted, in submission, or in the preparation process; and 16 intellectual property disclosures for licensing by our partners. And out of those 16, one technology has spun out into a company, Phenometrics LLC, which makes a small, lab-based photobioreactor technology.

Will the original partners stay fixed, or do you bring in new partners over the course of the project?

We’ve added a number of partners in the last year, including Phycal, SRS Energy, Idaho National Laboratories and Reliance Industries, from India. We’ve also entered into a number of memoranda of understanding for collaborative research between NAABB partners and a number of other organizations including Argonne National Laboratory, primarily in using their GREET model system. We’ve entered into an agreement with Advanced Biomass R&D Center, in South Korea, and another research collaborative agreement with Industrial Technologies Research Institute, in Taiwan. Another new partner on the industry side is Pan Pacific, out of Australia, and we’ve just added the Narional Renewable Energy Laboratory (NREL) as a partner to the consortium. So, quite a bit of activity in the partnership arena, and it’s still growing.

Solix Biofuels photobioreactor beds at New Mexico State University

Solix Biofuels photobioreactor beds at New Mexico State University

If you could extrapolate to the end of the project, what do you think will develop over the next year?

Our main goal is essentially to understand the techno-economic viability of algae as a feedstock for the biofuels industry. I think at the end of the third year we will have developed a complete analysis based on currently accepted technologies, and then added to those we’ll have a number of new technologies and innovations that we’ve been able to develop over the course of this project. And I believe we’ll show a much more favorable condition for algae as a feedstock for biofuels.

So, will it be at $2.00 a gallon that everybody would love to see? I don’t think so. But I think it will be much more favorable than we’ve been seeing in the last couple of years. We think we’ll be able to show that we can trim major cost and major risk out of algae as a biofuel.

Does this seem like a once only type of research project, or do you see this as something that will be more common from here on?

I think what we’re seeing is that more and more of these funding opportunities are calling out for consortia type arrangements, and overall the effect has been a very positive approach to developing and managing these kinds of efforts. I think we’ll see consortia as a major way of doing research in the future.

My vision is that we will see an NAABB-type consortium continue, and it will look a bit different beyond Year Three. I hope that the focus for the future will be more towards addressing the objectives of our industrial partners rather than the needs of the funding federal agencies. The funding federal agencies are a good way to get things started and to get some momentum going. But now that we’ve accomplished that, we need to shift our efforts to understanding the industry better, and the issues the industry is facing, and try to develop programs that can go after those things the industry feels are the most important to work on – those that a consortium like ours can take on.

Dr. Richard Sayre, your co-director of the consortium has recently relocated to your home base, LANL. What is he working on and how has this affected the NAABB operations?

Starting with the second question, it has helped tremendously by having him next door to bounce things off on a daily basis, which was not really possible before because of our physical locations. From an operations side it’s helped us gel a bit more. His focus has been primarily in the area of algal biology, so his team has been focusing on understanding contaminants, specifically bacterial contaminants in algal cultures and developing methods for controlling them. Just today he let me know that they have found a number of peptides that will control the bacterial content of algal cultures in a very positive way. I think we’re going to see some new approaches to algal cultivation coming out of this group.

From a LANL perspective, it’s been a great kick. He brings a stature and an energy with him that’s wonderful. Along with the New Mexico consortium universities, we’re developing a laboratory around the concept of biology for energy science, and he will be the first person moving into that laboratory. So he represents our starship for the biofuels development effort here at Los Alamos National Laboratory, and we’re very excited to have him here.

You have an upcoming conference, the 2nd International Conference on Algal Biomass, Biofuels and Bioproducts scheduled for June 10-13, in San Diego. What do you see happening at that conference?

Our primary focus is understanding the latest and greatest in technical and scientific achievements throughout the algal research community internationally.

What we’ll see, first of all, is that it’s going to be huge. We’ve received over 640 abstract submissions both for oral and poster presentations for this conference, which overwhelmed us. Last year the conference attracted 300 people, and it will probably double or triple this year.

Our scientific committee has worked very hard on developing the program, and we will have about 90 oral presentations primarily in two parallel sessions, and a little over four hundred posters. It’s going to be a very high quality scientific and technical conference and San Diego is going to be a great setting.

We’ll see a lot more participation internationally this year than we had last year. The submissions for presentations that came in were almost equal between the U.S. and international.

Final thoughts on NAABB, Year Two?

All in all, this has been a great year, and I hope we’re starting to see an impact from the work being done by the NAABB partners into the algal biofuels community.